CN102399713B - Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving - Google Patents
Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving Download PDFInfo
- Publication number
- CN102399713B CN102399713B CN 201110282657 CN201110282657A CN102399713B CN 102399713 B CN102399713 B CN 102399713B CN 201110282657 CN201110282657 CN 201110282657 CN 201110282657 A CN201110282657 A CN 201110282657A CN 102399713 B CN102399713 B CN 102399713B
- Authority
- CN
- China
- Prior art keywords
- soil
- subtilis
- phosphorus
- strain
- application
- 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.)
- Expired - Fee Related
Links
- 239000002689 soil Substances 0.000 title claims abstract description 57
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 11
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 11
- 229910019142 PO4 Inorganic materials 0.000 title abstract description 5
- 239000010452 phosphate Substances 0.000 title abstract description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 title abstract description 5
- 239000002893 slag Substances 0.000 claims abstract description 17
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 230000000813 microbial effect Effects 0.000 claims abstract description 12
- 230000008635 plant growth Effects 0.000 claims abstract description 7
- 230000001737 promoting effect Effects 0.000 claims abstract description 7
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 51
- 229910052698 phosphorus Inorganic materials 0.000 claims description 51
- 239000011574 phosphorus Substances 0.000 claims description 51
- 239000002068 microbial inoculum Substances 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 22
- 239000002609 medium Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 230000001580 bacterial effect Effects 0.000 claims description 18
- 244000005700 microbiome Species 0.000 claims description 17
- 241000876865 Piscidia Species 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 11
- 239000002054 inoculum Substances 0.000 claims description 8
- 238000011218 seed culture Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 235000015099 wheat brans Nutrition 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 5
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 4
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 2
- 239000002686 phosphate fertilizer Substances 0.000 abstract description 11
- 238000000855 fermentation Methods 0.000 abstract description 9
- 230000004151 fermentation Effects 0.000 abstract description 9
- 239000003337 fertilizer Substances 0.000 abstract description 7
- 239000000126 substance Substances 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 5
- 230000001976 improved effect Effects 0.000 abstract description 4
- 239000003795 chemical substances by application Substances 0.000 abstract description 3
- 241000508269 Psidium Species 0.000 abstract 1
- 210000003608 fece Anatomy 0.000 abstract 1
- 239000010871 livestock manure Substances 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 52
- 230000000694 effects Effects 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- 230000001954 sterilising effect Effects 0.000 description 12
- 238000004659 sterilization and disinfection Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 9
- 230000012010 growth Effects 0.000 description 8
- 241000726221 Gemma Species 0.000 description 7
- 240000008042 Zea mays Species 0.000 description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 7
- 229910000389 calcium phosphate Inorganic materials 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000001506 calcium phosphate Substances 0.000 description 6
- 235000011010 calcium phosphates Nutrition 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 5
- 235000005822 corn Nutrition 0.000 description 5
- 239000002594 sorbent Substances 0.000 description 5
- ABUGVBRDFWGJRD-CHOYNLESSA-N [9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-methyloxolan-2-yl]-2-(2,4-dinitrophenyl)sulfanylpurin-6-yl] [hydroxy(phosphonooxy)phosphoryl] hydrogen phosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](C)O[C@H]1N1C2=NC(SC=3C(=CC(=CC=3)[N+]([O-])=O)[N+]([O-])=O)=NC(OP(O)(=O)OP(O)(=O)OP(O)(O)=O)=C2N=C1 ABUGVBRDFWGJRD-CHOYNLESSA-N 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 239000006916 nutrient agar Substances 0.000 description 4
- 238000012856 packing Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 239000004411 aluminium Substances 0.000 description 3
- 229910052782 aluminium Inorganic materials 0.000 description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000003415 peat Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 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 2
- 238000003794 Gram staining Methods 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 2
- WYWFMUBFNXLFJK-UHFFFAOYSA-N [Mo].[Sb] Chemical compound [Mo].[Sb] WYWFMUBFNXLFJK-UHFFFAOYSA-N 0.000 description 2
- 239000002154 agricultural waste Substances 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000013401 experimental design Methods 0.000 description 2
- 235000011194 food seasoning agent Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 235000009973 maize Nutrition 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000003381 solubilizing effect Effects 0.000 description 2
- 230000028070 sporulation Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 108010013043 Acetylesterase Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 235000019890 Amylum Nutrition 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 241000195940 Bryophyta Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 244000301850 Cupressus sempervirens Species 0.000 description 1
- 241001057636 Dracaena deremensis Species 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 229910021536 Zeolite Inorganic materials 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QLULGSLAHXLKSR-UHFFFAOYSA-N azane;phosphane Chemical compound N.P QLULGSLAHXLKSR-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- YYRMJZQKEFZXMX-UHFFFAOYSA-L calcium bis(dihydrogenphosphate) Chemical compound [Ca+2].OP(O)([O-])=O.OP(O)([O-])=O YYRMJZQKEFZXMX-UHFFFAOYSA-L 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000005669 field effect Effects 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000019691 monocalcium phosphate Nutrition 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 239000010451 perlite Substances 0.000 description 1
- 235000019362 perlite Nutrition 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
Abstract
The invention discloses a bacillus subtilis HL-1 and an application thereof in the respect of soil phosphate dissolving. The strain is collected in China General Microbiological Culture Collection Center (CGMCC) on Aug 24th, 2011, with a collection number of CGMCC No.5175. The 16SrDNA nucleotide sequence of the strain is represented by SEQ ID NO:1. With added diatomite and guava slag adsorbing agent materials, a fermentation broth of the bacillus subtilis HL-1 can be prepared into microbial manure. With the strain provided by the invention, phosphate availability and phosphate fertilizer utilization rate of the soil can be improved, plant growth can be promoted, and soil microbe diversity can be improved. The strain has important significance in reducing chemical fertilizer application, modifying soil micro-ecology, and promoting soil sustainable utilization. The strain has wide application prospect in production practice.
Description
Technical field
The present invention relates to a kind of microorganism and application thereof, be specifically related to a kind of subtilis (Bacillus subtilis) HL-1 and decomposing the soil insoluble phosphorus and improving the application of the aspects such as soil micro-ecosystem structure and Promoting plant growth.
Background technology
Phosphorus is one of necessary nutritive element of growth and development of plants, synthetic and respiration important all to nitrogen fixation, photosynthesis, energy transfer, signal transduction, biomacromolecule.5,700,000,000 hm are arranged in the world
2Soil lacks phosphorus, and phosphorus concentration is from 0.1～10 μ mol/L in the most of soils, and in the ideal situation, the required phosphorus concentration of grass growth is 1-5 μ mol/L, and high to need phosphorus plant such as tomato, pea then be 5～60 μ mol/L.Do not reach under the perfect condition at phosphorus, plant is with the underproduction 5～15%.The use of traditional chemical fertilizer can alleviate the shortage of Soil Phosphorus to a certain extent, but as the principal item calcium superphosphate of Chinese phosphate fertilizer and phosphorus ammonium etc., no matter at northern calcareous soil or southern acid soil, because of the strong solid phosphorus effect of soil, this season utilization ratio less than 20% causes the wasting of resources.There is 74% the scarce phosphorus of arable soil in China, and the phosphorus in the soil more than 95% is the indissoluble form, and plant is difficult to absorb.And very easily fixed by chemistry after phosphate fertilizer is manured into soil, form the compounds such as the extremely low calcium phosphate of solvability, iron, aluminium, the plant utilization rate only is 5～25%.Even significantly do not improve through constantly using for many years phosphate fertilizer yet.Increasing the p application rate is the approach of a kind of " high investment, low output ".Simultaneously, the phosphate fertilizer scarcity of resources of China, throughput is difficult to satisfy the demands.Phosphoric in the soil also has no lack of, and through use the accumulation of chemical fertilizer throughout the year, the content of phosphoric in soil is very high, but plant can't absorb these phosphorus, will bring great variety for Chinese agriculture if improve the utilization ratio of Soil Phosphorus.Therefore, improve the phosphate fertilizer utilising efficiency, strengthen the activation of soil phosphorus and utilize the vital task that becomes scientific research.
Phosphate solubilizing microorganism is a class peculiar microorganism function colony that invalid phosphorus can be converted into the available phosphorus that plant can absorb in the soil, phosphate fertilizer be can improve and phosphate fertilizer utilization efficiency and dissolving soil indissoluble phosphorus fixed, improve, in the biological global chemical recycle of natural and agroecological phosphorus element, played keying action, by universally acknowledged for being a kind of environment protection type soil phosphorus activation biological control measure of Cheap highly effective.They are converted into invalid phosphorus by modes such as acidolysis, chelating, displacement and secretion Phosphoric acid esterases can (mainly be HPO by the available phosphorus of plant utilization
4 2-And H
2PO
4 -Form).Studies show that in a large number, use the microbial fertilizer that contains phosphate solubilizing bacteria to Promoting plant growth, to improve the Soil structure effect remarkable.Utilize molten phosphorus microorganism to the utilization ratio of the validity that improves phosphate fertilizer and soil indissoluble phosphorus, reduce that phosphate fertilizer drops into, production-increasing function and the agricultural sustainable development of the limited phosphorus ore of performance China are significant.In view of the molten phosphorus ability between different types of phosphorus-solubilizing bacteria or the different strains has larger difference, the work of efficient phosphorus-dissolution screening is particularly important.
The phosphorus decomposing microbial inoculum that is used at present the making bio-feritlizer generally uses vermiculite, the peat composed of rotten mosses, perlite and zeolite powder etc. as absorption carrier, these materials are all Nonrenewable resources, if seek a kind of renewable resources, make it can partially or completely replace Nonrenewable resources commonly used at present, not only play the effect that economizes on resources, and can promote the recycle of agricultural wastes, thereby reach the purpose of agricultural sustainable development.The piscidia slag is the byproduct of the piscidia course of processing, and fibre content is high, is obtaining unusual effect and application prospect as sorbent material aspect removal Wastewater Dyes and the removal heavy metal pollution.Piscidia slag outward appearance and microbial bacteria agent carrier peat are similar, and quality is more aobvious brown browner, and piscidia slag water content approximately 4～5% has advantages of lower than peat (about water content 30%) water content.Yet, using renewable resources---the piscidia slag replaces Nonrenewable resources commonly used as bio-fertilizer absorbent material, makes the microbial inoculum with efficient phosphate-solubilizing effect, does not appear in the newspapers.
Summary of the invention
The object of the invention is to provides a kind of subtilis according to above-mentioned deficiency of the prior art.
Another object of the present invention provides the application of above-mentioned bacterial strains.
The present invention is achieved through the following technical solutions above-mentioned purpose:
A kind of subtilis (Bacillus subtilis) HL-1 is called for short the HL-1 bacterium.The Classification And Nomenclature of this bacterial strain is: subtilis Bacillus subtilis, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on August 24th, 2011, deposit number is CGMCC No.5175, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
This bacterial strain screening is gram-positive microorganism in the soil of stove mountain, GuangZhou, Guangdong Province city, cultivate 12h at beef-protein medium, examine under a microscope thalline with single or paired arrangement, gemma quantity few (3～5%) is cultivated 24h at beef-protein medium, thalline is with single, paired or catenation, give birth in the gemma or near in give birth to, gemma is several 80～90%, thalline does not expand behind the sporulation, thalline is shaft-like, without pod membrane, mobility is arranged, aerobic.Cultivate the bacterium colony that forms behind the 24h at nutrient agar and be circle, little yellow, surface irregularity is opaque, and the edge is irregular.
Through sequencing, the 16SrDNA nucleotide sequence of this subtilis HL-1 is shown in SEQ ID NO:1.
The microbial inoculum of being made by above-mentioned subtilis HL-1.
The preparation method of subtilis HL-1 microbial inoculum, that the inoculation that will activate arrives seed culture medium, 30 ℃ of culture temperature, after shaking table is cultivated 24h under the rotating speed 113rpm condition, obtain primary seed solution, then be transferred to seed tank culture by 10～15% inoculum sizes, with secondary seed medium at 30 ℃, air flow 1000L/h, cultivate 24h under the rotating speed 70rpm condition, obtain secondary seed solution, secondary seed solution is inoculated into fermentor tank by 10～15% inoculum sizes, cultivate (being enlarged culturing) in the continuous fermentor tank deep layer of fermention medium relaying, culture condition is 30 ℃, air flow 10000L/h, rotating speed 70rpm cultivates 24h.Fermented liquid after the cultivation mixes fermented liquid with diatomite and piscidia slag: diatomite: the mass ratio of piscidia slag is 0.6: 1: 1; After stirring, pulverize, be prepared into described microbial inoculum.
Wherein every 1L seed culture medium contains extractum carnis 5g, peptone 5g, sodium-chlor 5g, and remainder is water, pH value 7.1～7.4; Seed culture medium is through 115～121 ℃ of sterilization 20min, and is for subsequent use.
Every 1L secondary seed medium and fermention medium contain bean cake powder 30g, wheat bran powder 10g, ammonium nitrate 1.5g, yeast extract paste 3g, sodium-chlor 2.5g, sal epsom 0.2g, dipotassium hydrogen phosphate 0.3g and manganous sulfate 0.05g, and remainder is water, pH value 7.1～7.4.
The concrete preparation process of subtilis HL-1 microbial inoculum is as follows:
(1) first order seed is cultivated
Seed culture based component: extractum carnis 5g/L, peptone 5g/L, sodium-chlor 5g/L, distilled water 1L, pH value 7.1～7.4.115～121 ℃ of sterilizations of substratum 20min, the HL-1 bacterium after the access activation, shaking table is cultivated.Culture condition: 30 ℃, rotating speed 113rpm, time 24h.
(2) secondary seed fermentation culture
Secondary seed medium composition: bean cake powder 30g, wheat bran powder 10g, ammonium nitrate 1.5g, yeast extract paste 3g, sodium-chlor 2.5g, sal epsom 0.2g, dipotassium hydrogen phosphate 0.3g, manganous sulfate 0.05g, water 1L, regulating the pH value is 7.1～7.4,121 ℃ of sterilization 20min, wait to be cooled to 30 ℃, the access primary seed solution, inoculative proportion is 1: 10 (10L secondary seed medium inoculation 1L primary seed solution).It is 30 ℃ that leavening temperature is set, and rotating speed is 70rpm, and air flow is 10000L/h, and tank pressure remains on 0.02～0.03MPa.
(3) fermentor tank enlarged culturing
The same secondary seed medium of fermentation culture based component.In the time of 30 ℃, the access secondary seed solution, inoculative proportion is 1: 10.It is 30 ℃ that leavening temperature is set, and rotating speed is 70rpm, and air flow is 10000L/h, and tank pressure remains on 0.02～0.03MPa.
(4) interpolation of absorption carrier
Bacterium liquid, diatomite, piscidia slag after step (3) cultivation stirrer in are mixed in mass ratio at 0.6: 1: 1, make microbial inoculum after the pulverizing.
(5) packing of microbial inoculum and storage
Take by weighing microbial inoculum and be sub-packed in the aluminium matter bag, air seasoning place is deposited in sealing.
The application of subtilis HL-1 of the present invention in decomposing insoluble phosphorus, Promoting plant growth and/or increase soil microbial community diversity and increase bacterial abundance.
Compared with prior art, the present invention has following beneficial effect:
The contriver filters out subtilis Bacillus subtilis HL-1 through a large amount of experimental studies.This bacterial strain not only can improve validity and the phosphate fertilizer utilization efficiency of indissoluble phosphorus in the soil, thereby Promoting plant growth improves diversity of soil microorganism, to reducing chemical fertilizer application, improves soil micro-ecosystem and promotes the soil sustainable use to have great importance.
Fermention medium take beans cypress wheat bran etc. as main component, have preparation technology simple, effective, low cost and other advantages, and use renewable resources---piscidia slag is as absorption carrier, has economizing on resources, turns waste into wealth, promotes the advantages such as recycle of agricultural wastes.
Description of drawings
Fig. 1 .HL-1 bacterium gramstaining Photomicrograph;
Fig. 2 .HL-1 bacterium spore staining Photomicrograph, blue portion is gemma among the figure;
Fig. 3. different strains produces the comparison diagram of water-soluble phosphorus content under the calcium phosphate substratum, X-coordinate is strain number, and ordinate zou is water-soluble phosphorus concentration;
Fig. 4. the comparison diagram of different strains producing microbial phosphorus content under the calcium phosphate substratum, X-coordinate are strain number, and ordinate zou is microbe-P concentration;
Fig. 5. for the HL-1 bacterium promotes corn growth field effect contrast figure;
Fig. 6. different strains is processed the graphic representation that soil microbial community metabolic activity AWCD value changes, and wherein HL-1 is the HL-1 bacterium, and CK is control group.
Embodiment
Separation, the Purification and Characterization of embodiment 1 subtilis HL-1
(1) separates
Preparation indissoluble inorganic phosphorus substratum: glucose 10.0g, ammonium sulfate 0.5g, sodium-chlor 0.3g, Repone K 0.3g, iron vitriol 0.03g, four water manganous sulfate 0.03g, yeast extract paste 0.4g, calcium phosphate 5g, distilled water 1L, pH value 7.0～7.5.
The soil that gathers on the stove mountain, Tianhe District, Guangzhou City, Guangdong Province is the screening soil sample, adopt spread plate accurately to take by weighing soil sample 10.0g to be measured, put into the 250mL triangular flask (adding granulated glass sphere) that the 90mL sterilized water is housed, shaking table vibration 30min, microorganism is fully disperseed, leave standstill 20～30s, get supernatant liquor and carry out 10 times of dilutions of successively decreasing, adopt 10
3～10
5Extent of dilution pipettes respectively diluent 0.1mL with liquid-transfering gun, is coated on the indissoluble inorganic phosphorus culture medium flat plate, is inverted for 28 ℃ and cultivates 7d, observes day by day molten phosphorus circle, record phosphorus-solubilizing bacteria bacterium colony (d) and molten phosphorus loop diameter (D).The bacterial isolates that filters out the phosphorus decomposing circle is numbered a-4, a-7, and a-15, and b-17, wherein the phosphorus decomposing effect of a-15 is the strongest.After molecular biology identification and Physiology and biochemistry evaluation, determine that the a-15 bacterial strain is subtilis (Bacillus subtilis).
(2) purifying
Nutrient agar: extractum carnis 3.0g, peptone 5.0g, sodium-chlor 5.0g, agar 18.0g, distilled water 1L, pH value 7.0～7.2.
After the bacterial strain that filters out utilized the plate streak purifying, be stored on the nutrient agar slant medium, be called for short the HL-1 bacterium among the present invention.
Embodiment 2 CHARACTERISTICS IDENTIFICATION
(1) morphological features
Gram-positive microorganism has gemma, and thalline does not expand behind the sporulation.Thalline is shaft-like, without pod membrane, mobility is arranged, and is aerobic.The gramstaining effect is seen Fig. 1, and thalline is dyed to bluish voilet, and gemma is not painted.The spore staining effect is seen Fig. 2, and thalline is yellowish brown, and gemma is light blue.
(2) colony morphology characteristic
The bacterium colony that forms at nutrient agar is circle, little yellow, and surface irregularity is opaque, and the edge is irregular.
(3) physio-biochemical characteristics
According to Biolog microorganism identification instrument, analyze this bacterial strain situation of utilizing to 31 kinds of different carbon sources under aerobic condition, the results are shown in Table 1.
Table 1HL-1 bacterium is to the situation of utilizing of 31 kinds of different carbon sources
Annotate :+be expressed as the positive maybe can utilize;-be expressed as feminine gender maybe can not utilize
(4) molecular biological characteristic
Adopt the test kit method to extract bacteria total DNA.Adopt bacterial 16 S rDNA universal primer F63 (CAG GCC TAA CACATG CAA GTC, SEQ ID NO:2) and R1087 (CTC GTTGCG GGA CTT ACC CC, SEQ ID NO:3), the 16S rDNA of pcr amplification bacterium, obvious band appears near 1000bp, to carry out sequencing after the pcr amplification product recovery, with the dna sequence dna input GenBank that obtains, with the Blast program all sequences in the database is compared analysis, found that bacterial strain of the present invention 16S rDNA sequence and with GenBank in subtilis have higher homology, its similarity is 99%.In conjunction with the result of above-mentioned flat-plate bacterial colony feature, Biolog microplate cultivation results, 16SrDNA sequence and with reference to " the outstanding Bacteria Identification handbook of uncle is carried out the Physiology and biochemistry experiment, identify that this bacterial strain is subtilis (Bacillus subtilis), called after Bacillus subtilis HL-1.
The growth characteristics of embodiment 3HL-1 bacterium under different carbon source environment
Beef-protein medium: extractum carnis 5g, peptone 5g, sodium-chlor 5g, water 1000ml, pH are 7.1～7.4.
Respectively with Semen Maydis powder, wheat bran, glucose, peanut press pulp, sucrose, rice bran and 7 kinds of materials of plant amylum as carbon source, concentration is 20g/L, substitutes the extractum carnis in the beef-protein medium, other components unchanged.Above-mentioned 7 kinds of substratum are boiled 10min, naturally be cooled to room temperature, regulating the pH value with the NaOH of 1mol/L or HCl is 7.1～7.5, is prepared into fermention medium.Substratum 45mL is respectively organized in packing in the 250mL triangular flask, sterilization, and each is organized substratum and does three repetitions.Inoculum size according to 10% is inoculated into fermention medium after the above-mentioned sterilization with the HL-1 bacterium, 33 ℃ of temperature, cultivates 24h under the rotating speed 113rpm condition, measures the viable count of respectively organizing in the substratum with the method for plate culture count.The result shows (table 2): the HL-1 bacterium is under the environment take wheat bran as carbon source, and its growth characteristics are best, and viable count is 5.44 ± 0.22 * 10
8Cfu/mL.
In the fermention medium take different substances as carbon source, the grow viable count (* 10 of 24h of table 2:HL-1 bacterium
8Cfu/mL
Annotate: data are 5 mean value ± standard errors that repeat, and the different alphabetical persons of colleague in the table are illustrated in 0.05 level difference significantly (DMRT method).
The growth characteristics of embodiment 4HL-1 bacterium under the different fermentations culture medium condition
Choose 6 kinds of different culture media compositions and carry out respectively fermentation culture, concrete experimental design sees Table 3.6 kinds of substratum in the table 3 are boiled respectively 10min, naturally be cooled to room temperature, regulating the pH value with the NaOH of 1mol/L or HCl is 7.1～7.4.Accurately measuring 45mL with graduated cylinder cultivates based on the 250mL triangular flask, at 115～121 ℃ of lower high-temperature sterilization 20min.Good HL-1 bacterium is inoculated in the 50mL seed culture medium will to activate (method is with the purifying the embodiment 1) from flat board, places shaking table to cultivate 33 ℃ of temperature, rotating speed 113rpm.Shaking table is seed liquor after cultivating 24h.Get the 5mL seed liquor, the inoculum size according to 10% is inoculated into the fermention medium after the sterilization among the embodiment 3, after shaking table is cultivated 24h, manages the fermented liquid viable count everywhere with the method for plate culture count mensuration, two dilution gradients, and each extent of dilution repeats for three times.Measurement result such as table 4, the result shows that the viable count of HL-1 bacterium in No. 4 substratum is the highest, the viable count of fermentation 24h is 15.03 ± 1.30 * 10
8Cfu/mL.
The experimental design of table 3 fermentation culture
In different culture media, the ferment viable count (* 10 of 24h of table 4:HL-1 bacterium
8Cfu/mL)
Annotate: data are 3 mean value ± standard errors that repeat, and the different alphabetical persons of colleague in the table are illustrated in 0.05 level difference significantly (DMRT method).
The preparation of embodiment 5HL-1 bacteria agent
(1) first order seed is cultivated
Seed culture based component: extractum carnis 5g/L, peptone 5g/L, sodium-chlor 5g/L, distilled water 1L, pH value 7.1～7.4.115～121 ℃ of sterilizations of substratum 20min, the HL-1 bacterium after the access activation, shaking table is cultivated.Culture condition: 30 ℃, rotating speed 113rpm, time 24h.
(2) secondary seed is cultivated
Secondary seed medium composition: bean cake powder 30g, wheat bran powder 10g, ammonium nitrate 1.5g, yeast extract paste 3g, sodium-chlor 2.5g, sal epsom 0.2g, dipotassium hydrogen phosphate 0.3g, manganous sulfate 0.05g, water 1L, regulating the pH value is 7.1～7.4,121 ℃ of sterilization 20min, wait to be cooled to 30 ℃, the access primary seed solution, inoculative proportion is 1: 10.It is 30 ℃ that leavening temperature is set, and rotating speed is 70rpm, and air flow is 1000L/h, and tank pressure remains on 0.02～0.03MPa.
(3) fermentor tank enlarged culturing
The same secondary seed medium of fermentation culture based component.In the time of 30 ℃, the access secondary seed solution, inoculative proportion is 1: 3.3.It is 30 ℃ that leavening temperature is set, and rotating speed is 70rpm, and air flow is 10000L/h, and tank pressure remains on 0.02～0.03MPa.
(4) interpolation of sorbent material
Bacterium liquid, diatomite and piscidia slag after step (3) cultivation stirrer in are mixed in mass ratio at 0.6: 1: 1, make microbial inoculum after the pulverizing.
(5) packing of microbial inoculum and storage
Take by weighing the 10.0g microbial inoculum and be sub-packed in the aluminium matter bag, air seasoning place is deposited in sealing.
Embodiment 6HL-1 bacterium is with the growth characteristics of different sorbent materials as absorption carrier
(1) with single diatomite as sorbent material
600mL fermented liquid (embodiment 5 step 3 gained bacterium liquid) evenly is mixed with into the dry powder microbial inoculum with 2kg sterilization diatomite, is positioned over shady and cool sections temperature and preserves.Respectively at the 7th, 14,20,26,31,37d takes by weighing microbial inoculum 10.00g and joins in the 100mL sterilized water that granulated glass sphere is housed, and leaves standstill 20min, the 200rpm 30min that fully vibrates on rotary shaking table measures the viable count of different number of days with the method for plate culture count.The result shows (table 5): the bacterium number is on a declining curve along with the increase of fate, and the viable count of 37d is 0.49 ± 0.06 * 10
8Cfu/mL.
Table 5 diatomite is cultivated the HL-1 bacterium at the viable count (* 10 of different number of days
8Cfu/mL)
| Fate (d) | 7 | 14 | 20 | 26 | 31 | 37 |
| Viable count | 1.61±0.09 | 0.91±0.06 | 0.80±0.11 | 0.61±0.03 | 0.62±0.03 | 0.49±0.06 |
Annotate: data are 3 mean value ± standard errors that repeat.
(2) with piscidia slag and diatomite mixture as sorbent material.
600mL fermented liquid (embodiment 5 step 3 gained bacterium liquid), 1kg sterilization diatomite and 1kg sterilization piscidia slag evenly are mixed with into the dry powder microbial inoculum, are positioned over shady and cool sections temperature and preserve.Respectively at the 7th, 14,20,26,31,37d takes by weighing microbial inoculum and gets the 10.00g sample and join in the 100mL sterilized water that granulated glass sphere is housed, leave standstill 20min, the 200rpm 30min that fully vibrates on rotary shaking table, make into the mother liquor bacteria suspension, measure the viable count of different number of days with the method for plate culture count.The result shows (table 6): as absorption carrier, the viable count of HL-1 bacterium is also more satisfactory with piscidia slag and diatomite mixture, and the viable count of 37d is 0.29 ± 0.04 * 10
8Cfu/mL.
Table 6 diatomite and piscidia slag are cultivated the viable count (* 10 of HL-1 bacterium different number of days
8Cfu/mL)
| Fate (d) | 7 | 14 | 20 | 26 | 31 | 37 |
| Viable count | 1.80±0.16 | 0.54±0.04 | 0.56±0.08 | 0.48±0.04 | 0.32±0.04 | 0.29±0.04 |
Annotate: data are 3 mean value ± standard errors that repeat.
Embodiment 7HL-1 fermented liquid is at the viable count of different number of days
(3) HL-1 fermented liquid among the embodiment 5 is packed in the aseptic plastic bottle, and sealing is placed in 4 ℃ of refrigerators, respectively the thalline quantity the 1st, 5,10,18, in the 31d analytical unit volume of liquid microbial inoculum.The result shows (table 7): fermented liquid is 2.24 ± 0.07 * 10 at the viable count of 31d
9Cfu/mL.
Table 7 liquid HL-1 microbial inoculum is at the viable count (* 10 of different number of days
9Cfu/mL)
| Fate (d) | 1 | 5 | 10 | 18 | 31 |
| Viable count | 2.76±0.10 | 2.47±0.05 | 2.07±0.06 | 2.29±0.12 | 2.24±0.07 |
Annotate: data are 3 mean value ± standard errors that repeat.
The effect of the product water-soluble phosphorus of embodiment 8HL-1 bacterium under insoluble inorganic phosphorus culture medium culturing is tested the sterilized indissoluble inorganic phosphorus of the 50mL substratum (concentration of calcium phosphate is 5.0g/L) of packing in the triangular flask, add respectively a-4, a-7, a-15 (being the HL-1 bacterium) and b-17 bacterium (inoculum size 5 rings) that the separating step among the embodiment 1 filters out, make behind the bacterium liquid 28 ℃, 70rpm, shaking table is cultivated 7d.With the bacterium liquid after cultivating at 10000rpm, 4 ℃ of centrifugal 15min, (unit is mg/L with molybdenum antimony resistance colorimetric method mensuration water-soluble phosphorus content to get supernatant liquor, the milligram number that namely represents contained water-soluble phosphorus in every liter of sample), establish the substratum that does not connect the HL-1 bacterium and be contrast, each is processed and repeats 3 times.Test-results is seen Fig. 3.The result shows: the water-soluble phosphorus content of HL-1 bacterium is the highest, reaches 63.84mg/L.
The effect experiment of the producing microbial phosphorus of embodiment 9HL-1 bacterium under insoluble inorganic phosphorus culture medium culturing is processed (the 5mg N,O-Diacetylmuramidase joins in the 1mL TEN solution) with the precipitation behind embodiment 8 medium centrifugals with lysate, 37 ℃ of water-bath 1h, then 4 ℃, the centrifugal 15min of 10000rpm, get supernatant liquor and measure microbe-P content (unit is mg/L, namely represents the milligram number of contained microbe-P in every liter of sample) with molybdenum antimony resistance colorimetric method.Test-results is seen Fig. 4.The result shows: the microbe-P content of HL-1 bacterium is the highest, reaches 32.58mg/L.
The effect experiment of embodiment 10HL-1 microbial inoculum promoting growth of plants field
Adopt HL-1 bacterium and blank to carry out Piglet s colibacillosis, being divided into 2 treatment group (gets 3.0kg soil for the 1st group and adds HL-1 fermented liquid 3mL, represent with HL-1, get 3.0kg soil for the 2nd group and add the HL-1 fermented liquid 3mL of deactivation as blank, represent with CK), each treatment group repeats 4 times.Each treatment group soil adds respectively urea 326mg, calcium phosphate 145mg and Repone K 238mg.Corn washing vernalization is soaked into preservation moisture with filter paper, is placed in 30 ℃ of incubators, exposes two days later the bud sowing to the seedbed.Approximately 25cm is high when seedling, transplants seedlings to basin, 2 seedlings of every basin.Water every day in right amount, the water yield of every basin is identical, evenly, notes not making water to flow out at the bottom of the basin in order to avoid fertilizer loss and cause error.Measured corn plant height (height at straight posterior lobe tip is smoothed out with the fingers on corn ground to maize leaf) on May 11st, 2011.Measured maize leaf (comprising all blades that grow) on May 9th, 2011.The result shows (table 8): add the HL-1 bacterium and can significantly promote the release of soil available phosphorus and the growth of corn.Compared with the control, use HL-1 microbial inoculum soil available phosphorus content and increased by 48%.
Table 8 different strains phosphorus decomposing effect reaches the short fruit contrast table that comes into force to corn
Annotate: data are 4 mean value ± standard errors that repeat, and the different alphabetical persons of same column in the table are illustrated in 0.05 level difference significantly (DMRT method).
Embodiment 11HL-1 bacterium is to the improved effect test of soil microorganisms functional diversity
Concrete operations are as follows: take by weighing the fresh soil that is equivalent to 10g oven dry soil sample and join in the 250mL triangular flask that 100mL sterile saline (0.85%) is housed; Behind vortex oscillator concussion 1min, in ice-water bath, leave standstill 1min; Repeat 3 times; Leave standstill 2min, getting the above-mentioned soil extraction of 5mL adds in the 45mL sterile saline (0.85%), behind the mixing, repeat this step, bacterium liquid (the bacterium liquid after the embodiment 5 step 3 gained fermentation culture) after diluting 1000 times is added in the BiologEco plate, every hole adds 150 μ L the microplate of inoculation is cultivated at 30 ℃ of incubators, with the soil that does not add bacterial classification as blank, respectively at 0,24,48,72,96h calculates the multifarious formula of soil microbial community following (table 9) with the light absorption value that microplate reader reads under the 590nm.10): each process the AWCD value in time increase and increase, and processing (HL-1 group) soil microbial community diversity indices (the soil microorganisms metabolic activity AWCD that adds bacterial classification, diversity of soil microorganism index Shannon, soil microorganisms species richness R ichness) all is significantly higher than the soil (control group that does not add bacterial classification, represent with CK), reach significant difference (
P＜
0.05).Illustrate that the HL-1 bacterium improved diversity of soil microorganism significantly.See Fig. 6.
[0053] table 9 calculates the formula of Microbial Community Diversity index
Table 10 different treatment soil microbial community diversity index (72h)
| Process | AWCD | Shannon(H) | Richness |
| CK | 0.65±0.28 a | 1.22±0.09 a | 27.33±1.53 a |
| HL-1 | 0.89±0.09 b | 1.31±0.04 b | 30.33±0.58 b |
Annotate: data are 3 mean value ± standard errors that repeat, and the different alphabetical persons of same column in the table are illustrated in 0.05 level difference significantly (DMRT method).
SEQUENCE LISTING
＜110〉Agricultural University Of South China
＜120〉subtilis HL-1 and the application aspect the soil phosphorus decomposing thereof
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1021
<212> DNA
＜213〉artificial sequence
<400> 1
aggcgtgcat gacgacacca tgcaccacct gtcactctgc tcccgaagga gaagccctat 60
ctctagggtt ttcagaggat gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa 120
accacatgct ccaccgcttg tgcgggcccc cgtcaattcc tttgagtttc agccttgcgg 180
ccgtactccc caggcggagt gcttaatgcg ttaacttcag cactaaaggg cggaaaccct 240
ctaacactta gcactcatcg tttacggcgt ggactaccag ggtatctaat cctgtttgct 300
ccccacgctt tcgcgcctca gtgtcagtta cagaccagaa agtcgccttc gccactggtg 360
ttcctccata tctctacgca tttcaccgct acacatggaa ttccactttc ctcttctgca 420
ctcaagtctc ccagtttcca atgaccctcc acggttgagc cgtgggcttt cacatcagac 480
ttaagaaacc acctgcgcgc gctttacgcc caataattcc ggataacgct tgccacctac 540
gtattaccgc ggctgctggc acgtagttag ccgtggcttt ctggttaggt accgtcaagg 600
tgccagctta ttcaactagc acttgttctt ccctaacaac agagttttac gacccgaaag 660
ccttcatcac tcacgcggcg ttgctccgtc agactttcgt ccattgcgga agattcccta 720
ctgctgcctc ccgtaggagt ctgggccgtg tctcagtccc agtgtggccg atcaccctct 780
caggtcggct acgcatcgtt gccttggtga gccgttacct caccaactag ctaatgcgac 840
gcgggtccat ccataagtga cagccgaagc cgcctttcaa tttcgaacca tgcagttcaa 900
aatgttatcc ggtattagcc ccggtttccc ggagttaccc cagtcttatg ggcaggttac 960
ccacgtgtta ctcacccgtc cgccgctaac tcactcgagc atgctactag cttttgcccc 1020
g 1021
Claims (2)
- Subtilis ( Bacillus subtilis) HL-1, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No.5175 on August 24th, 2011.
- 2. described subtilis HL-1 according to claim 1 is characterized in that its 16S rDNA nucleotide sequence is shown in SEQ ID NO:1.3. the microbial inoculum that is prepared into by claim 1 or 2 described subtilis HL-1.4. microbial inoculum according to claim 3 it is characterized in that being comprised of subtilis HL-1, diatomite and piscidia slag.5. the preparation method of the described microbial inoculum of claim 4, it is characterized in that the bacterial strain of activation is inoculated into seed culture medium by 10 ~ 15 % inoculum sizes, 30 ℃ of culture temperature, under the rotating speed 113rpm condition, shaking table is cultivated 24h, obtain primary seed solution, then be transferred to seed tank culture by 10 ~ 15% inoculum sizes, with secondary seed medium at 30 ℃, air flow 1000L/h, cultivate 24h under the rotating speed 70rpm condition, obtain secondary seed solution, secondary seed solution is inoculated into fermentor tank by 10 ~ 15% inoculum sizes, in fermention medium, continue enlarged culturing, at 30 ℃, air flow 10000L/h, cultivate 24h under the rotating speed 70rpm condition, the fermented liquid after the cultivation adds diatomite and piscidia slag, stirs, pulverize, be prepared into described microbial inoculum;Wherein every 1L seed culture medium contains extractum carnis 5g, peptone 5g, sodium-chlor 5g, and remainder is water, pH value 7.1 ~ 7.4;Every 1L secondary seed medium or fermention medium contain bean cake powder 30g, wheat bran powder 10g, ammonium nitrate 1.5g, yeast extract paste 3g, sodium-chlor 2.5g, sal epsom 0.2g, dipotassium hydrogen phosphate 0.3g and manganous sulfate 0.05g, and remainder is water, pH value 7.1 ~ 7.4.6. preparation method according to claim 5, the fermented liquid after it is characterized in that cultivating: diatomite: the mass ratio of piscidia slag is 0.6:1:1.7. claim 1 or the 2 described subtilis HL-1 application in decomposing insoluble phosphorus.8. claim 1 or 2 application of described subtilis HL-1 in Promoting plant growth.9. claim 1 or the 2 described subtilis HL-1 application in increasing soil microbial community diversity and increase bacterial abundance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110282657 CN102399713B (en) | 2011-09-22 | 2011-09-22 | Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110282657 CN102399713B (en) | 2011-09-22 | 2011-09-22 | Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102399713A CN102399713A (en) | 2012-04-04 |
| CN102399713B true CN102399713B (en) | 2013-01-30 |
Family
ID=45882368
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110282657 Expired - Fee Related CN102399713B (en) | 2011-09-22 | 2011-09-22 | Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102399713B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333694B (en) * | 2013-06-05 | 2015-10-28 | 华南理工大学 | A kind of by soil inavailable phosphorus microbiobacterial agent being converted into available phosphorus and its preparation method and application |
| CN103773709B (en) * | 2013-10-12 | 2015-05-20 | 河北农业大学 | Bacillus subtilis with efficient phosphate solubilizing effect and application thereof |
| CN104263679B (en) * | 2014-09-03 | 2016-08-24 | 南京聚肽高科农业有限公司 | A kind of efficient phosphate-solubilizing bacterium and application thereof |
| CN109022318B (en) * | 2018-08-15 | 2021-08-20 | 龙海市农丰源肥料有限公司 | Bacillus subtilis L1 and application thereof in degrading residual lincomycin in lincomycin residues |
| CN109136149B (en) * | 2018-09-21 | 2019-07-30 | 云南星耀生物制品有限公司 | Application of the bacillus subtilis in terms of soil phosphorus decomposing and cellulose degradation |
| CN110079484B (en) * | 2019-05-28 | 2022-07-08 | 青岛力力惠生物科技股份有限公司 | Bacillus subtilis and application thereof in agricultural production |
| CN110734876B (en) * | 2019-11-04 | 2021-04-16 | 华中农业大学 | Bacillus megaterium FJW1 biocontrol preparation as well as preparation method and application thereof |
| CN112625947B (en) * | 2020-12-16 | 2021-08-03 | 江苏省中国科学院植物研究所 | Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis |
| CN114315482A (en) * | 2021-08-13 | 2022-04-12 | 北京市农林科学院 | Biological bacterial fertilizer for increasing roots and improving efficiency and preparation method and application thereof |
| CN113999837B (en) * | 2021-11-08 | 2023-06-09 | 浙江丰瑜生态科技股份有限公司 | Agricultural microbial agent and preparation process thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410528A (en) * | 2002-11-21 | 2003-04-16 | 浙江大学 | Construction method of bacillus phosphorus relieving engineering bacterial strain |
| CN101899405A (en) * | 2010-01-20 | 2010-12-01 | 辛伟成 | Bacillus subtilis GU and application in preparing efficient biological compound phosphate fertilizer thereof |
| CN101955897A (en) * | 2010-08-27 | 2011-01-26 | 中国林业科学研究院林业研究所 | Functional strain No.3 as well as culture method and application thereof |
| CN102061273A (en) * | 2010-11-05 | 2011-05-18 | 南开大学 | Bacillus subtilis with phosphate-solubilizing effect and application thereof |
-
2011
- 2011-09-22 CN CN 201110282657 patent/CN102399713B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410528A (en) * | 2002-11-21 | 2003-04-16 | 浙江大学 | Construction method of bacillus phosphorus relieving engineering bacterial strain |
| CN101899405A (en) * | 2010-01-20 | 2010-12-01 | 辛伟成 | Bacillus subtilis GU and application in preparing efficient biological compound phosphate fertilizer thereof |
| CN101955897A (en) * | 2010-08-27 | 2011-01-26 | 中国林业科学研究院林业研究所 | Functional strain No.3 as well as culture method and application thereof |
| CN102061273A (en) * | 2010-11-05 | 2011-05-18 | 南开大学 | Bacillus subtilis with phosphate-solubilizing effect and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| 一种解磷菌剂的研制及其应用效果;叶明等;《浙江大学学报》;20100630;第36卷(第6期);第657-661页 * |
| 优良解磷菌株诱变选育与解磷培养基优化;孙剑秋等;《核农学报》;20100131;第24卷(第1期);第41-45页 * |
| 叶明等.一种解磷菌剂的研制及其应用效果.《浙江大学学报》.2010,第36卷(第6期), |
| 孙剑秋等.优良解磷菌株诱变选育与解磷培养基优化.《核农学报》.2010,第24卷(第1期), |
| 江丽华等.解磷菌在有机无机生物复合肥中活性的研究.《山东农业科学》.2002,(第6期), |
| 游银伟等.高效解磷菌Bacillus subtilis P-1的N离子束诱变育种.《西南农业学报》.2009,第22卷(第4期), |
| 解磷菌在有机无机生物复合肥中活性的研究;江丽华等;《山东农业科学》;20020630(第6期);第35-37页 * |
| 高效解磷菌Bacillus subtilis P-1的N离子束诱变育种;游银伟等;《西南农业学报》;20090831;第22卷(第4期);第1020-1022页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102399713A (en) | 2012-04-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102399713B (en) | Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving | |
| CN104928212B (en) | Bacillus megaterium X3 and preparation method thereof, application | |
| CN104877937B (en) | A kind of bacillus amyloliquefaciens bacterial manure and its preparation method and application promoting chili growth | |
| CN102876608B (en) | Bacillus amyloliquefaciens and application thereof | |
| CN101914445B (en) | Indigenous probiotic microorganism solid fungicide and preparation method and application thereof | |
| CN102021118B (en) | Preparation method of liquid inoculant of aboriginal probiotic bacillus megaterium, bacillus mucilaginosus and azotobacter chroococcum | |
| CN101948780B (en) | Antagonist bacterium for preventing and treating continuous cropping hot pepper epidemic disease and microbial organic fertilizer thereof | |
| CN105925502B (en) | From the bacterial strain Bacillus megaterium OP6 of silkworm excrement and its application | |
| CN106635903A (en) | Growth-promoting bacteria combination for enhancing salt tolerance of crops in moderate-severe saline and alkaline lands | |
| CN105936881A (en) | Bacillus saccharophila used for degrading alginic acid, and application method thereof | |
| CN109868242B (en) | Salt-tolerant acetoin-producing bacillus subtilis and application thereof | |
| CN104818233A (en) | Bacillus vallismortis and functional vegetable seedling raising biological matrix prepared from bacillus vallismortis | |
| CN102321551B (en) | Application of bacillus thuringiensis GL-1 in phosphate solubilization and disease prevention of soil | |
| CN103374528A (en) | Aspergillus niger strain and application thereof | |
| CN101558766B (en) | Trichoderma solid granules for preventing and controlling tobacco soil-borne fungus diseases and preparation method thereof | |
| CN102816719B (en) | Growth-promoting rhizobacteria SXH-2 and application thereof | |
| CN103173380B (en) | Bacillus cereus for activating soil nutrient and application of bacillus cereus | |
| CN102827792B (en) | Plant growth-promoting rhizobacterium SXH-3 and application thereof | |
| CN104593301B (en) | One plant of wall bacillus G1 and its preparation method and application | |
| CN110981591A (en) | Special growth-promoting fertilizer for oil tea, and preparation and application thereof | |
| CN104789494B (en) | The method for improving turf salt-resistance using garbage compost microbial bacterial agent is strengthened | |
| CN102898197A (en) | Technology for producing growth promoting bioorganic fertilizer by using algae mud as additive and product | |
| CN105907399B (en) | A kind of cadmium pollution renovation agent and its with the application in plant combined reparation agricultural land soil | |
| CN104909846A (en) | Bacillus amyloliquefaciens HFJ-7-containing composite chicken manure decomposed starter and application thereof | |
| CN117165475A (en) | Bacillus cereus and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130130 |