CN111944714A - Siamese bacillus HZY-54 growing in anthurium and application thereof - Google Patents
Siamese bacillus HZY-54 growing in anthurium and application thereof Download PDFInfo
- Publication number
- CN111944714A CN111944714A CN202010730157.5A CN202010730157A CN111944714A CN 111944714 A CN111944714 A CN 111944714A CN 202010730157 A CN202010730157 A CN 202010730157A CN 111944714 A CN111944714 A CN 111944714A
- Authority
- CN
- China
- Prior art keywords
- hzy
- strain
- anthurium
- percent
- fermentation
- 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.)
- Granted
Links
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 48
- 241001312221 Anthurium Species 0.000 title claims description 81
- 238000000855 fermentation Methods 0.000 claims abstract description 117
- 230000004151 fermentation Effects 0.000 claims abstract description 117
- 230000012010 growth Effects 0.000 claims abstract description 73
- 241000196324 Embryophyta Species 0.000 claims abstract description 48
- 230000001737 promoting effect Effects 0.000 claims abstract description 20
- 239000013543 active substance Substances 0.000 claims abstract description 15
- 240000000467 Carum carvi Species 0.000 claims abstract description 14
- 241000278457 Bacillus siamensis Species 0.000 claims abstract description 7
- 235000005747 Carum carvi Nutrition 0.000 claims abstract description 7
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims abstract description 7
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- 230000000694 effects Effects 0.000 claims description 136
- 240000003768 Solanum lycopersicum Species 0.000 claims description 114
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 107
- 201000010099 disease Diseases 0.000 claims description 61
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 61
- 238000011282 treatment Methods 0.000 claims description 61
- 239000011550 stock solution Substances 0.000 claims description 59
- 239000007788 liquid Substances 0.000 claims description 45
- 241000894006 Bacteria Species 0.000 claims description 39
- 239000001963 growth medium Substances 0.000 claims description 39
- 239000003085 diluting agent Substances 0.000 claims description 35
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 230000001580 bacterial effect Effects 0.000 claims description 29
- 230000003385 bacteriostatic effect Effects 0.000 claims description 27
- 230000002265 prevention Effects 0.000 claims description 25
- 244000209526 Anthurium andraeanum Species 0.000 claims description 24
- 238000002474 experimental method Methods 0.000 claims description 24
- 239000003814 drug Substances 0.000 claims description 23
- 239000002609 medium Substances 0.000 claims description 20
- 230000035784 germination Effects 0.000 claims description 19
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 claims description 18
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 18
- 239000005760 Difenoconazole Substances 0.000 claims description 18
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 18
- 239000006013 carbendazim Substances 0.000 claims description 18
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 claims description 18
- 229910052799 carbon Inorganic materials 0.000 claims description 18
- BQYJATMQXGBDHF-UHFFFAOYSA-N difenoconazole Chemical compound O1C(C)COC1(C=1C(=CC(OC=2C=CC(Cl)=CC=2)=CC=1)Cl)CN1N=CN=C1 BQYJATMQXGBDHF-UHFFFAOYSA-N 0.000 claims description 18
- 238000012360 testing method Methods 0.000 claims description 18
- 229920002472 Starch Polymers 0.000 claims description 17
- 229910052757 nitrogen Inorganic materials 0.000 claims description 17
- 239000008107 starch Substances 0.000 claims description 17
- 235000019698 starch Nutrition 0.000 claims description 17
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 12
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 11
- 239000000126 substance Substances 0.000 claims description 11
- 238000011068 loading method Methods 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 9
- PVTHJAPFENJVNC-MHRBZPPQSA-N kasugamycin Chemical compound N[C@H]1C[C@H](NC(=N)C(O)=O)[C@@H](C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)[C@@H]1O PVTHJAPFENJVNC-MHRBZPPQSA-N 0.000 claims description 9
- 241000222291 Passalora fulva Species 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 239000012137 tryptone Substances 0.000 claims description 8
- 238000000354 decomposition reaction Methods 0.000 claims description 6
- 230000003247 decreasing effect Effects 0.000 claims description 6
- 238000006460 hydrolysis reaction Methods 0.000 claims description 6
- PLXMOAALOJOTIY-FPTXNFDTSA-N Aesculin Natural products OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)[C@H]1Oc2cc3C=CC(=O)Oc3cc2O PLXMOAALOJOTIY-FPTXNFDTSA-N 0.000 claims description 5
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 5
- 238000012404 In vitro experiment Methods 0.000 claims description 5
- 239000001913 cellulose Substances 0.000 claims description 5
- 229920002678 cellulose Polymers 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 claims description 5
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 claims description 5
- 230000004899 motility Effects 0.000 claims description 5
- 239000004382 Amylase Substances 0.000 claims description 4
- 102000013142 Amylases Human genes 0.000 claims description 4
- 108010065511 Amylases Proteins 0.000 claims description 4
- 102000016938 Catalase Human genes 0.000 claims description 4
- 108010053835 Catalase Proteins 0.000 claims description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 4
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 4
- 235000019418 amylase Nutrition 0.000 claims description 4
- 235000013527 bean curd Nutrition 0.000 claims description 4
- 210000003495 flagella Anatomy 0.000 claims description 4
- 230000015784 hyperosmotic salinity response Effects 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 4
- 150000004965 peroxy acids Chemical class 0.000 claims description 4
- 239000002932 luster Substances 0.000 claims description 3
- 238000011161 development Methods 0.000 abstract description 9
- 239000000575 pesticide Substances 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 8
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 description 38
- 244000052616 bacterial pathogen Species 0.000 description 32
- 238000012216 screening Methods 0.000 description 21
- 230000002401 inhibitory effect Effects 0.000 description 19
- 239000000243 solution Substances 0.000 description 18
- 241000223221 Fusarium oxysporum Species 0.000 description 16
- 238000000034 method Methods 0.000 description 16
- 239000008223 sterile water Substances 0.000 description 16
- 230000001717 pathogenic effect Effects 0.000 description 15
- 244000052769 pathogen Species 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 13
- 241000123650 Botrytis cinerea Species 0.000 description 12
- 240000008067 Cucumis sativus Species 0.000 description 11
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 11
- 238000012258 culturing Methods 0.000 description 11
- 238000010790 dilution Methods 0.000 description 11
- 239000012895 dilution Substances 0.000 description 11
- 239000000706 filtrate Substances 0.000 description 11
- 241000223600 Alternaria Species 0.000 description 9
- 244000053095 fungal pathogen Species 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 241000219109 Citrullus Species 0.000 description 8
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 8
- 241000845082 Panama Species 0.000 description 8
- 241000813090 Rhizoctonia solani Species 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 230000004763 spore germination Effects 0.000 description 8
- 241000213004 Alternaria solani Species 0.000 description 7
- 241000221696 Sclerotinia sclerotiorum Species 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 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
- 241000223218 Fusarium Species 0.000 description 6
- 239000012880 LB liquid culture medium Substances 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 241000233614 Phytophthora Species 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- 230000000443 biocontrol Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000009630 liquid culture Methods 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 241000223208 Curvularia Species 0.000 description 5
- 241000223211 Curvularia lunata Species 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000000306 component Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000011835 investigation Methods 0.000 description 5
- 230000002262 irrigation Effects 0.000 description 5
- 238000003973 irrigation Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 230000007226 seed germination Effects 0.000 description 5
- 239000002689 soil Substances 0.000 description 5
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 4
- 206010059866 Drug resistance Diseases 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241000508192 Fusarium oxysporum f. sp. niveum Species 0.000 description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 4
- 229930195725 Mannitol Natural products 0.000 description 4
- 241000509558 Phytophthora melonis Species 0.000 description 4
- 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 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- 241000589634 Xanthomonas Species 0.000 description 4
- 235000015278 beef Nutrition 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000000227 grinding Methods 0.000 description 4
- 239000008101 lactose Substances 0.000 description 4
- 230000003902 lesion Effects 0.000 description 4
- 239000000594 mannitol Substances 0.000 description 4
- 235000010355 mannitol Nutrition 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 4
- 230000008635 plant growth Effects 0.000 description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 3
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 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 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 3
- 206010027146 Melanoderma Diseases 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 244000088415 Raphanus sativus Species 0.000 description 3
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 3
- 235000002560 Solanum lycopersicum Nutrition 0.000 description 3
- 244000061458 Solanum melongena Species 0.000 description 3
- 235000002597 Solanum melongena Nutrition 0.000 description 3
- 241000209149 Zea Species 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 230000003042 antagnostic effect Effects 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000003912 environmental pollution Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 238000005507 spraying Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 241001677259 Acanthophoenix rubra Species 0.000 description 2
- 240000007124 Brassica oleracea Species 0.000 description 2
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 2
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 2
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 2
- 241001610411 Fourraea alpina Species 0.000 description 2
- 241000223224 Fusarium oxysporum f. cucumerinum Species 0.000 description 2
- 241000233654 Oomycetes Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241000588702 Pectobacterium carotovorum subsp. carotovorum Species 0.000 description 2
- 241000172199 Phyllospora Species 0.000 description 2
- 241000521936 Pseudomonas amygdali pv. lachrymans Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 206010039509 Scab Diseases 0.000 description 2
- 241001558929 Sclerotium <basidiomycota> Species 0.000 description 2
- 241001123668 Verticillium dahliae Species 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000013401 experimental design Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- MLFHJEHSLIIPHL-UHFFFAOYSA-N isoamyl acetate Chemical compound CC(C)CCOC(C)=O MLFHJEHSLIIPHL-UHFFFAOYSA-N 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000035040 seed growth Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000010455 vermiculite Substances 0.000 description 2
- 235000019354 vermiculite Nutrition 0.000 description 2
- 229910052902 vermiculite Inorganic materials 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- RXZBMPWDPOLZGW-XMRMVWPWSA-N (E)-roxithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=N/OCOCCOC)/[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 RXZBMPWDPOLZGW-XMRMVWPWSA-N 0.000 description 1
- OICGFSUWXCJLCO-UHFFFAOYSA-N 1-ethylsulfonylsulfanylethane Chemical compound CCSS(=O)(=O)CC OICGFSUWXCJLCO-UHFFFAOYSA-N 0.000 description 1
- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 description 1
- 241000223602 Alternaria alternata Species 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 235000011331 Brassica Nutrition 0.000 description 1
- 241000193417 Brevibacillus laterosporus Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 239000005944 Chlorpyrifos Substances 0.000 description 1
- WNBCMONIPIJTSB-BGNCJLHMSA-N Cichoriin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1)c1c(O)cc2c(OC(=O)C=C2)c1 WNBCMONIPIJTSB-BGNCJLHMSA-N 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 206010052804 Drug tolerance Diseases 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241001555627 Melonis Species 0.000 description 1
- 241000228347 Monascus <ascomycete fungus> Species 0.000 description 1
- 241001602876 Nata Species 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 241000232299 Ralstonia Species 0.000 description 1
- 241001448530 Rohdea japonica Species 0.000 description 1
- 241000221662 Sclerotinia Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 235000002634 Solanum Nutrition 0.000 description 1
- 241000207763 Solanum Species 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 241000836034 Westindia Species 0.000 description 1
- 241000194062 Xanthomonas phaseoli Species 0.000 description 1
- 241000321040 Xanthomonas phaseoli pv. dieffenbachiae Species 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- KIPLYOUQVMMOHB-MXWBXKMOSA-L [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O Chemical compound [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O KIPLYOUQVMMOHB-MXWBXKMOSA-L 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940111121 antirheumatic drug quinolines Drugs 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- SBPBAQFWLVIOKP-UHFFFAOYSA-N chlorpyrifos Chemical compound CCOP(=S)(OCC)OC1=NC(Cl)=C(Cl)C=C1Cl SBPBAQFWLVIOKP-UHFFFAOYSA-N 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 229940093496 esculin Drugs 0.000 description 1
- AWRMZKLXZLNBBK-UHFFFAOYSA-N esculin Natural products OC1OC(COc2cc3C=CC(=O)Oc3cc2O)C(O)C(O)C1O AWRMZKLXZLNBBK-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 230000009546 growth abnormality Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 229940117955 isoamyl acetate Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- OGJPXUAPXNRGGI-UHFFFAOYSA-N norfloxacin Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNCC1 OGJPXUAPXNRGGI-UHFFFAOYSA-N 0.000 description 1
- 229960001180 norfloxacin Drugs 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000008659 phytopathology Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000004382 potting Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003248 quinolines Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000002336 repolarization Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 229960005224 roxithromycin Drugs 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229940063650 terramycin Drugs 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 150000003557 thiazoles Chemical class 0.000 description 1
- 235000012184 tortilla Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
Images
Classifications
-
- 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
- C12N1/205—Bacterial isolates
-
- 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
- C12R2001/07—Bacillus
-
- 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
- A01N63/22—Bacillus
-
- 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
- C12N1/02—Separating microorganisms from their culture media
-
- 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
Abstract
The invention relates to a caraway endogenous Siamese bacillus HZY-54 separated from a caraway plant, wherein the HZY-54 strain is named as follows: HZY-54, class name: siamese Bacillus (Bacillus siamensis), the collection number is: CGMCC No.20159, preservation date: 29/6/2020, depository: china general microbiological culture Collection center, West Lu No. 1 Hospital No. 3, Beijing, Chaoyang, the area of the republic of China. The strain HZY-54 can be stably colonized in a plant body, and the fermentation liquor contains growth promoting active substances and broad-spectrum antibacterial active substances, so that a foundation is laid for the development and research of rich natural resources of biological pesticides and microbial pesticides.
Description
Technical Field
The invention belongs to the field of biological control, and particularly relates to a monascus siamensis HZY-54 in a anthurium and application thereof.
Background
The anthurium andraeanum has a peculiar and attractive appearance, is full of vitality and vitality, is a hotspot for people to choose in the early stage, and brings considerable development prospect and economic value to the economic industry of anthurium andraeanum flowers in China. In the 70 th of the 20 th century, anthurium varieties are introduced for the first time in China, and after the 90 th of the China begins to formally develop the anthurium system in industrial scale, more and more farmers participate in the anthurium system. However, the failure or unreasonable management work such as planting, cultivation, maintenance and the like causes the large-area outbreak of the anthurium disease, particularly the leaf blight.
The frequent anthurium diseases cannot be effectively prevented and controlled, more and more high-disease-resistant varieties can be selected by people in the flower market, and the supply of anthurium is greater than the demand, so that the economic industry is weakened and seriously damaged.
Among them, the occurrence and prevalence of leaf blight of anthurium are the most serious. The disease has two infection modes. The disease starts from the leaf part, invades from the edge of the leaf, stomata or wound, and after the infection succeeds, water stain-like disease spots are formed on the back of the leaf, and the disease spots are deepened and necrotized in the later period. Another pathogen spreads to other parts through the vascular bundles of the stem of the plant. The disease is fond of warm and humid and strong-light growing environment, and has rapid development and spread and strong infectivity.
The disease started in 1960 in brazil and then developed in a large area in america. It began to flood the islands of the west india, france in 1989 and was determined to be cold-tolerant pseudomonad (Pseudomonas sp.). In 1999, the American scholars determined the disease pathogenic bacteria to be ralstonia (Psedomonas) solanacearum. After 2000 years, the disease begins to spread widely in China. Originally popular in southern wet areas, the pathogenic bacteria causing the anthurium bacterial blight was preliminarily identified by experts and scholars in China as a pathogenic variant of Rohdea japonica (Xanthomonas phaseoli pv. reeffenbach)
After determining the pathogeny of the disease, China quickly develops the prevention and treatment work. In the current work of disease control, chemical preparations are the first choice of people as the most economical and rapid prevention and control means, and among them, copper preparations, agricultural antibiotics, thiazoles, quinolines and other preparations are widely used.
The prevention and treatment work aiming at the bacterial leaf blight of anthurium andraeanum is no exception, the early stage Jiang Gui Zhi and the like find that terramycin has better prevention and treatment effects on anthurium andraeanum, the old and new stage also find that the pathogenic bacteria are sensitive to 25 ten thousand U medical tetracycline, the recent period Zhang Shipeng and the like use household medicines to carry out bacteriostasis experiments to verify that the roxithromycin capsules and norfloxacin capsules have better inhibition effects on xanthomonas, and then Wang Ying 35678 and the like carry out greenhouse experiments on the leaf blight of anthurium andraeanum, and finally, the result shows that ethylicin and kasugamycin have better prevention and treatment effects on anthuri.
If the chemical preparation is used properly, the disease can be effectively solved or alleviated to some extent. However, with the great increase of the input amount of chemical substances in the environment and the unscientific use method, the drug resistance and drug tolerance of pathogens are continuously improved, and the prevention and treatment effect is gradually reduced. In addition, the chemical substances remained in the environment seriously pollute the atmosphere, natural resources such as soil and water resources, the ecological environment is forced to be damaged, and the daily life loss of people is serious.
Although effective, chemical agents are irreversible. The biological control method can effectively control diseases, relieve the problems of drug resistance, environmental pollution and the like, and has the advantages of wide sources, rich varieties and economy.
Biological control is characterized in that organisms or metabolites of certain organisms are used for replacing chemical substances to inhibit the growth and development of pathogens. Chaiqingka et al found that when the fermentation stock solution of biocontrol strain bacillus amyloliquefaciens LJ02 and 100-fold diluent are used for root irrigation treatment of cucumber, LJ02 can effectively inhibit gray mold of cucumber within 7 days, and fermentation supernatant can induce various gene expressions of pathogenic bacteria. Fang and other research results show that the bacillus laterosporus can effectively degrade the content of chlorpyrifos remained in soil and vegetables, and the problem of environmental pollution is relieved. Guo sea and the like find that the bacillus amyloliquefaciens 262AG6 has good bacteriostatic effect and good performance on plant growth and ecological environment. Arrebella et al isolated a strain of Bacillus amyloliquefaciens which has certain antibacterial activity against 7 pathogenic fungi of harvested citrus. Besides the advantages of a plurality of endophytes, the bacillus has stronger stress resistance than most of biocontrol bacteria, and the stress resistance of host plants is also improved. But the research on the biological control method of the leaf blight of anthurium andraeanum is very little at home and abroad.
The disease of anthurium andraeanum leaf blight is quick, no specific medicine exists, the flower industry is easy to culture, high-disease-resistant varieties are more and more, and the system form of the anthurium andraeanum industry is extremely severe.
The problems of environmental pollution, drug resistance and drug resistance are probably considered while the anthurium disease is solved, and the research, development, popularization and implementation of the biological preparation with a severe form, high efficiency and no toxicity are not slow.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a Siamese bacillus HZY-54 growing in a anthurium and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a caraway endogenous Siamese bacillus HZY-54 separated from a caraway plant, wherein the HZY-54 strain is named as: HZY-54, class name: siamese Bacillus (Bacillus siamensis), the collection number is: CGMCC No.20159, preservation date: 29/6/2020, depository: china general microbiological culture Collection center, West Lu No. 1 Hospital No. 3, Beijing, Chaoyang, the area of the republic of China.
Moreover, the HZY-54 strain is milky round, and the surface of a fresh colony is wet, smooth and sticky. Along with the increase of the culture time, the surface of a Siamese bacillus HZY-54 colony in a anthurium becomes rough, the middle part of the colony is inwards sunken, no luster exists, the texture is dry and astringent, and the colony is in a bean curd residue shape; the endophytic Siamese bacillus HZY-54 in the anthurium can be clearly seen in an electron microscope picture, the thallus is in a short rod shape, two flagella are fine and long, the thallus is full, and the surface is smooth.
Moreover, the HZY-5 strain is a gram-positive bacterium; the starch hydrolysis reaction is positive and can produce amylase; can grow in the temperature of 4-45 ℃, wherein the temperature of 37 ℃ is the most suitable temperature for Y-54 to grow; the pH value is 3.0, the pH value is 12.0, namely the peracid and the alkali can not grow, and the pH value is 7.0, and the neutral can normally grow; the methyl red reaction is negative; the decomposition reaction of the leucine is negative; cellulose cannot be decomposed; the drying resistance is strong; has motility; the salt tolerance is strong; the citrate utilization culture medium is alkaline; the V-P experiment shows negative; the aesculin is positive in water receiving display; no fluorescence was observed on KB plates; positive in catalase test.
Moreover, the culture medium of the HZY-5 strain is LB culture medium: the carbon, nitrogen source and inorganic salt are preferably starch, tryptone and magnesium chloride; the fermentation conditions are that the rotating speed is 200r/min, the pH is 8, the liquid loading amount is 75mL/250mL, the inoculation amount is 3 percent, the fermentation time is 72 hours, the viable count of the HZY-54 strain reaches 2.43 multiplied by 109cfu/mL。
The application of the caraway endogenous Siamese bacillus HZY-54 separated from the caraway plant in the aspects of stable colonization in the plant body and/or production of broad-spectrum bacteriostatic active substances and/or growth promoting substances.
Moreover, the Siamese bacillus HZY-54 growing in the anthurium can be colonized in the tomatoes and the anthurium, the general growth trend of increasing first and then decreasing is shown in the tomatoes, and the colonized bacteria amount is always root>Leaf part>Stem part, and the maximum colonization amount of the root part, stem part and leaf part of the tomato at 11d, and the maximum colonization amount of the stem part, stem part and leaf part of the tomato at 9-11d can be respectively 2.93 multiplied by 105cfu/mL、1.13×105cfu/mL and 1.53X 105cfu/mL; in the colonization period 15d of anthurium andraeanum, the colonization bacteria amount is as follows from large to small: root of a tree>Leaf part>The stem part of the plant grows in the anthurium andraeanum plant body and then expands, and the 7 th d colonization amount tends to be the mostLarge, continuing until 11 d.
Moreover, the maximum IAA production amount of the fermentation liquor of the Siamese bacillus HZY-54 growing in the anthurium is 97.23 mu g/L at the 3 rd time, and the germination index of the tomato seeds treated by the stock solution is improved by 28.11 percent; the plant height, root length, fresh weight, stem thickness, leaf number and leaf area of tomato seedlings are respectively increased by 198.85mm, 130.28mm, 147.48g, 2.63mm, 38 pieces and 12.70cm2。
Moreover, the growth promoting active substances and the broad spectrum antibacterial active substances are derived from fermentation liquor obtained after the HZY-54 strain is fermented and cultured.
The culture conditions of the HZY-54 strain fermentation culture are that the rotation speed is 200r/min, the pH value is 8, the liquid loading amount is 75mL/125mL, the inoculation amount is 3%, and the culture time is 72 h; the basic fermentation medium is LB, the used carbon source is soluble starch, the used nitrogen source is tryptone, and the inorganic salt is MgCl2。
Moreover, the original solution of the fermentation liquid has the best effect on indoor in vitro experiments of Cladosporium fulvum, the disease indexes of the tomato leaves pretreated by the original solution are only 2.00 percent, and the disease indexes of the tomato leaves pretreated by 10X, 20X, 50X and 100X diluent are respectively 2.67 percent, 14.00 percent, 24.67 percent, 38.89 percent and 45.33 percent;
or the control effects of the fermentation liquor stock solution and the medicament difenoconazole are as follows: the control effect of 14d is respectively 79.14 percent and 71.07 percent, and the effect of the carbendazim medicament is as follows: the prevention effect of 14d is 56.04%, the effect of 50X is the worst, namely the prevention effect of 14d is 35.25%, and in the same time, the leaves of the plant in the medicament difenoconazole and strain HZY-54 stock solution treatment group are complete, the freshness is better, the small area of the leaves of the plant in the carbendazim treatment group is green, yellow and fresh, the disease of the 50X treatment group and the CK group is serious, the large area of the leaves of the plant is yellow, wilted and poor in freshness;
or the control effect of the 50X diluted fermentation liquid and kasugamycin is as follows: the 14d control effect is 66.95 percent and 68.29 percent, the stock solution control effect is the worst, and the 14d control effect is 36.65 percent.
The invention has the advantages and positive effects that:
1. the invention relates to a Siamese bacillus HZY-54 growing in a anthurium, which is a strain with broad bacteriostatic spectrum and remarkable bacteriostatic effect obtained from leaves after being separated and purified from different tissues of the anthurium and screened; the strain HZY-54 can be stably colonized in a plant body, and the fermentation liquor contains growth promoting active substances and broad-spectrum antibacterial active substances, thereby laying a foundation for the development and research of enriching natural resources of biological pesticides and microbial pesticides.
2. The siamese bacillus HZY-54 growing in the anthurium can be stably colonized in the anthurium and tomato plants, the fermentation liquor contains active substances which can promote the growth of plants and have bacteriostatic effects, and the bacteriostatic active substances which are high-efficiency, broad-spectrum, low-toxicity and environment-friendly can play a good bacteriostatic role, so that references can be provided for the research of the development and utilization of abundant natural biological pesticide resources and microbial pesticides.
3. The 50X fermentation stock solution of the siamesed bacillus HZY-54 growing in the anthurium can effectively prevent and treat bacterial leaf blight of the anthurium and leaf mold diseases of tomatoes.
Drawings
FIG. 1 is a graph showing the results of primary screening of a red palm endophytic Siamese bacillus strain with biocontrol properties in accordance with the present invention;
FIG. 2 is a diagram showing the result of rescreening a red palm endogenic Siamese bacillus with biocontrol properties in the present invention;
FIG. 3 is a diagram showing the inhibitory effect of the Siamese bacillus HZY-54 strain in the anthurium on 10 kinds of pathogenic bacteria; wherein, the first row of pictures are sequentially Rhizoctonia solani (Rhizoctonia solani), watermelon Fusarium oxysporum (Fusarium oxysporum f.sp.niveum), cucumber Phytophthora (Phytophthora melonis), eggplant Sclerotinia sclerotiorum (sclerotirotinia sclerotiorum), radish black spot (Alternaria brassicca), and the second row of pictures are sequentially solanum solani (Alternaria solani), cucumber Fusarium oxysporum (Fusarium oxysporum f.sp.cuumbeum), tomato leaf mold (Cladosporium fulvum), tomato Botrytis cinerea (Botrytis cinerea) and maize Curvularia tortilla (Curvularia lunata) from left to right;
FIG. 4 is a graph showing the inhibitory effect of Siamese bacillus HZY-54 fermentation broth on 10 kinds of pathogenic bacteria in the anthurium of the present invention; wherein, the first row of pictures are sequentially rhizoctonia solani (R.solani), fusarium oxysporum (F.oxysporum f.sp.niveum), phytophthora cucumeri (P.melonicium), sclerotinia solani (S.sclerotiorum), alternaria carota (A.brassica), and the second row of pictures are sequentially solanum lycopersicum (A.solani), fusarium cucumerinum (F.cuumber f.sp.cuumber), botrytis cinerea (C.fulvum), botrytis cinerea (B.cinerea) and curvularia zea (C.lunata) from left to right;
FIG. 5 is a graph showing the inhibitory effect of Siamese bacillus HZY-54 fermentation filtrate on 10 pathogenic bacteria in the anthurium of the present invention; wherein, the pictures are sequentially verticillium dahliae (R.solani), fusarium oxysporum (F.oxysporum f.sp.niveum), phytophthora cucumeri (P.melonis), sclerotinia sclerotiorum (S.sclerotiorum), alternaria carota (A.brassica), and the pictures in the second row are sequentially solanum lycopersicum (A.solani), fusarium cucumerinum (F.culorum), botrytis cinerea (C.fulvum), botrytis cinerea (B.cinerea) and curvularia zea (C.lucata) from left to right;
FIG. 6 is a blank control chart of the inhibitory effect of 10 pathogenic bacteria according to the present invention; wherein, the pictures are sequentially verticillium dahliae (R.solani), fusarium oxysporum (F.oxysporum.sp.niveum), phytophthora cucumeri (P.melonis), sclerotinia sclerotiorum (S.sclerotiorum), alternaria solani (A.brassiccus), and the pictures in the second row are sequentially solanum lycopersicum (A.solani), fusarium oxysporum (F.oxysporum.sp.cuumbeum), botrytis cinerea (C.fulvum), botrytis cinerea (B.cinerea) and curvularia zea (C.lunata) from left to right;
FIG. 7 is a graph showing the inhibitory effect of Siamese bacillus HZY-54 fermentation broth on 3 pathogenic bacteria in the anthurium of the present invention; wherein, from left to right, the soft rot bacterium (Erwinia carotovora subsp. carotovora Ecc), the pseudomonad (Pseudomonas syringae pv. lachrymans Psl), the Xanthomonas (Xanthomonas axonodis pv. dioffenbachiae Xad) are arranged in sequence;
FIG. 8 is a graph showing the effect of Siamese bacillus HZY-54 fermented filtrate in anthurium on germination of tomato leaf mold germ spores; wherein the upper graph is the spore inhibition rate, and the lower graph is the spore germination rate;
FIG. 9 is a graph showing the effect of Siamese bacillus HZY-54 fermented filtrate in anthurium on tomato leaf mold germ hyphae; wherein the first row of pictures are sequentially HZY-54 sterile filtrate, 50 times of diluent, difenoconazole and carbendazim from left to right, and the second row of pictures are blank control groups;
FIG. 10 is a drawing of a Siamese bacillus HZY-54 colony and an electron microscope in the anthurium of the present invention; wherein, the left image is a colony image, and the right image is an electron microscope image;
FIG. 11 is a graph showing the result of electrophoresis of the 16SrDNA amplification product of Siamese bacillus HZY-54 in the anthurium of the present invention;
FIG. 12 is a phylogenetic tree of the Siamese bacillus HZY-54 growing in the anthurium of the present invention;
FIG. 13 is a graph showing the effect of different basal media on the viable count of the HZY-54 strain in the present invention;
FIG. 14 is a graph showing the effect of different C sources on the viable count of the HZY-54 strain in the present invention;
FIG. 15 is a graph showing the effect of different N sources on the inhibition rate of the viable count of the HZY-54 strain in the present invention;
FIG. 16 is a graph showing the effect of different inorganic salts on the bacterial inhibition rate of the viable count of HZY-54 in the present invention;
FIG. 17 is a graph of the colonization cycle of various parts of a tomato plant by the strain HZY-54 of the present invention;
FIG. 18 is a graph showing the change in the production of HZY-54IAA of the strain of the present invention;
FIG. 19 is a data chart of the effect of fermentation broth of HZY-54 strain on tomato seed germination in the present invention;
FIG. 20 is a graph showing the effect of fermentation broth of HZY-54 strain on tomato seed germination in the present invention; wherein, the pictures are sequentially stock solution, 10 times of diluent, 20 times of diluent, 50 times of diluent, 100 times of diluent and a blank control group from left to right;
FIG. 21 is a data graph of the strain fermentation broth of HZY-54 according to the present invention on the growth of tomato seedlings in plant height;
FIG. 22 is a data plot of the HZY-54 strain broth versus the growth of tomato seedlings in root length in accordance with the present invention;
FIG. 23 is a data plot of fermentation broth of HZY-54 strain of the present invention on stem thickness growth of tomato seedlings;
FIG. 24 is a data plot of the growth of the HZY-54 strain broth versus the number of leaves of a tomato seedling in accordance with the present invention;
FIG. 25 is a data plot of the broth of the HZY-54 strain of the present invention versus the fresh weight growth of tomato seedlings;
FIG. 26 is a data plot of the HZY-54 strain broth versus growth of tomato seedling leaf length in accordance with the present invention;
FIG. 27 is a data plot of the HZY-54 strain broth versus leaf width growth of tomato seedlings in accordance with the present invention;
FIG. 28 is a graph showing the effect of fermentation broth of HZY-54 strain in the present invention on the growth of tomato seedlings; wherein, the pictures are sequentially stock solution, 10 times of diluent, 20 times of diluent, 50 times of diluent, 100 times of diluent and a blank control group from left to right;
FIG. 29 is a graph showing the effect of fermentation broth of HZY-54 strain in the present invention on the growth of anthurium andraeanum seedlings; wherein, the pictures are sequentially stock solution, 50 times of diluent and a blank control group from left to right;
FIG. 30 is a graph showing the in vitro control effect of the fermentation broth of HZY-54 strain on tomato leaves in the present invention; wherein, the pictures are sequentially stock solution, 10 times of diluent, 20 times of diluent, 50 times of diluent, 100 times of diluent and a blank control group from left to right;
FIG. 31 is a diagram showing the effect of fermentation broth of HZY-54 strain on indoor potting of tomato in the present invention; wherein the pictures are sequentially difenoconazole, carbendazim, stock solution, 50-time diluent and a blank control group from left to right;
FIG. 32 is a diagram showing greenhouse effect of fermentation broth of HZY-54 strain on anthurium in the present invention; wherein, the pictures are a control group, 50 times of diluent, stock solution and difenoconazole from left to right in sequence.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
The raw materials used in the invention are all conventional commercial products if no special description is provided, the method used in the invention is all conventional methods in the field if no special description is provided, and the mass of all the materials used in the invention is the conventional use mass.
A caraway endogenous Siamese bacillus HZY-54 separated from a caraway plant, wherein the HZY-54 strain is named as: HZY-54, class name: siamese Bacillus (Bacillus siamensis), the collection number is: CGMCC No.20159, preservation date: 29/6/2020, depository: china general microbiological culture Collection center, West Lu No. 1 Hospital No. 3, Beijing, Chaoyang, the area of the republic of China.
Preferably, the HZY-54 strain is milky round, and the surface of a fresh colony is wet, smooth and sticky. Along with the increase of the culture time, the surface of a Siamese bacillus HZY-54 colony in a anthurium becomes rough, the middle part of the colony is inwards sunken, no luster exists, the texture is dry and astringent, and the colony is in a bean curd residue shape; the endophytic Siamese bacillus HZY-54 in the anthurium can be clearly seen in an electron microscope picture, the thallus is in a short rod shape, two flagella are fine and long, the thallus is full, and the surface is smooth.
Preferably, the HZY-5 strain is a gram-positive bacterium; the starch hydrolysis reaction is positive and can produce amylase; can grow in the temperature of 4-45 ℃, wherein the temperature of 37 ℃ is the most suitable temperature for Y-54 to grow; the pH value is 3.0, the pH value is 12.0, namely the peracid and the alkali can not grow, and the pH value is 7.0, and the neutral can normally grow; the methyl red reaction is negative; the decomposition reaction of the leucine is negative; cellulose cannot be decomposed; the drying resistance is strong; has motility; the salt tolerance is strong; citrate utilization medium is alkaline (dark blue); the V-P experiment shows negative; the aesculin is positive in water receiving display; no fluorescence was observed on KB plates; positive in catalase test.
Preferably, the culture medium of the HZY-5 strain is an LB culture medium: the carbon, nitrogen source and inorganic salt are preferably starch, tryptone and magnesium chloride; the fermentation conditions are that the rotating speed is 200r/min, the pH is 8, the liquid loading amount is 75mL/250mL, the inoculation amount is 3 percent, the fermentation time is 72 hours, the viable count of the HZY-54 strain reaches 2.43 multiplied by 109cfu/mL。
The application of the caraway endogenous Siamese bacillus HZY-54 separated from the caraway plant in the aspects of stable colonization in the plant body and/or production of broad-spectrum bacteriostatic active substances and/or growth promoting substances.
Preferably, the Siamese bacillus HZY-54 growing in the anthurium can be colonized in the tomatoes and the anthurium, the general growth trend of increasing first and then decreasing is presented in the tomatoes, and the colonized bacteria amount is always root>Leaf part>Stem part, and the maximum colonization amount of the root part, stem part and leaf part of the tomato at 11d, and the maximum colonization amount of the stem part, stem part and leaf part of the tomato at 9-11d can be respectively 2.93 multiplied by 105cfu/mL、1.13×105cfu/mL and 1.53X 105cfu/mL; in the colonization period 15d of anthurium andraeanum, the colonization bacteria amount is as follows from large to small: root of a tree>Leaf part>The stem part is increased in the anthurium plant body firstly and then expands, the colonization amount of the 7 th d tends to be maximum, and the colonization amount lasts until the 11 th d.
Preferably, the maximum IAA production amount of the fermentation liquor of the Siamese bacillus HZY-54 growing in the anthurium is 97.23 mu g/L at the 3 rd time, the effect of the stock solution is good, and the germination index of the tomato seeds treated by the stock solution is improved by 28.11 percent; the plant height, root length, fresh weight, stem thickness, leaf number and leaf area of tomato seedlings are respectively increased by 198.85mm, 130.28mm, 147.48g, 2.63mm, 38 pieces and 12.70cm2。
Preferably, the growth promoting active substance and the broad spectrum bacteriostatic active substance are derived from fermentation liquor obtained after the HZY-54 strain is fermented and cultured.
Preferably, the culture conditions of the HZY-54 strain fermentation culture are that the rotating speed is 200r/min, the pH value is 8, the liquid loading capacity is 75mL/125mL, the inoculation amount is 3%, and the culture time is 72 h; the basic fermentation medium is LB, the used carbon source is soluble starch, the used nitrogen source is tryptone, and the inorganic salt is MgCl2。
Preferably, the stock solution of the fermentation liquor has the best effect on the indoor in vitro experiment of C.fulvum, the disease index of the tomato leaves pretreated by the stock solution is only 2.00 percent, and the disease indexes of the tomato leaves pretreated by the 10X, 20X, 50X and 100X diluent are respectively 2.67 percent, 14.00 percent, 24.67 percent, 38.89 percent and 45.33 percent;
or the control effects of the fermentation liquor stock solution and the medicament difenoconazole are as follows: the control effect of 14d is respectively 79.14 percent and 71.07 percent, and the effect of the carbendazim medicament is as follows: the prevention effect of 14d is 56.04%, the effect of 50X is the worst, namely the prevention effect of 14d is 35.25%, and in the same time, the leaves of the plant in the medicament difenoconazole and strain HZY-54 stock solution treatment group are complete, the freshness is better, the small area of the leaves of the plant in the carbendazim treatment group is green, yellow and fresh, the disease of the 50X treatment group and the CK group is serious, the large area of the leaves of the plant is yellow, wilted and poor in freshness;
or the control effect of the 50X diluted fermentation liquid and kasugamycin is as follows: the 14d control effect is 66.95 percent and 68.29 percent, the stock solution control effect is the worst, and the 14d control effect is 36.65 percent; the fermentation stock solution of the HZY-54 strain has almost no prevention effect at first, but the prevention effect is improved under the condition that each treatment group has no any supplementary application along with the increase of time.
More specifically, the preparation and detection are as follows:
1. screening of Strain HZY-54
1.1 materials
A sample to be tested: fresh and healthy anthurium, a variety of panama and vitamin are provided by a flower demonstration center in Tianjin, and 30 pots are provided respectively.
Test strains: cucumber Fusarium oxysporum (Fusarium ox-ysporaum. f.sp.cucumerium), Rhizoctonia solani Kuhn, watermelon Fusarium oxysporum (Fusarium oxysporum f.sp.niveum), cucumber Phytophthora blight (Phytophthora melonis), eggplant sclerotium (sclerotium sclerotiorum), Alternaria nigra (Alternaria brassica), tomato early blight (Alternaria solani), tomato leaf mold (Cladosporium fulvum), tomato gray mold (Botrytis cinerea), corn Curvularia lunata (Curvularia lunata nata); anthurium bacterial leaf blight (Xanthomonas axonopodis pv. dieffenbachiae Xad), cucumber bacterial angular leaf blight (Pseudomonas syringae pv. lachrymans Psl), and cabbage soft rot (Erwinia carotovora subsp. carotovora Ecc), all provided by the institute of Otsu Phytopathology laboratory.
Culture medium: YDC for culture Xad; LB for Ecc and B.endophytic bacteria; LB liquid culture medium is used for Xad, Ecc, Psl and fermentation culture of endophytic bacillus; KB for Psl culture; PDA was used to culture fungi and perform bacteriostatic tests.
1.2 methods
1.2.1 isolation, purification and preservation of endophytes
1. Cleaning healthy anthurium andraeanum material with ultrasonic cleaner for 10min, cutting root, stem, leaf, etc. parts of plant with sterilized scissors into pieces of about 1cm, sterilizing once every time, and standing separately;
2. soaking the treated sample in 75% alcohol for 2-3 min; after cleaning, sterilizing again by using sodium hypochlorite (NaClO) solution with the mass concentration of 3%; then taking out and putting into sterile water for washing for 3 times, and leaving the washing liquid when washing for 3 times.
3. Putting the cleaned crushed material into a sterilized mortar, grinding the crushed material into slurry, standing for 5min, sucking a small amount of supernatant of grinding fluid by a fluid-moving gun, beating the supernatant onto an LB culture medium, separating by using two methods of spreader coating and inoculating loop scribing, and culturing for 72h at 28 ℃ with CK as cleaning fluid for the 3 rd time; the representative strain was again selected for transfer purification and cultured at 28 ℃ for 48 hours.
4. The purified endophyte is subjected to shaking culture at 28 ℃ for 24h, then is mixed with 50% of glycerol, and is subpackaged in a centrifuge tube, and the mixture can be stored for a long time after being frozen in a refrigerator at-20 ℃.
1.2.2 prescreening
Watermelon wilt germs are used as primary screening indicator bacteria. The endophyte is shake cultured in a shaking table at 30 ℃ and 180r/min for 24h to obtain fermentation liquor. Inoculating pathogenic fungi in the center of PDA culture medium by plate confronting culture method. Four filter paper sheets are placed at a position 1.5cm away from the periphery of pathogenic bacteria, 10 mu L of different endophyte fermentation liquid is inoculated on the filter paper sheets respectively, the process is repeated for 3 times, the culture is carried out for 7d at 28 ℃ with the sterile water being CK, and the bacteriostasis rate is calculated.
1.2.3 double sifting
The alternaria philoxeroides is used as a secondary screening target bacterium. The experimental procedure was consistent with 1.2.2.
1.2.4 inhibition of pathogenic bacteria
1.2.4.1 inhibition of pathogenic fungi and Oomycota
(1) Inhibition of endophyte fermentation liquor on pathogenic bacteria
And (3) carrying out full shake culture on the endophyte in a shaking table at 30 ℃ and 180r/min for 24 hours to obtain fermentation liquor. The pathogenic fungi are inoculated in the midpoint of the PDA culture medium by adopting a four-point confrontation culture method. Four filter paper sheets are placed at a position 1.5cm away from the periphery of the pathogenic bacteria, 10 mu L of endophyte fermentation liquor is inoculated on the filter paper sheets respectively, the process is repeated for 3 times, the culture is carried out for 7d at the temperature of 28 ℃, and the bacteriostasis rate is calculated.
(2) Inhibitory Effect of endophytic bacterial strains on pathogenic bacteria
Inoculating pathogenic fungi to the middle position of a PDA (personal digital assistant) plate, streaking and inoculating endophytes at a position 1.5cm away from the periphery of the pathogenic fungi, repeating for 3 times, culturing at 28 ℃ for 7d with sterile water as CK, and calculating the bacteriostasis rate.
(3) Inhibition of pathogenic bacteria by endophyte secondary biomass
Preparation of sterile filtrate:
the 3d endophytes were prepared and inoculated into LB liquid medium and cultured on a shaker for 48 h. Subpackaging the fermentation liquid, centrifuging at 12000r/min for 20min at 4 deg.C, collecting supernatant, filtering the supernatant with 0.22 μm disposable microporous membrane, and storing at 4 deg.C.
And (3) fully mixing the endophyte sterile filtrate with a PDA culture medium (the volume ratio is 1:10), wherein the temperature of the culture medium is not suitable to be too high so as to prevent the endophyte secondary substance from not resisting high temperature and inactivating. Inoculating the pathogenic bacteria to the center of the culture medium, culturing at 2 deg.C for 7d, and repeating for 3 times, wherein the sterile water is CK.
Preparation of spore suspension:
transferring fresh spores of pathogenic bacteria of tomato leaf mold into a centrifuge tube with a proper amount of sterile water, and filtering to obtain spore suspension.
After the spore suspension is diluted to 1 × 104 times by sterile water, 1mL of the spore suspension, 1mL of the stock solution of the sterile filtrate of HZY-54, 50X diluent, 1000X difenoconazole and 500X carbendazim medicament are sucked and fully mixed, sterile water is used as CK, each treatment is repeated for 3 times, and the culture is carried out for 24 hours. Appropriate amount of each treated mixed solution is absorbed and placed on a clean glass slide without impurities, and the spore germination rate (based on the growth of a germ tube) is observed.
Spore germination rate ═ number of spores/total spores) × 100% (smith et al, 2019; lium et al, 2016).
The spore germination inhibition rate is (control spore germination rate-treated spore germination rate)/the control spore germination rate is multiplied by 100%.
Selecting hypha of tomato leaf mold disease pathogen, adding 1mL of HZY-54 sterile filtrate, 50X diluent, 1000X difenoconazole and 500X carbendazim into a sterile centrifuge tube respectively, fully soaking, taking sterile water as CK, and observing the morphological change of the hypha under a microscope after 24 hours.
1.2.4.2 inhibitory Effect on pathogenic bacteria
Xad, Psl and Ecc are subjected to shaking culture at 28 ℃ and 180r/min fully to obtain a zymogen liquid. And (3) carrying out gradient dilution on the endophyte fermentation liquor by 10X, 50X and 100X. Respectively and uniformly mixing the pathogenic bacteria fermentation liquor with an LB solid culture medium (1:10), pouring the mixture into a flat plate, and when the mixture is mixed, the temperature of the LB solid culture medium is not suitable to be too high, so that Xad is prevented from being killed at high temperature. Sterile filter paper sheets with the size of about 5mm are placed in the middle of a culture medium, 10 mu L of fermentation liquor of endophytes and diluent thereof are inoculated to each filter paper sheet, the sterile water is CK, the culture is carried out at 28 ℃, the diameter of a bacteriostasis zone is measured after 7d, and the treatment is repeated for 3 times.
Inhibition rate (control colony diameter-treatment colony diameter)/control colony diameter (zhangguohui et al, 2017).
1.2.5 morphological characteristics and culture Properties
A sterilized slide glass is obliquely inserted into the fresh bacterial plate, and the bacterial body is taken out when growing on the slide glass. Appropriate amount of osmic acid 1% was added to the slide and fumigated for 2 h. Rinsing with phosphate buffer solution, fixing the sample with 2.5% glutaric acid solution again, rinsing with phosphate buffer solution after 2h, performing gradient dehydration with ethanol for about 15min each time, and finally naturally drying in isoamyl acetate. After sample pretreatment, scanning and photographing are carried out in an electron microscope at a Chinese academy of agricultural sciences.
The strain is inoculated into LB culture medium for culture observation.
1.2.6 physiological and biochemical characterization of HZY-54 Strain
According to the morphological characteristics of endophyte colonies and indexes of selection (1) temperature (2) aerobic and anaerobic properties (3) gram stain (4) dryness resistance (5) methyl red experiment (6) starch hydrolysis (7) salt resistance (8) citrate utilization (9) whether to generate fluorescence reaction on KB culture medium (10) contact enzyme experiment (11) optimal pH growth (12) esculin hydrolysis (13) motility and the like in Bergey's bacteria identification handbook, physiological and biochemical identification is carried out on the HZY-54 strain. (14) Cellulose decomposition (15), tyrosine decomposition (16), V-P measurement (17), ultraviolet measurement (18) glucose oxidation fermentation measurement.
1.2.7 molecular characterization
After extracting the DNA of HZY-54 by using a method of a bacterial genome DNA extraction kit, amplifying by using a universal primer (27F/1492R)16SrDNA, wherein the reaction system is shown in Table 1, and a PCR amplification system comprises the following steps: pre-denaturation at 94 ℃ for 5min, and extension at 72 ℃ for 10min for 35 cycles (denaturation at 94 ℃ for 1min, annealing at 60 ℃ for 1min, and extension at 72 ℃ for 1 min). The obtained PCR product was sent to Huada Gene Co., Ltd for sequencing. And (4) carrying out sequence alignment on the sequencing result in an NCBI website, and constructing a phylogenetic tree by using MEGA 7.0.
TABLE 1 PCR amplification System for HZY-54 Strain
1.3 results
1.3.1 preliminary separation results
237 endophytes are separated from the root, stem, leaf, inflorescence and other different parts of 2 healthy anthurium of different varieties, 128 endophytes are separated from the panama variety, and 109 endophytes are separated from the panama variety; wherein the endophytic bacteria separated from the root of anthurium andraeanum are at most 107 strains, which account for 45.15%; the second is 72 leaves, accounting for 30.38%; the stems are fewer than 3 plants and account for 12.66%; there was little above the stem.
1.3.2 preliminary screening results of endophytes
The preliminary screening results using the watermelon fusarium wilt bacteria as target bacteria show that: among 237 endophytes, 52 strains have inhibition effect on fusarium oxysporum f.sp.citrulli with an inhibition rate of more than 50%, wherein 39 strains of Panama and 13 strains of Victoria varieties exist. 39 strains separated from roots account for 28.47 percent of the total endophytes, only 3 strains separated from stems are obtained as the endophytes, the number of the endophytes is extremely small, 0 strain with bacteriostasis on fusarium oxysporum f.sp.citrulli is obtained, and 13 strains separated from leaves account for 13.40 percent. Wherein Y-24, HZY-54 and Y-36 are screened from the leaves of the Panama variety; y-92 and Y-114 of multi-variety leaf screening; g-52, G-27, G-57 and G-82 screened from the roots of the panama species have the prevention and treatment effect of more than 65 percent. As shown in fig. 1 and table 2.
TABLE 2 preliminary screening of antagonistic action of strains on Fusarium oxysporum F.sp.sp.melonis
1.3.3 rescreening results of endophytes
The results of the rescreening with the alternaria philoxeroides as the target bacteria show that: the endophytes Y-24, Y-36, HZY-54, Y-92, Y-114, G-25, G-27, G-57 and G-82, wherein the HZY-54 has the best bacteriostasis with the bacteriostasis rate of 81.79 percent, and the second is Y-24 with the bacteriostasis rate of 66.23 percent, and the endophytes Y-36, Y-92 and G-57 have the general bacteriostasis capacities of 49.33 percent, 40.05 percent and 49.50 percent respectively. The endophytes G-52, G-27 and G-82 have almost no bacteriostatic ability on alternaria solani. From the bacteriostatic rate of watermelon fusarium oxysporum and radish black spot pathogen, HZY-54 separated from the leaves of the Panama variety has strong bacteriostatic ability on two pathogenic fungi, endophytes Y-92 and Y-114 separated from the leaves of the vitamin multi-variety have good inhibitory effect on watermelon fusarium oxysporum, and the inhibitory effect on radish black spot pathogen is general. In conclusion, the subsequent studies were mainly conducted with the HZY-54 strain. As shown in fig. 2 and table 3.
TABLE 3 rescreening of antagonistic action of the strains on alternaria terebrata
1.3.4 inhibitory Effect on pathogenic bacteria
1.3.4.1 bacteriostatic action of HZY-54 Strain on 9 fungi and 1 oomycete
Experiment, HZY-54 was cultured in fermentation broth and the supernatant was extracted to prepare sterile filtrate, and the inhibition of the HZY-54 strain, fermentation broth and sterile filtrate on ten pathogens was investigated by plate confrontation. Experimental results show that the three treatments have an inhibiting effect on pathogenic bacteria, and the inhibiting effect is obvious, wherein the inhibiting effect of the fermentation liquor treatment group is more prominent. The HZY-54 strain is proved to have better inhibiting effect on 10 pathogenic bacteria and broad-spectrum bacteriostatic ability. The inhibitor has obvious inhibition effect on alternaria alternata, eggplant sclerotinia sclerotiorum, tomato early blight, cucumber blight and tomato leaf mold, and the inhibition rate of bacteria is more than 80%. Especially, the inhibition effect on tomato leaf mold is optimal, the inhibition rate is up to 86.42%, the inhibition effect on watermelon fusarium wilt, tomato gray mold, corn curvularia and cotton rhizoctonia solani is slightly weak, but the inhibition rate of the HZY-54 strain can also reach 75% or more. The inhibition effect on cucumber fusarium wilt bacteria is the worst, but the prevention and treatment effect can also reach more than 50 percent. In conclusion, the HZY-54 strain, the fermentation liquor and the sterile filtrate have obvious inhibition effects on ten pathogenic fungi, but the fermentation liquor has stronger bacteriostatic ability; in inhibition tests of ten kinds of fungi, the inhibition effect on the tomato leaf mold pathogen is the best and can reach 86.42%, so that the subsequent research mainly takes the tomato leaf mold pathogen as the main material. As shown in fig. 3, 4, 5, 6 and table 4.
TABLE 4 inhibitory Effect of HZY-54 on pathogenic bacteria
Note: the data in the table are the average number +/-standard error, the different small letters represent the significance of the difference of the bacteriostasis rate among the strains, and the lower case letters in the table are the same.
1.3.4.2 Effect on pathogenic spores
As shown in Table 5 and FIG. 8, when the stock solution of HZY-54, the 50X diluent, the 1000X difenoconazole solution and the carbendazim solution were mixed with the spore suspension in equal volumes, the germination rates of the spores after 24 hours of culture were 8.14%, 25.00%, 12.43% and 29.71%, respectively; the inhibition rates were 73.25%, 17.84%, 59.15%, and 2.4%, respectively. HZY-54 had a significant effect on inhibiting the germination of the Phyllospora lycopersici spores even higher than that of the medicament difenoconazole, while the medicament carbendazim had little effect on Phyllospora lycopersici, as shown in FIG. 8.
TABLE 5 Effect of HZY-54 Strain on the Germination of pathogenic spores
1.3.4.3 Effect on pathogenic hyphae
The normal hyphae should be uniform in thickness, full in shape and complete in structure. However, for the supernatant of HZY-54 and 1000X difenoconazole with different concentrations, the growth and development of the treated carbendazim are seriously influenced after the 500X carbendazim is treated, and the hypha of the treated group is deformed and bent and is not straightened; the front end of the mycelium expands to be spherical, and the middle section shrinks to form a node; the substances in the hyphae seep out to cause the hyphae to break hollow, and the like. The hypha growth abnormality of the medicament 1000X difenoconazole and HZY-54 stock solution is most obvious. As shown in fig. 9.
1.3.4.5 inhibition of bacterial pathogens
In the preliminary screening and the inhibition experiments on 10 pathogenic bacteria, the HZY-54 strain shows better bacteriostatic activity. In the inhibition test of pathogenic bacteria, the fermentation stock solution of the HZY-54 strain has poor inhibition effect. However, after the fermentation liquor is diluted in a gradient way, the inhibiting effect of the HZY-54 strain gradient dilution liquid is obvious, and 50X is optimal. The maximum inhibition zone for Xad can reach 34.00mm, the average inhibition zone for Psl is 30.67mm, the inhibition effect for Ecc is slightly weak, and the average inhibition zone is only 24.33mm (as shown in figure 7). In conclusion, the 50X diluent of the HZY-54 strain also has better bacteriostatic activity on 3 pathogenic bacteria. As shown in fig. 7.
1.3.5 morphological characteristics and culture Properties
The HZY-54 strain colony separated from the healthy anthurium leaves is milky round, the surface of a fresh colony is smooth, the surface of the colony becomes rough along with the growth of culture time, the middle part of the colony is inwards sunken, the colony is not glossy, and the texture of the colony is in a bean curd residue shape, so that the strain can be preliminarily judged to be bacillus. The purified strains are shown in FIG. 10. From the electron micrographs: the thallus is in a short rod shape, has two flagella, is slender and long, and is full and clear.
1.3.6 physiological and Biochemical Properties
The physiological and biochemical experiment results of the HZY-54 strain show that the HZY-54 strain is gram-positive; the starch hydrolysis reaction is positive, and amylase can be generated; can grow in the temperature of 4-45 ℃, wherein the temperature of 37 ℃ is the most suitable temperature for the growth of HZY-54; the pH value is 3.0, the pH value is 12.0, namely the peracid and the alkali can not grow, and the pH value is 7.0, and the neutral can normally grow; the methyl red reaction is negative; the decomposition reaction of the leucine is negative; cellulose cannot be decomposed; the drying resistance is strong; has motility; the salt tolerance is strong; citrate utilization medium is alkaline (dark blue); the V-P experiment shows negative; the aesculin is positive in water receiving display; no fluorescence was observed on KB plates; positive in catalase test. Some physiological and biochemical results are shown in Table 6.
TABLE 6 physiological and biochemical experimental results
Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.
Note:"+"indicates positive；growth,"-"indicates negative；no growth.
1.3.7 sequencing results and analysis of 16rSDNA of endophytes
The PCR product obtained was subjected to DNA detection using 1% agarose gel. The gel was photographed in a gel imager by a software system, and the electrophoresis results are shown in FIG. 11. Nucleotide sequence comparison analysis is carried out on the strains through a BLAST program in NCBI, strains with higher similarity are selected from sequence comparison results for development analysis, and then a MEGA software is utilized to establish a corresponding phylogenetic tree, wherein the phylogenetic tree of the HZY-54 strain is shown in figure 12. In the figure, the HZY-54 strain and Siamese Bacillus (B.siamensis) are found to be the same branch, and in combination with the prior morphological colony observation and various physiological and biochemical characteristics, the gene coverage rate and homology comparison of the HZY-54 strain and Siamese Bacillus (B.siamensis) reach 99.5 percent at the same time, and the HZY-54 strain can be determined to be Siamese Bacillus (B.siamensis).
2. Screening of optimal growth conditions for Strain HZY-54
2.1 materials
The strain is as follows: HZY-54 Strain
Test medium: LB liquid medium, NA liquid medium, PDB (PDA liquid medium), KB liquid medium, Gao's I liquid medium.
Blood counting plate (25X 16)
2.2 methods
2.2.1 screening of basal Medium
The bacterial strains are inoculated in each sterilized basal culture medium (A: LB liquid culture medium (CK), B: NA liquid culture medium, C: PDB (PDA liquid culture medium), D: KB liquid culture medium and E (Gao's I liquid culture medium), the viable bacteria number of different fermentation basal culture media under the same culture conditions is measured 24 hours at 28 ℃, and the optimal fermentation basal culture medium is determined.
2.2.2 selection of optimal carbon sources
Respectively replacing the C source in a basic culture medium with equal amounts of soluble starch, maltose, sucrose, lactose, mannitol and glucose, keeping other components unchanged, and determining the number of viable bacteria when different C sources are measured by taking a fermentation basic culture medium as CK to determine the optimal carbon source.
2.2.3 selection of optimal Nitrogen Source
Selecting the optimum C source as C source, and adding peptone, yeast powder, beef extract, urea and (NH) in equal amount4)2SO4、KNO3Replacing the original nitrogen source, measuring the viable count under different N source conditions, and determining the optimal nitrogen source.
2.2.4 screening of optimal inorganic salts
Adding KCl and CaCl in equal amount to the selected optimum inorganic salt2、NH4Cl、MgSO4、MgCl2And determining the number of viable bacteria under different inorganic salt conditions to determine the optimal inorganic salt.
2.2.5 orthogonal optimization of fermentation conditions
To further study the effect of fermentation conditions on the viable count of HZY-54 strain, an orthogonal test L was used16(45) (see table 7) the optimum fermentation conditions of the HZY-54 strain were selected by optimizing the rotation speed, pH, liquid loading amount, inoculum size, and fermentation time.
TABLE 7 fermentation conditions orthogonal experimental design
2.3 results
2.3.1 optimal basal Medium screening
A, B, C, D, E (A: LB liquid medium (CK), B: NA liquid medium, C: PDA liquid medium, D: Gao's I liquid medium (GS), E: KB liquid medium) 4 basic media are screened to carry out fermentation culture on the HZY-54 strain, and the optimal growth basic medium is selected according to the final growth viable bacteria number. Under the same culture conditions, the number of viable bacteria in LB culture medium is at most 1.67X 108cfu/mL, followed by NA: 1.31X 108cfu/mL, the difference between the two is not obvious, and the unit of viable count can reach 1 multiplied by 108cfu/mL or more. The growth of the thallus in PDA, GS and KB plates is general, and the unit of viable count reaches 1 × 107cfu/mL or more, respectively: 6.61X 107，3.67×107，3.50×107cfu/mL. In conclusion, the LB and NA plates were the best growth media for the HZY-54 strain, and there was no significant difference between them. As shown in fig. 13 and table 8.
TABLE 8 Effect of different basal media on HZY-54 Strain
2.3.2 optimal carbon Source screening
Under the same culture conditions, maltose, mannitol, starch, lactose, sucrose, etc. are used to replace carbon source in the original culture medium, wherein when soluble starch is used as C source, the viable count is maximum and can reach 3.71 × 108cfu/mL, lactose, mannitol and sucrose, respectively, the viable count reaches 2.29 × 108，2.23×108And 1.35X 108cfu/mL, when the four substances are used as carbon sources to culture the HZY-54 strain, the unit of viable count can reach 1 × 108The above. When the HZY-54 strain is cultured by using maltose, CK and glucose as carbon sources, the number of viable bacteria is small, and the unit is 1 × 107cfu/mL or more. The viable count is 7.31 × 107，2.34×107，2.11×107cfu/mL, and the number of viable bacteria is reduced by one order of magnitude when the soluble starch, lactose, mannitol and sucrose are taken as carbon sources comprehensively, so that different carbon sources can generate larger influence on the growth of the strain HZY-54. In conclusion, soluble starch was the best carbon source for the growth of the HZY-54 strain in this experiment. As shown in fig. 14 and table 9.
TABLE 9 Effect of different carbon sources on HZY-54 Strain
2.3.3 optimal Nitrogen Source selection
Under the same culture conditions, using equal amount of peptone, tryptone, yeast powder, beef extract, urea, (NH)4)2SO4、KNO3The beef extract with the strongest strain activity after replacing the original nitrogen source is 6.39 multiplied by 108Yeast powder, urea, peptone (NH)4)2SO4，KNO3When the strain is used as a nitrogen source for culture, the strain activity is weaker, and the viable count is respectively as follows: 5.83X 108，3.51×108，1.95×108，1.23×108，0.93×108cfu/mL, wherein, when KNO3When the strain HZY-54 is cultured as nitrogen source, the number of viable bacteria is reduced by one order of magnitude compared with other treated groups, and KNO is obtained3Compared with other carbon sources, the growth of the strain HZY-54 is not suitable. But no CK group cultured strains have strong activity, and the number of CK group live bacteria is 6.79 multiplied by 108cfu/mL. In conclusion, the original nitrogen source is still the best nitrogen source for culturing the HZY-54 strain, and then beef extract is used. As shown in fig. 15 and table 10.
TABLE 10 Effect of different Nitrogen sources on the HZY-54 Strain
2.3.4 screening of optimal inorganic salts
Adding KCl and CaCl in equal amount under the same culture condition2、NH4Cl、MgSO4、MgCl2And determining the activity of the HZY-54 strain by calculating the viable count under the condition of measuring different inorganic salts. Wherein the most active is MgCl2The bacterial strain cultured as the inorganic salt component has the following viable count: 7.80X 108cfu/mL, the others are KCl: 6.35X 108cfu/mL，MgSO4：6.90×108cfu/mL，CaCl2：4.33×108cfu/mL，CK：1.30×108cfu/mL，NH4Cl：1.22×108cfu/mL. I.e. MgCl2Is the best inorganic salt for the growth of the HZY-54 strain. By combining the data, although the difference between different inorganic salt components is obvious, the viable count unit does not change in magnitude. As shown in fig. 16 and table 11.
TABLE 11 Effect of different inorganic salts on HZY-54 Strain
2.3.5 orthogonal optimization of fermentation conditions
The results of the optimal combination of culture conditions of the HZY-54 strain based on the selected optimal medium components are shown in Table 12, wherein the culture conditions of group 9 are optimal, and the viable count cultured under the combined conditions reaches 2.43X 109The corresponding level combination was A4B3C2D1E 2.
As is known from the tables, the various culture conditions have different effects on the growth and activity of the strains, the order of the extent of the effects being the fermentation time>Rotational speed>Liquid loading amount>pH>The inoculation amount is optimized through an orthogonal test, the fermentation conditions of the HZY-54 strain are 200r/min of rotation speed, 8 of pH, 75mL/250mL of liquid loading amount, 3 percent of inoculation amount and 72 hours of fermentation time, and the number of viable bacteria can reach 2.43 multiplied by 109cfu/mL. The number of viable bacteria is 1.67X 10 before the initial culture medium components and culture conditions are not optimized8cfu/mL, when the components of the culture medium and the culture conditions are optimized, the viable count can reach 2.43 multiplied by 109cfu/mL, compared with the viable count of the strain cultured by the basal culture medium under the initial condition, the viable count of the optimized strain is improved by 2.26 multiplied by 109cfu/mL, increased by one order of magnitude.
TABLE 12 orthogonal table of fermentation conditions for HZY-54 Strain
3. HZY-54 strain colonization, growth promotion and prevention and treatment effects
3.1 materials
Fulvum pathogen is provided by plant pathology laboratory of Tianjin academy of agriculture;
tomato seeds: spring field, top-quality L400 (produced by Liaoning green field agricultural science and technology Co., Ltd., and produced and operated by Fenugreek seed Co., Ltd.);
nutrient soil, vermiculite, 50-hole plug seedling trays and 10 multiplied by 8.5 multiplied by 7 small flowerpots, wherein the trays are purchased from Cao Zhua flower market in the western Qing district in Tianjin;
anthurium andraeanum: provided by flower center in Tianjin city, the variety is Panama;
HZY-54 strain fermentation broth (1X 10)6cfu/mL)
Xad bacterial suspension (1X 10)6cfu/mL)
PDA liquid culture medium
3.2 methods
3.2.1 Germination of tomato seeds
Mixing nutrient soil and vermiculite (mass ratio of 2:1) into a seedling tray, sterilizing tomato seeds with hot water, burying the tomato seeds in the soil at a position of about 1cm, placing 2 seeds in each hole, placing the seedling tray in a tray, watering with proper amount of clear water, keeping moist and draining waterlogging in time, and culturing in a light incubator at 25 ℃. Transplanting the tomato seedlings into a small flowerpot when the tomato seedlings grow to about 7cm, and moving the seedlings to a place with sufficient sunlight and warmth after the seedlings are revived in a dark warm area for 2 d.
3.2.2 Strain rifampicin resistance screening and colonization ability determination
Inoculating a fresh HZY-54 strain into an LB liquid culture medium containing 0.5 mu g/LRif by using a Rif labeling method, performing shake culture for 24 hours until the culture solution is turbid, sucking 1mL of seed solution, transferring the seed solution into the LB liquid culture medium containing 1 mu g/LRif, and continuing the shake culture for 24 hours until the culture solution is turbid. By analogy, until the strain can still grow in LB liquid culture medium containing 200 mug/LRif, and the colony morphology and the bacteriostatic ability are the same as those of the original strain, the success of the HZY-54 strain marked by Rif is proved at this time, and the mark is recorded as HZY-54R。
Selecting 90 pots of tomato seedlings with 3-4 true leaves and consistent growth vigor, and using HZY-54RThe fermentation stock solution is treated by root irrigation, and the fermentation liquor is ensured not to fly to the leaves. The root, stem and leaf of tomato seedling are different every 2 days
Tissue sampling was performed by mixing random samples 5 times according to the sampling site. And after the sample is washed clean by clear water, the ultrasonic cleaning instrument is cleaned for 10min again. And all samples were separately diluted (1X 10) by grinding according to different sites4cfu/mL), spread on LB plate containing 50. mu.g/LRif, and cultured at 28 ℃ for 72h, each treatment was repeated 3 times. The colonization of the HZY-54 strain at different sites was determined by recording the number of colonies.
The detection method for verifying the colonization ability of the HZY-54 strain on anthurium andraeanum is the same as the tomato colonization method.
3.2.3 detection of IAA-producing ability of HZY-54 Strain
The method of a Salkowski's reagent colorimetric method is adopted, after the strain is shake-cultured in a King-B culture medium for 15d, the strain is centrifuged at 12000r/min for 5min, the supernatant is taken and added into colorimetric solution, the mixture is uniformly mixed and then is treated in dark for 30min, and the color change is observed.
Preparing an IAA mother solution, preparing 10, 20, 30, 40, 50 and 60 mu g/mL IAA aqueous solution on the basis of the IAA mother solution, using sterile water as a control, measuring the absorbance value of the IAA aqueous solution at OD530 at each concentration, and drawing an IAA standard curve. Inoculating the strain into an LB liquid culture medium, culturing for 15d, centrifuging the turbid bacterial liquid at 12000r/min for 5min each day of 15d, taking 1mL of supernatant, adding a proper amount of colorimetric solution, mixing uniformly, carrying out dark treatment at normal temperature for 30min, detecting the light absorption value at 530nm, calculating the IAA yield by using a standard curve, and repeating for 3 times.
3.2.4 Effect of HZY-54 Strain on the Germination of tomato seeds
Selecting tomato seeds with uniform and plump sizes, sterilizing, and pretreating in fermentation liquor stock solution, 5X dilution, 10X dilution, 20X dilution, 50X dilution and 100X dilution for 24h respectively, and treating with sterile water to obtain CK. Placing sterile filter paper in a culture dish, wetting with sterile water, uniformly placing the treated seeds in the culture dish, culturing 15 seeds in dark at 25 ℃, keeping the surfaces of the seeds moist, repeating the treatment for 3 times, performing the test for 2 times, and observing and measuring the germination rate after 7 days every day.
3.2.5HZY-54 Strain Effect on plant growth
3.2.5.1HZY-54 Strain Effect on tomato seedlings
Selecting 60 pots of tomato seedlings with similar size and plant height and 3-4 true leaves, respectively irrigating roots with fermentation liquor stock solution, 10X, 20X, 50X and 100X diluted fermentation liquor, 10ml of each plant, applying again every 10d, culturing at room temperature by taking sterile water as CK, treating 3 pots of seedlings each time, repeating each treatment for 3 times, and measuring plant height, fresh weight, leaf length, leaf width and root length after sowing for 30 d.
3.2.5.2 Effect of HZY-54 Strain on Anthurium seedlings
Under the condition of the results of the indoor flat plate bacteriostasis experiments, the anthurium seedlings are subjected to root irrigation treatment by selecting a fermentation stock solution with the best effect and the worst concentration of 50X, each plant is 10mL, the root irrigation treatment is carried out every ten days, sterile water is CK, the plant is cultured at room temperature, and the plant height, the fresh weight, the leaf length, the leaf width and the heel length are measured after 10d, 20d and 30d respectively.
1.2.6HZY-54 Strain in vitro leaf prevention and efficacy determination
Selecting healthy leaves with similar growth conditions from similar positions on different tomato plants, after surface disinfection, respectively spraying fermentation liquor stock solution and 5X, 10X, 20X, 50X and 100X diluent on the leaves, wherein the sterile water is CK, after pretreatment for 24 hours, inoculating a tomato leaf mold germ cake on the leaves, then placing the leaves in a culture dish containing sterile water-wetted sterile filter paper, treating 3 leaves for each time, repeating the test for 3 times, alternately culturing at 28 ℃ in a moisture-preserving dark state, respectively observing the disease condition of the leaves after 7 days, and carrying out disease investigation.
And (3) disease index investigation: Σ (number of diseased leaves at each stage × the disease level value)/(total number of investigated leaves × highest level value) × 100 ].
3.2.7HZY-54 Strain against potted tomato leaf mold
Inoculating Phytophthora lycopersici into PDA liquid culture medium, culturing at 28 deg.C for 7 days at 180r/min to obtain bacterial suspension, and diluting to 1 × 106And the cfu/mL is reserved. Tomato seedlings of about 15cm are selected for carrying out the control test of the HZY-54 strain on the tomato leaf mold germs, and a blank control is set at the same time. Firstly, using HZY-54 strain fermentation liquor (1X 10)6cfu/mL) of tomato seedlings were pretreated by root irrigation, 10mL per plant, one week later inoculated with pathogenic bacteria, and shaken pathogenic bacteria (1X 10) were inoculated by needle back-grafting6cfu/mL), stabbing the leaves of the plants 2-5 times with an aseptic needle so as to make the plants stabbed but not penetrated, dipping bacterial liquid with a brush pen, uniformly smearing the bacterial liquid on the wounds of the leaves, culturing in an illumination incubator at 25 ℃, treating 10 plants each, and repeating for 3 times. And (3) carrying out pesticide application treatment after the disease is developed, investigating the disease condition of the tomato leaves at 7d and 14d respectively, and recording and investigating disease index and control effect, wherein the pesticide, the HZY-54 strain fermentation liquor and the diluent thereof are supplemented again after the 7d investigation is finished.
7d, carrying out disease index investigation after the first medicine; 14d, the disease condition after the medicine is investigated again. Recording and investigating the disease occurrence condition of all tomato leaves, the total number of plant leaves, the number of diseased leaves and disease occurrence grade, and calculating the disease index and the prevention and treatment effect of anthurium andraeanum after medicament treatment.
Grading standards
Level 0: no disease spots;
level 1: lesion area < 5% of total area;
and 2, stage: the area of the lesion is more than or equal to 5 percent and less than 20 percent of the total area
And 3, level: the area of the lesion is more than or equal to 20 percent and less than 35 percent of the total area;
4, level: the area of the lesion is more than or equal to 35 percent and less than 50 percent of the total area;
and 5, stage: the area of the scab is less than or equal to 50 percent.
3.2.8 greenhouse prevention effect of anthurium bacterial leaf blight
A single colony is picked up and inoculated into 250mL LB liquid medium, cultured for 72h under the conditions of 28 ℃ and 200r/min, and taken out for standby.
Preparing kasugamycin into a solution with the optimal concentration of 1000 times by using sterile water, and diluting the fermentation stock solution of HZY-54 by 50X to prepare 50X.
The experiment is carried out in a greenhouse of a flower demonstration center in Tianjin, and 4 treatments are arranged in total: 6% kasugamycin WP 100mg/L, HZY-54 of 50X, HZY-54 stock solution and clear water control CK group. Each treatment zone adopts random zone-level arrangement. Each treatment was repeated 4 times, 4m per cell2And selecting anthurium seedlings with the same growth and morbidity as test materials in about 200 pots, wherein the plant height is about 15-25 cm. The treatment group control area is sprayed with 6% kasugamycin WP 600 times liquid (100mg/L), 50X of HZY-54 and stock solution by a spraying method; a clear water control area is arranged, no treatment is carried out after germs are inoculated, the interval between each treatment group is 50cm, and mutual influence among medicaments is prevented. The control group spray clear is CK.
And observing the control effect every 7 days after spraying, recording the disease condition and disease grade of the plant, and calculating the disease index of the anthurium andraeanum plant and the control effect of each treatment on the disease.
Disease grading standard: the grading standard is as same as 1.2.7.2
3.3 data statistics and analysis
Disease index ∑ (number of disease stages × number of diseased plants at each stage) × 100/(number of investigated plants × number of highest stage).
The control effect/% ([ 1- (pre-drug disease index of control region x post-drug disease index of control region/post-drug disease index of control region x pre-drug disease index of application region) ] × 100.
The test data are statistically analyzed by adopting SPSS and Excel 2007 software, and the significance of the difference between treatments is tested by applying Duncan's new repolarization method.
3.4 results and analysis
3.4.1 colonization of the Strain
Irrigating root with fermentation stock solution to treat tomato seedling, sampling according to different parts every other day, grinding and diluting (1 × 10)4) After plating on LB plates containing 50. mu.g/L of Rif and incubation at 28 ℃ for 72h, the Rif-tagged HZY-54 strain could be detected in tomato until complete digestion at 15 d. HZY-54RThe tomato body generally shows a growth trend of increasing first and then decreasing, and the graph can show that the colonized bacteria amount always remains at the root>Leaf part>A stem portion. The strain reaches the maximum colonization amount of 2.93 multiplied by 10 at the tomato root at 11d5cfu/mL, and the maximum colonization can be maintained after treatment at 9-11d (no significant difference between 9d and 11d, P)<0.05); the colonization amount of the stem and the leaf of the tomato reaches the peak value on the same day, and is respectively 1.13 multiplied by 105cfu/mL and 1.53X 105cfu/mL, both peaks retained 5d (no significant difference at 7d, 9d and 11d, P)<0.05)。HZY-54RThe colonization amount of the strain in the tomato in vivo colonization period at roots is obviously higher than that of stems and leaves most of the time (the colonization amount of the roots and the leaves of 3d and 7d after treatment is not obviously different, and P is<0.05) and the colonization amount of the stem and the leaf part is not obviously different all the time.
The colonization effect on anthurium is similar to that of tomato, the colonization period is 15d, and the colonization bacterial quantity is as follows from large to small: root of a tree>Leaf part>A stem portion. In anthurium plants, the colonization amount of the 7d tends to be maximum and continues to the 11 d. LB at 15d containing 50. mu.g/L RifWhen plate detection is carried out, a germ-free colony grows, and the strain HZY-54RThe digestion is complete, and the colonization amount of the strains in the whole period is always the maximum root, the second leaf part and the last stem part. As shown in fig. 17.
3.4.2 IAA result detection
Reference is made to the Salkowski colorimetric method in the literature to verify whether the HZY-54 strain has the ability to produce IAA. After comparison, the supernatant of the HZY-54 strain fermentation liquor mixed with the colorimetric solution turns red, and the HZY-54 strain is proved to have the capability of producing auxin and promoting the growth of plants. The absorbance at 530nm of an IAA solution in water is evaluated by the standard curve y of 0.0131x +0.0433, R20.9913. The experiment respectively determines the IAA yield of the HZY-54 strain within 15d, and the experiment finds that the IAA content in the supernatant of the HZY-54 detected at the 3d basically tends to be maximum, the yield is 97.23 mu g/L (shown in figure 18) according to a standard curve, and then the supernatant is gradually digested. That is, when the growth rate of the cells is gradually decreased, the rate of generation of IAA is also rapidly decreased, and even digestion is started.
3.4.3 Effect on the Germination of tomato seeds
From experimental results, the germination rate of tomato seeds is in direct proportion to the concentration of the HZY-54 bacterial liquid, and the HZY-54 bacterial strain is proved to have a remarkable promoting effect on seed germination. The germination rate of the tomato seeds treated by the fermentation stock solution is highest and reaches 92 percent. The accelerating effect of 10X, 20X and 50X in the gradient dilution of HZY-54 strain fermentation liquor on seed germination is obvious, the germination rate is over 80 percent, and the germination rate of 100X treatment group seeds is weaker to 76.42 percent. Although the germination rate is in a descending trend along with the increase of the dilution times of the fermentation liquid, the germination rate of the fermentation diluent treatment group is obviously greater than that of the CK control group in the experimental result. The HZY-54 strain and the gradient diluent thereof are proved to have obvious growth promoting effect on tomato seeds, and the germination rate can reach 92 percent to the maximum. Can be used as a better seed growth promoting biological agent to continue further research in the future. As shown in fig. 19, 20 and table 13.
TABLE 13 tomato seed Germination
3.4.4 Effect on plants
3.4.4.1 Effect on tomato seedlings
And (3) irrigating the roots of the tomato seedlings by using the stock solution of the fermentation liquor of the HZY-54 strain and the gradient diluent thereof. As shown in FIGS. 21 to 28 and the results in Table 14, the height of the tomato seedlings irrigated with the fermentation stock solution reaches 286mm, the root length reaches 210mm, the fresh weight reaches 228g, the stem thickness reaches 5.14mm, the leaf width is 3.67cm, the leaf length is 4.59cm, and 49 leaves are obtained. Compared with the CK group, the result of each index is obviously greater than that of the CK group. Tomato seedlings from the other gradient dilution treatment groups showed significantly enhanced growth compared to the CK group. But all indexes are lower than those of the fermentation stock solution treatment group, and the higher the concentration of the bacterial solution is, the more beneficial the tomato seedlings are to grow. The HZY-54 strain is proved to have better growth promoting effect on physiological indexes of tomato seedling root system, plant height, fresh weight, thick stem, dense plant leaves, leaf width, leaf length and the like, and the growth promoting capability of the strain is in inverse proportion to the concentration of fermentation liquor.
TABLE 14 Effect of different part growth of tomato seedlings tested under different treatment conditions
3.4.4.2 Effect on Anthurium plants
The experimental results show that, different from tomato seedlings, the anthurium seedlings treated by the HZY-54 fermentation stock solution have poor growth conditions, weak seedlings, yellowing, short plants, softer plant leaves, thinner mesophyll and the like, and the growth vigor of the anthurium seedlings is even lower than that of the clear water control group. The 50X diluent has good effect and obvious growth promotion effect on anthurium plants, the height of the anthurium plants reaches 33.60cm after 30d, the leaf length is 133.39mm, the leaf width is 73.72mm, and the number of leaves is 19. Under the condition of not applying the fermentation liquor and the diluent thereof, the growth vigor of the anthurium plants of the stock solution treatment group becomes stronger, the growth promoting effect of 50X on the anthurium plants is relatively weakened, but the growth promoting effect is still remarkable compared with that of a clear water control group. In conclusion, the HZY-54 strain fermentation stock solution is unfavorable for anthurium growth at first, but the growth vigor gradually rises along with the reduction of the concentration, and 50X has stronger growth promoting effect on anthurium plants at first, but the growth promoting effect is relatively weakened along with the reduction of the concentration of the bacterial liquid. That is, 50X was an ideal concentration for demonstrating that the HZY-54 strain had a growth promoting effect on anthurium growth. The growth was as shown in FIG. 29.
TABLE 15 Effect of different treatments on anthurium growth
3.4.5 Effect on in vitro leaves
The control effect of HZY-54 on in vitro leaves on tomato leaf mold is shown in Table 16 and FIG. 30, and similar to the result of the confrontation experiment, the fermentation liquid of HZY-54 can effectively inhibit the growth of the scab of tomato leaf mold, and the dilution liquid with different times has different inhibiting effects on tomato leaf mold. Wherein, the fermentation stock solution, 10X, 20X, 50X and 100X diluent and CK group have significant difference, the disease indexes are respectively 2.00, 2.67 and 14.00, the 100X diluent and CK group have no significant difference, and the disease indexes are respectively 38.89 and 45.33. In conclusion, the in vitro experiment of indoor tomato leaves shows that the concentration of the fermentation liquid of the strain HZY-54 is in positive correlation with the disease index.
TABLE 16 in vitro control of tomato leaves
3.4.6 indoor inhibition and prevention effects on tomato leaf mold
From the results of the in-house leaf exsomatization experiments, the stock solution with the best effect and 50X with the intermediate effect and two representative agents were selected for the efficacy control test (as shown in fig. 31 and table 17). The control effect of different treatments is obviously different. After investigation, the control effect of the difenoconazole is the best, the control effect is 70.38 percent and 79.14 percent respectively, and the control effect is obviously higher than that of other treatments; secondly, stock solution with the prevention and treatment effects of 68.04% and 71.07%; the effect of the carbendazim is general, and the control effect reaches about 50 percent; the 50X effect is the worst, the highest effect can only reach 35.25%, and the prevention and treatment effect is obviously lower than that of the above 3 treatments. In the same time, the leaves of the plant of the difenoconazole and HZY-54 fermentation stock solution treatment group are complete and have better freshness, the leaves of the plant of the carbendazim treatment group lose green and yellow in small area, the freshness is common, the 50X treatment group and the CK group have serious diseases, and the leaves of the plant have yellow, wilting and poor freshness in large area.
TABLE 17 control of tomato leaf mold by HZY-54 Strain
3.4.7 greenhouse control of Anthurium bacterial leaf blight
From the results of greenhouse experiments, the kasugamycin and HZY-54 have the best 50X effect, the HZY-54 has the worst inhibition effect on the fermentation stock solution, and the HZY-54 has the same inhibition effect on xanthomonas as the indoor bacteriostasis experiment. And the two have no significant difference, and the highest prevention and treatment effect can reach more than 65 percent. The control effect of the HZY-54 fermentation stock solution is almost not good at first, but in the statistical process of experimental results, the HZY-54 stock solution is found to be too high in concentration, so that the control effect is poor, and the disease is even more serious. But with the time being longer and under the condition of no application, the number of new leaves of the anthurium andraeanum seedlings in the stock solution treatment group is obviously increased, and the control effect is improved along with the increase of the number of new leaves of the anthurium andraeanum seedlings. As shown in fig. 32 and table 18.
TABLE 18 control of bacterial leaf blight of anthurium by HZY-54 Strain
4. Conclusion
4.1 separation, screening and identification of endophytic antagonistic bacteria of Anthurium andraeanum
The invention adopts a tissue homogenate method to separate endophyte 237 strains from different tissue parts of anthurium andraeanum plants; watermelon fusarium wilt bacteria are used as primary screening indicator bacteria, and 9 strains with the bacteriostasis rate of over 65 percent exist. And (3) re-screening 9 endophytes in the primary screening result by taking the alternaria terebrata as an indicator bacterium, wherein the HZY-54 has the strongest bacteriostatic ability, and the bacteriostatic rate reaches 81.79%. Among the bacteriostatic actions of the HZY-54 strain on 10 pathogenic fungi, the bacteriostatic effect on tomato leaf mold pathogen is optimal, the bacteriostatic rate is 86.42%, the effect on cucumber fusarium wilt pathogen is poor, and the bacteriostatic rate is 56.56%. The HZY-54 stock solution influences the pathogenic spore germination and hypha growth of the tomato leaf mold pathogen, the spore inhibition rate can reach 73.25 percent, and the spore inhibition rate is obviously higher than that of the 50X solution and the carbendazim solution, so that hypha has deformity. 50X liquid of HZY-54 strain can effectively inhibit anthurium bacterial leaf spot, cucumber angular leaf spot and cabbage soft rot bacteria, and the inhibition diameters are 34.00mm, 30.67mm and 24.33mm respectively. The HZY-54 strain is determined to be the Siamese Bacillus (Bacillus simensis) through the morphology, physiological and biochemical characteristics, 16SrDNA sequencing result and NCBI sequence analysis of the HZY-54 strain.
4.2 study of fermentation conditions of HZY-54 Strain
The invention determines the optimal fermentation medium and optimal growth conditions of the HZY-54 strain. Adopting a one-factor orthogonal experimental design, and screening an optimal culture medium suitable for the growth of the HZY-54 strain as an LB culture medium: the carbon, nitrogen source and inorganic salt are preferably starch, tryptone and magnesium chloride; the optimal fermentation conditions are selected through orthogonal design, wherein the rotation speed is 200r/min, the pH is 8, the liquid loading is 75mL/250mL, the inoculation amount is 3%, the fermentation time is 72h, and the viable count of the HZY-54 strain can reach 2.43 multiplied by 109cfu/mL。
4.3 study of colonization, growth promotion and biocontrol effects of HZY-54 Strain
The invention determines that the HZY-54 strain has stronger colonization ability, is easy to colonize in roots and leaves of plants such as tomatoes, anthurium and the like, and has a colonization peak at the 11d, and then the colonization ability is rapidly reduced. The HZY-54 strain has stronger capability of producing IAA. The IAA content in the HZY-54 supernatant was 97.23. mu.g/L at the maximum yield at 3d, and then gradually digested.
According to the invention, fermentation liquor of the strain HZY-54 with different concentrations is adopted to pretreat tomato seeds for seed growth promotion test, and the result proves that the germination rate of the tomato seeds after pretreatment of the strain HZY-54 fermentation stock solution is the highest, 92.00 percent, and is obviously higher than that of a control group by 28.00 percent.
Growth promotion experiments on tomato plants prove that tomato seedlings pretreated by HZY-54 strain fermentation stock solution are 286.08mm high, 210.18mm long, 5.14mm thick, 40 leaves, 3.67cm wide, 4.59mm long and 228.20g fresh weight which are all obviously higher than those of a control group. Different from tomato seedlings, 50X diluent of HZY-54 strain has the best growth promotion effect on anthurium plants, the height of the anthurium plants reaches 33.60cm at 30d, the leaf length is 133.39mm, the leaf width is 73.72mm, and the leaf number is 19. The fermentation stock solution has a certain inhibiting effect on the growth of anthurium plants, and the overall growth vigor is lower than that of a control group. The strain HZY-54 is proved to have stronger growth promoting capability.
In an in vitro experiment of leaves of tomato leaf mold, the prevention and treatment effects of the stock solution and 10X disease are the best, and the disease index does not exceed 3 percent and is obviously lower than that of a control. In pot experiment, the control effect of the difenoconazole is optimum and reaches 70.38%, and the control effect of the stock solution also reaches more than 70%. 50X is inferior to carbendazim in control effect, and is only 35.25 percent. The greenhouse control effect of the anthurium bacterial leaf blight shows that: the 50X control effect of the HZY-54 strain is the best and reaches 68.29%, the control effect is not obvious compared with that of kasugamycin 66.95%, and the control effect of the HZY-54 fermentation stock solution is poor and only reaches 36.65%.
In conclusion, the Siamese bacillus HZY-54 grown in the anthurium andraeanum not only can effectively prevent and treat bacterial leaf blight, but also has bacteriostasis experiments on other diseases to prove the broad spectrum of bacteriostasis performance. Shows high-efficiency broad-spectrum bacteriostatic activity, and shows that the compound has strong biocontrol potential.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Claims (10)
1. A caraway endogenic Siamese bacillus HZY-54 separated from a caraway plant is characterized in that: the name of the HZY-54 strain is as follows: HZY-54, class name: siamese Bacillus (Bacillus siamensis), the collection number is: CGMCC No.20159, preservation date: 29/6/2020, depository: china general microbiological culture Collection center, West Lu No. 1 Hospital No. 3, Beijing, Chaoyang, the area of the republic of China.
2. The isolated anthurium endophytic siameses HZY-54 from the anthurium plant of claim 1, wherein: the HZY-54 strain is milky round, and the surface of a fresh bacterial colony is wet, smooth and sticky. Along with the increase of the culture time, the surface of a Siamese bacillus HZY-54 colony in a anthurium becomes rough, the middle part of the colony is inwards sunken, no luster exists, the texture is dry and astringent, and the colony is in a bean curd residue shape; the endophytic Siamese bacillus HZY-54 in the anthurium can be clearly seen in an electron microscope picture, the thallus is in a short rod shape, two flagella are fine and long, the thallus is full, and the surface is smooth.
3. The isolated anthurium endophytic siameses HZY-54 from the anthurium plant of claim 1, wherein: the HZY-5 strain is a gram-positive bacterium; the starch hydrolysis reaction is positive and can produce amylase; can grow in the temperature of 4-45 ℃, wherein the temperature of 37 ℃ is the most suitable temperature for the growth of HZY-54; the pH value is 3.0, the pH value is 12.0, namely the peracid and the alkali can not grow, and the pH value is 7.0, and the neutral can normally grow; the methyl red reaction is negative; the decomposition reaction of the leucine is negative; cellulose cannot be decomposed; the drying resistance is strong; has motility; the salt tolerance is strong; the citrate utilization culture medium is alkaline; the V-P experiment shows negative; the aesculin is positive in water receiving display; no fluorescence was observed on KB plates; positive in catalase test.
4. The anthurium endogenous siameses HZY-54 isolated from a anthurium plant according to any one of claims 1 to 3, wherein: the culture medium of the HZY-5 strain is LB cultureBase: the carbon, nitrogen source and inorganic salt are preferably starch, tryptone and magnesium chloride; the fermentation conditions are that the rotating speed is 200r/min, the pH is 8, the liquid loading amount is 75mL/250mL, the inoculation amount is 3 percent, the fermentation time is 72 hours, the viable bacteria number of the Y-54 strain reaches 2.43 multiplied by 109cfu·mL-1。
5. Use of a caraway endogenous siamese bacillus HZY-54 isolated from a caraway plant according to any one of claims 1 to 4 for stable colonization in the plant and/or for the production of broad spectrum bacteriostatic active substances and/or for the production of growth promoting substances.
6. Use according to claim 5, characterized in that: the siameses siamensis HZY-54 in the anthurium can be colonized in tomatoes and anthurium, the general growth tendency of increasing first and then decreasing is shown in the tomatoes, and the colonized bacteria amount is root all the time>Leaf part>Stem part, and the maximum colonization amount of the root part, stem part and leaf part of the tomato at 11d, and the maximum colonization amount of the stem part, stem part and leaf part of the tomato at 9-11d can be respectively 2.93 multiplied by 105cfu/mL、1.13×105cfu/mL and 1.53X 105cfu/mL; in the colonization period 15d of anthurium andraeanum, the colonization bacteria amount is as follows from large to small: root of a tree>Leaf part>The stem part is increased in the anthurium plant body firstly and then expands, the colonization amount of the 7 th d tends to be maximum, and the colonization amount lasts until the 11 th d.
7. Use according to claim 5, characterized in that: the maximum IAA production amount of the fermentation liquor of the Siamese bacillus HZY-54 growing in the anthurium at the 3d is 97.23 mu g/L, and the germination index of the stock solution after the tomato seeds are treated is improved by 28.11 percent; the plant height, root length, fresh weight, stem thickness, leaf number and leaf area of tomato seedlings are respectively increased by 198.85mm, 130.28mm, 147.48g, 2.63mm, 38 pieces and 12.70cm2。
8. Use according to claim 5, characterized in that: the growth promoting active substance and the broad spectrum antibacterial active substance are derived from fermentation liquor obtained after the HZY-54 strain is fermented and cultured.
9. Use according to claim 8, characterized in that: the culture conditions of the HZY-54 strain fermentation culture are that the rotating speed is 200r/min, the pH value is 8, the liquid loading amount is 75mL/125mL, the inoculation amount is 3%, and the culture time is 72 h; the basic fermentation medium is LB, the used carbon source is soluble starch, the used nitrogen source is tryptone, and the inorganic salt is MgCl2。
10. Use according to claim 5, characterized in that: the original liquid of the fermentation liquid has the best effect on indoor in vitro experiments of Cladosporium fulvum Cooke, the disease indexes of the tomato leaves pretreated by the original liquid are only 2.00 percent, and the disease indexes of the tomato leaves pretreated by 10X, 20X, 50X and 100X diluent are respectively 2.67 percent, 14.00 percent, 24.67 percent, 38.89 percent and 45.33 percent;
or the control effects of the fermentation liquor stock solution and the medicament difenoconazole are as follows: the control effect of 14d is respectively 79.14 percent and 71.07 percent, and the effect of the carbendazim medicament is as follows: the prevention effect of 14d is 56.04%, the effect of 50X is the worst, namely the prevention effect of 14d is 35.25%, and in the same time, the leaves of the plant in the medicament difenoconazole and strain HZY-54 stock solution treatment group are complete, the freshness is better, the small area of the leaves of the plant in the carbendazim treatment group is green, yellow and fresh, the disease of the 50X treatment group and the CK group is serious, the large area of the leaves of the plant is yellow, wilted and poor in freshness;
or the control effect of the 50X diluted fermentation liquid and kasugamycin is as follows: the 14d control effect is 66.95 percent and 68.29 percent, the stock solution control effect is the worst, and the 14d control effect is 36.65 percent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010730157.5A CN111944714B (en) | 2020-07-27 | 2020-07-27 | Siamese bacillus HZY-54 growing in anthurium and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010730157.5A CN111944714B (en) | 2020-07-27 | 2020-07-27 | Siamese bacillus HZY-54 growing in anthurium and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111944714A true CN111944714A (en) | 2020-11-17 |
| CN111944714B CN111944714B (en) | 2021-04-27 |
Family
ID=73338154
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010730157.5A Active CN111944714B (en) | 2020-07-27 | 2020-07-27 | Siamese bacillus HZY-54 growing in anthurium and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111944714B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112940991A (en) * | 2021-04-21 | 2021-06-11 | 安徽农业大学 | Antibacterial activity of Siamese bacillus and application thereof |
| CN113151059A (en) * | 2021-03-19 | 2021-07-23 | 北京林业大学 | Multifunctional Siamese bacillus and application thereof |
| CN113980863A (en) * | 2021-11-26 | 2022-01-28 | 南京工业大学 | Bacillus siamensis and application thereof |
| CN114134086A (en) * | 2021-12-21 | 2022-03-04 | 东北林业大学 | Siamese bacillus YW17 strain, biocontrol microbial inoculum and application thereof in soil-borne disease control and/or plant growth promotion |
| CN115029286A (en) * | 2022-07-21 | 2022-09-09 | 东北农业大学 | Biocontrol bacterium compound microbial inoculum and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034158A (en) * | 2017-04-27 | 2017-08-11 | 中国科学院微生物研究所 | Siam bacillus B 3 and its application |
| CN108034618A (en) * | 2018-01-24 | 2018-05-15 | 吉林省农业科学院 | Siam's Bacillus strain and its application |
| CN109022320A (en) * | 2018-08-21 | 2018-12-18 | 山东省花生研究所 | Siam bacillus and its application in root rot and southern blight prevention and treatment |
| CN109207404A (en) * | 2018-10-23 | 2019-01-15 | 山西农业大学 | Siam bacillus YJ15 and its application |
| CN110305813A (en) * | 2019-07-10 | 2019-10-08 | 福建省亚热带植物研究所 | A kind of Lyceum bacillus, preparation method and its usage |
| CN110564651A (en) * | 2019-09-30 | 2019-12-13 | 中国农业科学院烟草研究所 | bacillus siamensis and application thereof |
| CN110964654A (en) * | 2018-09-29 | 2020-04-07 | 福建省农业科学院农业生物资源研究所 | Bacillus antagonistic to fusarium wilt and application thereof |
| CN111073839A (en) * | 2020-01-16 | 2020-04-28 | 中化农业（临沂）研发中心有限公司 | Siam bacillus, microbial inoculum and application thereof |
| KR102125456B1 (en) * | 2017-10-20 | 2020-07-08 | 대한민국 | Bacillus siamensis strain promoting resistance of plants against biotic and abiotic stress and use thereof |
-
2020
- 2020-07-27 CN CN202010730157.5A patent/CN111944714B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034158A (en) * | 2017-04-27 | 2017-08-11 | 中国科学院微生物研究所 | Siam bacillus B 3 and its application |
| KR102125456B1 (en) * | 2017-10-20 | 2020-07-08 | 대한민국 | Bacillus siamensis strain promoting resistance of plants against biotic and abiotic stress and use thereof |
| CN108034618A (en) * | 2018-01-24 | 2018-05-15 | 吉林省农业科学院 | Siam's Bacillus strain and its application |
| CN109022320A (en) * | 2018-08-21 | 2018-12-18 | 山东省花生研究所 | Siam bacillus and its application in root rot and southern blight prevention and treatment |
| CN110964654A (en) * | 2018-09-29 | 2020-04-07 | 福建省农业科学院农业生物资源研究所 | Bacillus antagonistic to fusarium wilt and application thereof |
| CN109207404A (en) * | 2018-10-23 | 2019-01-15 | 山西农业大学 | Siam bacillus YJ15 and its application |
| CN110305813A (en) * | 2019-07-10 | 2019-10-08 | 福建省亚热带植物研究所 | A kind of Lyceum bacillus, preparation method and its usage |
| CN110564651A (en) * | 2019-09-30 | 2019-12-13 | 中国农业科学院烟草研究所 | bacillus siamensis and application thereof |
| CN111073839A (en) * | 2020-01-16 | 2020-04-28 | 中化农业（临沂）研发中心有限公司 | Siam bacillus, microbial inoculum and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| HAEYOUNG JEONG ET AL.: "Draft Genome Sequence of the Plant Growth-Promoting Bacterium Bacillus siamensis KCTC 13613T", 《JOURNAL OF BACTERIOLOGY》 * |
| 杨亚男: "番茄根际促生菌的筛选及其培养基优化", 《中国学位论文全文数据库》 * |
| 林志楷等: "暹罗芽孢杆菌研究进展", 《亚热带植物科学》 * |
| 许本宏等: "带鱼肠道中芽孢杆菌的分离鉴定及其发酵液抗菌性质研究", 《水产科学》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151059A (en) * | 2021-03-19 | 2021-07-23 | 北京林业大学 | Multifunctional Siamese bacillus and application thereof |
| CN112940991A (en) * | 2021-04-21 | 2021-06-11 | 安徽农业大学 | Antibacterial activity of Siamese bacillus and application thereof |
| CN112940991B (en) * | 2021-04-21 | 2022-04-05 | 安徽农业大学 | Antibacterial activity of Siamese bacillus and application thereof |
| CN113980863A (en) * | 2021-11-26 | 2022-01-28 | 南京工业大学 | Bacillus siamensis and application thereof |
| CN113980863B (en) * | 2021-11-26 | 2022-05-31 | 南京工业大学 | Bacillus siamensis and application thereof |
| CN114134086A (en) * | 2021-12-21 | 2022-03-04 | 东北林业大学 | Siamese bacillus YW17 strain, biocontrol microbial inoculum and application thereof in soil-borne disease control and/or plant growth promotion |
| CN115029286A (en) * | 2022-07-21 | 2022-09-09 | 东北农业大学 | Biocontrol bacterium compound microbial inoculum and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111944714B (en) | 2021-04-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111944714B (en) | Siamese bacillus HZY-54 growing in anthurium and application thereof | |
| CN106591185B (en) | Bacillus amyloliquefaciens plant subspecies and preparation and application of microbial inoculum thereof | |
| CN1325635C (en) | Endogenetic polymexa bacillus of plant for prophyiaxis and promoting growth and application thereof | |
| CN101851597B (en) | Streptomyces griseoflavus for resisting alfalfa diseases and screening method thereof | |
| CN108034618A (en) | Siam's Bacillus strain and its application | |
| CN108148794A (en) | A kind of the bacillus subtilis DYr3.3 and preparation method and application of broad-spectrum antibacterial activity | |
| CN110452832A (en) | One plant of acid resistance bacillus amyloliquefaciens Kc-5 and its application | |
| CN111925963A (en) | Application of Siamese bacillus B11 in prevention and/or treatment of cotton bamboo blast tip disease | |
| US20060018883A1 (en) | Microbial preparation & method for preventing and curing the bacterial wilt the plant and its use | |
| CN109112069B (en) | Biocontrol endophytic fungus and application thereof | |
| CN114134068A (en) | Paenibacillus polymyxa microbial agent, preparation method and application thereof | |
| CN106701623A (en) | Bacillus atrophaeus antagonistic to lycium chinensis root rot and application of bacillus atrophaeus | |
| CN110982725A (en) | Bacillus for antagonizing fusarium wilt and promoting growth and application thereof | |
| CN109749953B (en) | Bacillus cereus, microbial inoculum and preparation method and application thereof | |
| CN113151101B (en) | Serratia marcescens and application thereof | |
| CN110317747A (en) | A kind of bacillus amyloliquefaciens JT68 and its application in prevention and treatment tea anthracnose | |
| CN117165494A (en) | Kiwi fruit canker biocontrol strain Wq-1 and application thereof | |
| CN109303067B (en) | Streptomyces composition for preventing and treating potato scab and application thereof | |
| CN115058358B (en) | Salt-tolerant bacillus and application thereof | |
| CN104593284B (en) | One plant of Endophyte of walnut and its application | |
| CN116004419A (en) | Bacillus atrophaeus CY-2, microbial inoculum, preparation method and application thereof | |
| CN114456973B (en) | Streptomyces rochei in tobacco and application thereof in prevention and control of tobacco diseases | |
| CN113817642B (en) | Bacillus bailii YJ02, microbial preparation and application thereof | |
| CN109609403A (en) | A kind of biocontrol microorganisms and its application in terms of crops downy mildew prevention and treatment | |
| CN111979151B (en) | Biocontrol strain 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 | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240326 Address after: 300000 Tianjin Binhai New Area Economic and Technological Development Zone Binhai Zhongguancun Science and Technology Park Rongchuang Rongsheng Plaza 202-2 Patentee after: Tiankeda (Tianjin) Science Park Co.,Ltd. Country or region after: China Address before: 300384 No. 22, Jing Jing Road, Xiqing District, Tianjin Patentee before: TIANJIN AGRICULTURAL University Country or region before: China |