CN116536239A - Phosphate-dissolving microbial agent, and preparation and application thereof - Google Patents
Phosphate-dissolving microbial agent, and preparation and application thereof Download PDFInfo
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- CN116536239A CN116536239A CN202310412927.5A CN202310412927A CN116536239A CN 116536239 A CN116536239 A CN 116536239A CN 202310412927 A CN202310412927 A CN 202310412927A CN 116536239 A CN116536239 A CN 116536239A
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- phosphate
- culture medium
- penicillium
- aspergillus niger
- solid propagation
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- 230000000813 microbial effect Effects 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 239000007787 solid Substances 0.000 claims abstract description 65
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 63
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 63
- 239000010452 phosphate Substances 0.000 claims abstract description 63
- 239000001963 growth medium Substances 0.000 claims abstract description 58
- 230000003381 solubilizing effect Effects 0.000 claims abstract description 54
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 48
- 239000002689 soil Substances 0.000 claims abstract description 43
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 39
- 241000228143 Penicillium Species 0.000 claims abstract description 39
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 35
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 33
- 239000011574 phosphorus Substances 0.000 claims abstract description 33
- 239000000843 powder Substances 0.000 claims abstract description 27
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 23
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- 238000012258 culturing Methods 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 15
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- 238000002156 mixing Methods 0.000 claims description 4
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- 239000001506 calcium phosphate Substances 0.000 description 4
- 229910000389 calcium phosphate Inorganic materials 0.000 description 4
- 235000011010 calcium phosphates Nutrition 0.000 description 4
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- 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 4
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- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical compound [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 description 3
- 241000519895 Penicillium janczewskii Species 0.000 description 2
- 241000063980 Periphanes delphinii Species 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
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- 229910000398 iron phosphate Inorganic materials 0.000 description 2
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- 229910021645 metal ion Inorganic materials 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
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- 239000011591 potassium Substances 0.000 description 2
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- 238000012216 screening Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
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- 241000228212 Aspergillus Species 0.000 description 1
- 101000765308 Aspergillus niger N-(5'-phosphoribosyl)anthranilate isomerase Proteins 0.000 description 1
- 239000005955 Ferric phosphate Substances 0.000 description 1
- 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 1
- 241000059563 Hemicrepidius niger Species 0.000 description 1
- HSNVNALJRSJDHT-UHFFFAOYSA-N P(=O)(=O)[Mo] Chemical compound P(=O)(=O)[Mo] HSNVNALJRSJDHT-UHFFFAOYSA-N 0.000 description 1
- 241000918585 Pythium aphanidermatum Species 0.000 description 1
- 101100213970 Schizosaccharomyces pombe (strain 972 / ATCC 24843) ypt3 gene Proteins 0.000 description 1
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- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
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- 238000012851 eutrophication Methods 0.000 description 1
- 229940032958 ferric phosphate Drugs 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
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- 239000008103 glucose Substances 0.000 description 1
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- 239000012535 impurity Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical group 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910000399 iron(III) phosphate Inorganic materials 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 239000002245 particle Substances 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 239000002367 phosphate rock Substances 0.000 description 1
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical compound OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 1
- 230000008979 phosphorus utilization Effects 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
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- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- 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
- C12N3/00—Spore forming or isolating processes
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- 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
- C12N1/04—Preserving or maintaining viable microorganisms
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- 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
- C12N1/14—Fungi; Culture media therefor
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- C09K2101/00—Agricultural use
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/685—Aspergillus niger
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/80—Penicillium
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
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Abstract
The invention provides a phosphate-solubilizing microbial agent, and preparation and application thereof. The phosphate solubilizing microbial inoculum consists of penicillium spore powder and aspergillus niger spore powder which are obtained by culturing by a specific method (especially using a specific component and a solid propagation culture medium with the dosage of the specific component), the two strains play a role in synergy in dissolving indissolvable phosphorus sources in soil and the like, the utilization rate of phosphorus in the soil is obviously improved, the problems of resource waste, environmental pollution and the like caused by using excessive phosphate fertilizer are relieved, the plant growth is effectively promoted, and the phosphate solubilizing microbial inoculum is suitable for preparing bacterial fertilizers.
Description
Technical Field
The invention belongs to the technical field of microbial preparations. More particularly relates to a phosphate solubilizing microbial agent, and preparation and application thereof.
Background
Phosphorus is a major element necessary for plant growth, soil is the only source of phosphorus that plants acquire, and only soluble phosphorus can be directly absorbed and utilized by plants. In the actual production process, a considerable part of phosphorus and Ca are in the soil 2+ 、Al 3+ 、Fe 3+ The plasma metal ions form insoluble matters such as Ca-P, al-P, fe-P and the like in various forms, and cannot be directly utilized by plants, namely, the soil phosphorus element becomes an important limiting factor in the agriculture and forestry production process, so that the effectiveness of the soil phosphorus element and the fertilizer efficiency of the phosphate fertilizer are limited, and further, the landscape effect and ecological value of the garden green land are restricted. Therefore, in order to overcome the problem, a large amount of phosphate fertilizer is usually used in the current agriculture and forestry production process, but the phosphate fertilizer is not only a resource waste, but also brings environmental problems such as soil hardening and water eutrophication, and is not a good strategy.
The phosphate-dissolving bacteria can secrete small molecular organic acid, indissolvable phosphate in soil is released by means of reducing the pH value of the soil, chelating metal ions, ion exchange and the like and is re-absorbed and utilized by root systems of ground plants, so that the phosphate-dissolving bacteria gradually become one of hot tools for dissolving indissolvable phosphate sources in the soil, such as rhizobia with efficient phosphate-dissolving capability and the like provided by the prior art, but related reports of commonly using penicillium and aspergillus niger for phosphate-dissolving are not seen at present, and a scheme for carrying out specific selection on a solid propagation culture medium of a phosphate-dissolving bacteria agent prepared by the penicillium and the aspergillus niger is not seen.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the phosphate-dissolving microbial inoculum which can effectively dissolve indissolvable phosphorus sources in soil, remarkably improve the utilization rate of phosphorus in the soil and relieve the problems of resource waste, environmental pollution and the like caused by excessive use of phosphate fertilizer.
The first object of the invention is to provide a preparation method of a phosphate solubilizing microbial inoculum.
The second purpose of the invention is to provide the phosphate solubilizing microbial inoculum prepared by the method.
The third purpose of the invention is to provide the application of the phosphate solubilizing microbial inoculum in improving the utilization rate of phosphorus in soil.
The fourth object of the present invention is to provide a method for preserving the phosphate solubilizing microbial agent.
The fifth object of the invention is to provide a solid propagation medium.
The sixth object of the invention is to provide an application of the solid propagation medium in preparing a phosphate solubilizing microbial inoculum.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a preparation method of a phosphate solubilizing microbial inoculum, which comprises the following steps:
s1, inoculating penicillium into a PDA solid culture medium, culturing until spores cover the surface of the culture medium, and collecting penicillium spore suspension after washing; inoculating the strain into a solid propagation culture medium, culturing until spores cover the surface of the culture medium, and collecting penicillium spore powder;
s2, inoculating aspergillus niger into a PDA solid culture medium, culturing until spores cover the surface of the culture medium, and collecting aspergillus niger spore suspension after washing; inoculating the strain into a solid propagation culture medium, culturing until spores cover the surface of the culture medium, and collecting Aspergillus niger spore powder;
s3, uniformly mixing the S1 penicillium spore powder and the S2 aspergillus niger spore powder to obtain the phosphate solubilizing microbial inoculum;
s1 and S2 are not limited in sequence;
the solid propagation culture medium comprises the following components in percentage by mass of 19.5-20.5: 9.5 to 10.5:4.5 to 5.5:14.5 to 15.5 percent of corn cob, rice husk, wheat bran and water.
The terms "S1 and S2 are not limited to sequential order" means that S1 may be performed first, S2 may be performed first, or S1 and S2 may be performed simultaneously.
Preferably, in S1 and S2, the temperature of the culture is 25 to 30℃and most preferably 28 ℃.
Preferably, in S1 and S2, the flushing is with water.
Preferably, in S1 and S2, the mycelium is also removed after the rinsing, such as by filtration.
Preferably, in S1 and S2, the spore suspension is diluted and then inoculated into a solid propagation medium.
Further preferably, the dilution is to 800-1200 cfu/mL with water, most preferably 1000cfu/mL.
Preferably, in S1 and S2, the solid propagation medium is also sterilized, e.g.at 110-130℃for 25-35 min.
Preferably, in S1, the dosage ratio of the penicillium spore suspension to the solid propagation medium is 800-1200 cfu/g, and most preferably 1000cfu/g.
Preferably, in S2, the dosage ratio of the aspergillus niger spore suspension to the solid propagation medium is 800-1200 cfu/g, and most preferably 1000cfu/g.
Preferably, in S3, the mass ratio of the S1 penicillium spore powder to the S2 aspergillus niger spore powder is 0.8-1.2: 0.8 to 1.2, most preferably 1:1.
preferably, the mass ratio of the corn cob to the rice husk to the wheat bran to the water is 20:10:5:15.
preferably, the solid propagation medium further comprises agar.
Further preferably, the agar solution at 55-65 ℃ is sprayed on the surface of the solid propagation medium to form an adhesion layer with the thickness of 2-3 mm.
Agar is a common experimental culture medium formula, and is usually premixed with each component of the culture medium, and then sterilized, cooled and solidified to form a plane; in addition, the agar is attached to the surface of the solid propagation culture medium with irregular shape, so that the specific surface area of the culture medium is obviously increased, and a large number of spores are more favorably generated.
Further preferably, the concentration of agar in the agar solution is 1.5% (w/v) to 2.0% (w/v), most preferably 2% (w/v).
Further preferably, the agar solution is also sterilized, such as at 110-130℃for 15-25 min.
Further preferably, the speed of the agar solution spraying on the surface of the solid propagation medium is 20-40 mL/min, so that the agar is rapidly cooled and uniformly attached to the surface of the solid propagation medium, and the agar solution does not generate a plane.
Further preferably, the volume of the agar solution is 1/3 to 1/2 of the volume of the solid propagation medium.
Preferably, the phosphate solubilizing microbial agent is further covered with an organic cover.
Further preferably, the organic cover is a branch crushed material, such as pine bark crushed material dried and crushed to a particle size of 1-3 cm.
In the phosphate solubilizing microbial inoculum, the penicillium spore powder and the aspergillus niger spore powder play a role in synergism in dissolving indissolvable phosphorus sources in soil and the like, so that the utilization rate of phosphorus in the soil is remarkably improved, the environmental problem caused by using phosphate fertilizer is relieved, the plant growth can be effectively promoted, and the phosphate solubilizing microbial inoculum is suitable for preparing bacterial fertilizer. Therefore, the phosphate solubilizing microbial inoculum prepared by the method and the application of the phosphate solubilizing microbial inoculum in improving the utilization rate of the phosphorus in the soil are all within the protection scope of the invention.
Preferably, the phosphate-solubilizing bacterial agent is used at a concentration of 10 5 ～10 7 cfu/mL, most preferably 10 6 cfu/mL。
In addition, the invention also provides a preservation method of the phosphate solubilizing microbial inoculum, and the phosphate solubilizing microbial inoculum is dried to a water content of 9-39% and preserved at 3-5 ℃. Most preferably to a moisture content of 13% and stored at 4 ℃.
Preferably, the drying temperature is 35-40 ℃.
The invention specifically selects the components of the solid propagation culture medium, precisely controls the dosage proportion of each component, and can obviously improve the spore production capacity of aspergillus niger and penicillium when being used for culturing the aspergillus niger and penicillium, thereby being capable of rapidly obtaining a large amount of spore materials suitable for preparing the phosphate-solubilizing microbial inoculum. Thus, a composition comprising the following components in a mass ratio of 19.5 to 20.5:9.5 to 10.5:4.5 to 5.5:14.5 to 15.5 percent of corn cob residues, rice hulls, wheat bran and water, and the application of the solid propagation culture medium in preparing a phosphate solubilizing microbial inoculum are all within the protection scope of the invention.
Preferably, the penicillium (Penicillium brocae) is a strain S523, deposited in the China general microbiological culture collection center (CGMCC) with a deposit number of 40522 at a deposit address of 2023, and 03 and 09 days: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Preferably, the aspergillus niger (Aspergillus niger) is a strain S626, which is preserved in China general microbiological culture Collection center (CGMCC No. 40520) at a preservation address of 2023 and 03 and 09: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
The invention has the following beneficial effects:
1. in the phosphate solubilizing microbial inoculum, the penicillium spore powder and the aspergillus niger spore powder play a role in synergism in dissolving insoluble phosphorus sources in soil and the like, so that the utilization rate of phosphorus in the soil is remarkably improved, the environmental problem caused by using phosphate fertilizer is relieved, the growth of plants is effectively promoted, and the phosphate solubilizing microbial inoculum is suitable for preparing bacterial fertilizer.
2. The invention selects specific components, precisely controls the dosage proportion of each component, the prepared solid propagation medium takes agricultural product processing leftover materials as main materials, the cost is low, the water holding capacity is good, the specific surface area is large, and the medium is used for culturing aspergillus niger and penicillium, so that the spore production capacity of the two bacteria can be obviously improved, and a large amount of spore materials suitable for preparing phosphate solubilizing bacteria can be obtained in a short time.
3. The phosphate-solubilizing microbial inoculum of the invention has excellent stability and is suitable for long-term storage because the phosphate-solubilizing microbial inoculum takes fungus spores which are in a dormant state and have strong environmental tolerance as main components.
4. Compared with the traditional chemical fertilizer, the phosphate-dissolving microbial inoculum provided by the invention has the advantages of green, environment-friendly and strong lasting effect, and is free from pollution and residue to the environment.
Drawings
FIG. 1A shows the colony and the phosphate solubilizing ring of the strain S523, and FIG. 1B shows the colony and the phosphate solubilizing ring of the strain S626.
FIG. 2 is a phylogenetic tree of strain S523.
FIG. 3 is a phylogenetic tree of strain S626.
FIG. 4 shows the sporulation of strain S523 and strain S626 on T8 and T10 medium.
FIG. 5 shows the effect of different stoving time on the water content of phosphate decomposing microbial inoculum.
FIG. 6 shows the effect of different preservation times on spore germination rate of phosphate-solubilizing bacteria.
FIG. 7 is a graph showing the dry weight statistics of petunia.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 isolation, identification and preservation of P.brocae and A. Niger
(1) Test materials
Farmland soil and woodland red soil, PVK medium (glucose 10g, (NH) 4 ) 2 SO 4 0.5g，Ca 3 (PO 4 ) 2 5.0g，NaCl 0.2g，KCl 0.2g，MgSO 4 ·7H 2 O0.1 g, yeast extract 0.5g, mnSO 4 ·H 2 O 0.002g，FeSO 4 ·7H 2 O0.002 g, distilled water to 1000mL, and 2% (w/v) agar was added to the solid medium.
(2) Fungal isolation
Respectively collecting 10g farmland soil and 10g forest land red soil in the suburb of Guangzhou province of Guangdong, and carrying out subsequent treatment: adding 100mL of sterile water, fully shaking and uniformly mixing to obtain a soil suspension, and carrying out gradient dilution on the soil suspension to 10 volume percent -4 And g/mL, 100 mu L of the strain is coated on a PVK solid plate (containing 5g/L of calcium phosphate, 50 mu g/L of chloramphenicol and 100 mu g/L of streptomycin), colonies and the formation of phosphate solubilizing rings on the plate are observed after continuous culture for 5 days at 28 ℃, fungus strains with the maximum phosphate solubilizing ring radius/colony radius are selected, and the strain is continuously purified and cultured for 3 generations on the PVK plate, and is preserved in a refrigerator at 4 ℃ for standby.
The colony and the phosphate solubilizing ring on the plate are shown in FIG. 1, wherein FIG. 1A shows the colony and the phosphate solubilizing ring of the phosphate solubilizing fungus (designated S523) isolated from the farmland soil, and FIG. 1B shows the colony and the phosphate solubilizing ring of the phosphate solubilizing fungus (designated S626) isolated from the red soil of the woodland. Therefore, the phosphorus dissolving capability of the two strains is stronger.
(3) Fungus identification
3mL of S523 liquid PDA culture medium (OD 600 is 0.8-1.0) and 3mL of S626 liquid PDA culture medium (OD 600 is 0.8-1.0) are respectively taken for subsequent treatment: and (3) centrifugally collecting mycelia (8000 rpm,10 min), washing 3 times with clear water, extracting fungi DNA (Omega fungi DNA kit), amplifying fungus ITS fragments by using ITS4 and ITS5, recovering PCR products for sequencing analysis, and carrying out homology comparison with ITS1-5.8S-ITS2 of fungi in GenBank to construct a fungus phylogenetic tree.
The analysis results are shown in FIGS. 2 and 3, wherein FIG. 2 is the phylogenetic tree of strain S523 and FIG. 3 is the phylogenetic tree of strain S626. As can be seen, strain S523 is Penicillium (Penicillium brocae) (sequence similarity.gtoreq.97.4%) and strain S626 is Aspergillus niger (Aspergillus niger) (sequence similarity.gtoreq.97.7%).
(4) Determination of phosphorus-decomposing Capacity of fungi
Bacterial strain S523 single colony and bacterial strain S626 single colony are respectively picked and respectively inoculated into 3mL PVK liquid culture medium taking 5g/L calcium phosphate, 5g/L aluminum phosphate and 5g/L ferric phosphate as unique phosphorus sources, 1mL PVK liquid culture medium is taken after 3 days of culture at 28 ℃, the content of available phosphorus in the supernatant is detected by a phosphomolybdenum blue colorimetric method after 5 days of culture at 28 ℃, and the phosphate dissolving amount of the bacterial strain is calculated.
As a result, as shown in table 1, it was found that both strain S523 and strain S626 can effectively dissolve calcium phosphate, aluminum phosphate and iron phosphate, but S626 has a preference for dissolution of iron phosphate and aluminum phosphate, and S523 can better dissolve calcium phosphate.
TABLE 1 phosphate solubilizing ability of Strain S523 and Strain S626
(5) Preservation of fungi
Penicillium (Penicillium brocae) S523 strain is preserved in China general microbiological culture Collection center (CGMCC) at a preservation number of CGMCC No.40522 and a preservation address of 2023 and 03 and 09: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Aspergillus niger (Aspergillus niger) S626 strain is preserved in China general microbiological culture Collection center (CGMCC) at a preservation number of CGMCC No.40520 and a preservation address of 2023 and 09: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Example 2 solid propagation Medium formulation screening
(1) Test materials
Aspergillus niger (strain S626), penicillium phosphate (strain S523), bagasse, rice hulls, peanut bran, corn steep liquor and wheat bran.
(2) Test method
The mass ratio in table 1 is 1:1:1, adding water (the dosage of water in the culture medium is 30 wt%) to obtain solid propagation culture medium with 1-10 formulas, each formula contains 5 repetitions, and screening the formula of the solid propagation culture medium with the strongest spore-producing capability by taking the spore-producing capability of penicillium and aspergillus niger as indexes.
Firstly, respectively carrying out Z-shaped streaking on the surface of a PDA solid culture medium on the phosphate-solubilizing penicillium and phosphate-solubilizing aspergillus niger, culturing at 28 ℃ until spores cover the surface of the culture medium, washing spores from the solid plate with 0.1% (v/v) Tween 80 solution, collecting spore suspension, and filtering spore suspension with sterile double-layer gauze to remove doped mycelium according to 10 3 Inoculating cfu/g culture medium into the solid propagation culture medium (sterilized at 120deg.C for 30 min) of the above formula 1-10 (the usage ratio of strain to solid propagation culture medium is 1000cfu:1g), placing into a 28 deg.C incubator, continuously culturing until spores cover the surface of the culture medium, calculating spore production capacity, namely taking 1g of homogenized solid propagation culture medium with spores, adding 100mL of 0.02% (v/v) Tween solution, stirring with a magnetic stirrer for 10min, filtering with sterile double-layer gauze to remove impurities such as culture medium and mycelium, and sterilizing with sterile water according to 10: -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 10 -9 Performing gradient dilution, coating 100 mu L of the culture medium onto a PDA plate, placing the plate in a 28 ℃ incubator for culturing until colonies are generated, counting the number of the colonies, converting the number of the colonies into the number of spores (cfu/g) contained in a solid propagation culture medium per unit mass, and selecting a culture medium formula with the highest spore production capacity according to the counting result.
TABLE 1 solid propagation Medium formulation table
(3) Test results and analysis
The spore-producing ability of the P.delphinii and P.nigricans on the solid propagation medium of the formulation 1-10 is shown in Table 2.
TABLE 2 spore production ability on different solid propagation media
As can be seen from table 1:
(1) the sporulation quantity of the penicillium on the formula 1 is 10 5 On the order of cfu/g, the sporulation yield on formulas 3, 4, 5, 8, 9 was 10 6 On the order of cfu/g, the sporulation yield on formulas 2, 6 was 10 7 On the order of cfu/g, the sporulation on formulas 7 and 10 was 10 8 cfu/g order of magnitude;
(2) the spore yield of Aspergillus niger in formulas 1, 2, 4, 5, 8, 9 and 10 is 10 7 On the order of cfu/g, the sporulation on formulas 3, 6, 7 was 10 8 On the order of cfu/g.
The solid propagation culture medium prepared from different components is used for culturing the phosphate-dissolving aspergillus niger and phosphate-dissolving penicillium, the influence degree of the solid propagation culture medium on the spore production capacity of the strain is obviously different, and the solid propagation culture medium of the formula 7 is used for culturing the phosphate-dissolving aspergillus niger and phosphate-dissolving penicillium, so that the spore production amounts of the two phosphate-dissolving bacteria can reach 10 simultaneously 8 The cfu/g order of magnitude has the effect obviously better than that of formulas 1-6 and formulas 8-9, so that the solid propagation culture medium of formula 7 (namely rice husk, wheat bran, corn residue and water) is used for culturing aspergillus niger and penicillium, and the spore production capacity of the two bacteria can be obviously improved.
Example 3 optimization of solid propagation Medium ratio
(1) Test materials
Aspergillus niger (strain S626), penicillium phosphate (strain S523), testa oryzae, testa Tritici, corn residue and agar.
(2) Test method
Corn cob residues, rice hulls and wheat bran in the mass ratios of the groups T1 to T6 in Table 3 are uniformly mixed, and water (the water content in the culture medium is 30 wt%) is added to obtain the solid propagation culture medium with the formulas T1 to T6. In addition, in order to further improve the water retention capacity of the solid propagation medium and lock the flowing water in the medium, the agar solution accounting for 2% (w/v) of 1/3 volume of the solid propagation medium is added on the basis of the formula of the solid propagation medium of T1-T6 to obtain a formula T7-T12; wherein, the method for adding 2% (w/v) agar solution comprises the following steps: sterilizing 2% (w/v) agar solution at high temperature (120 ℃ for 20 min), cooling to 60 ℃, uniformly spraying on the surface of a solid propagation culture medium cooled to 25 ℃ (the agar spraying speed is 20-40 mL/min), and forming an adhesion layer with the thickness of 2-3 mm on the surface.
Each formulation contained 3 replicates, the spore-producing ability of Penicillium and Aspergillus niger was measured as in example 2, and the solid propagation medium formulation with the strongest spore-producing ability was screened out and photographed, and the results are shown in FIG. 4.
TABLE 3 solid propagation Medium formulation table
(3) Test results and analysis
The spore-producing ability of the P.delphinii and P.nigricans on the solid propagation medium of the formulations T1 to T12 is shown in Table 4.
TABLE 4 spore production ability on different solid propagation media
As can be seen from Table 4, the sporulation ability of Penicillium phosphate and Aspergillus phosphate in the T8 and T10 medium was significantly higher than that of the other treatment groups, and the addition of agar increased the sporulation ability of Penicillium phosphate by an order of magnitude (compared with the optimal results of example 2, from 10 8 Lifting cfu/g to 10 9 cfu/g)。
The sporulation of P.delbrueckii and P.delbrueckii on T8 and T10 medium is shown in FIG. 4. The spore growth area basically covers the surface and the gap of the whole solid propagation culture medium, so that the formula of the solid propagation culture medium is very suitable for producing spores of phosphate-dissolving penicillium and phosphate-dissolving aspergillus niger. In addition, considering that the production cost and price of wheat bran are higher than those of corn cob, T8 medium was selected as propagation medium used in the following examples.
EXAMPLE 4 preservation time optimization of phosphate solubilizing microbial inoculum
(1) Test materials
The P.phosphate-solubilizing penicillium spore powder and P.niger spore powder obtained by the culture in the T8 medium of example 3 were obtained, and the mixture was dried in an oven at 37℃and PDA (potato dextrose agar) plates were used.
(2) Test method
The penicillium spore powder and the aspergillus niger spore powder are mixed according to the proportion of 1:1, and obtaining the phosphate-dissolving microbial inoculum. 7 equal parts of a phosphate-solubilizing microbial inoculum having a mass of 0.5g was prepared, and the obtained mixture was packed in 1.5mL centrifuge tubes, treated in the manner shown in Table 5 (at a room temperature of 22 to 30 ℃) and the water contents (water content= (mass of fresh sample of treated solid fermentation product-mass of dry matter measured after complete drying of treated solid fermentation product at 65 ℃) of the CK, T1, T3 and T5 groups of phosphate-solubilizing microbial inoculum)/mass of fresh sample of treated solid fermentation product were measured, respectively. In the preservation process, 0.01g of the phosphate solubilizing bacterial agent is regularly taken 1 time per month, 20 mu L of the phosphate solubilizing bacterial agent is taken after being diluted by 200mL of water and coated on the surface of a PDA flat plate, and bacterial colony counting is carried out after 5 days of culture at 28 ℃, each group contains 3 repetitions, and the statistical value is spore germination rate = bacterial colony number of each stage/initial bacterial colony number multiplied by 100 percent, and the continuous statistics is carried out for 9 months.
TABLE 5 modes of treatment of phosphate solubilizing microbial agents
(3) Test results and analysis
The effect results of different drying time periods (corresponding to CK, T1, T3 and T5) on the water content of the phosphate-solubilizing microbial inoculum are shown in figure 5, and the effect results of different preservation time periods (corresponding to CK and T1-T6) on the spore germination rate of the phosphate-solubilizing microbial inoculum are shown in figure 6.
As can be seen from fig. 5: the moisture contents of the samples without drying, drying 1d, drying 3d and drying 5d were 39%, 29%, 13% and 9%, respectively.
As can be seen from fig. 6:
(1) the spore germination rate of CK and T1-T6 phosphate-dissolving microbial inoculum can be maintained to be more than 60% within 4 months, which shows that the phosphate-dissolving microbial inoculum has better stability and can be stored for a long time.
(2) The spore germination rate of T3 is obviously higher than that of T1 and T5, and the spore germination rate of T4 is obviously higher than that of T2 and T6. The spore activity of the phosphate solubilizing microbial agent treated by 3d is obviously better than that of the phosphate solubilizing microbial agent treated by 1d or 5d, which shows that the stability of the phosphate solubilizing microbial agent can be obviously improved after the phosphate solubilizing microbial agent is treated by 3d in a baking oven at 37 ℃, and the stability of the phosphate solubilizing microbial agent is optimal when the water content of the phosphate solubilizing microbial agent is 13 percent as shown in the figure 5.
(3) The spore germination rate of T2 is obviously higher than that of T1, the spore germination rate of T4 is obviously higher than that of T3, and the spore germination rate of T6 is obviously higher than that of T5; under the condition of normal temperature (T1, T3 and T5), the spore activity can be greatly reduced after 4-5 months, only T3 after being dried for 3 days can still maintain activity after 7 months (but the spore germination rate is only about 30 percent), and the T5 after being dried for 5 days has the worst preservation effect (completely losing activity at the 5 th month). The spore activity of the phosphate solubilizing microbial inoculum stored at 4 ℃ is obviously better than that of the phosphate solubilizing microbial inoculum stored at normal temperature, which shows that the stability of the phosphate solubilizing microbial inoculum can be obviously improved when the phosphate solubilizing microbial inoculum is stored at 4 ℃.
(4) The spore germination rate of T4 was significantly higher than other groups; under the condition (T4), the spore activity of the phosphate solubilizing microbial inoculum slowly decreases along with time, the spore germination rate within half a year can be maintained to be more than 80%, and the spore germination rate can also reach 75% after 9 months. The invention shows that after the phosphate-dissolving microbial inoculum is treated for 3 days in a baking oven at 37 ℃, the phosphate-dissolving microbial inoculum is preserved at 4 ℃, so that the optimal stability of the phosphate-dissolving microbial inoculum can be realized, the optimal preservation effect is achieved, and the preservation time of the phosphate-dissolving microbial inoculum is obviously prolonged.
Example 5 soil cultivation test
(1) Test materials
(1) The spore powder of P.P.aphanidermatum obtained by culturing in the T8 medium of example 3 was prepared into a concentration by using sterile waterIs 10 5 The spore suspension of cfu/mL is used for obtaining the penicillium fungus solution;
(2) taking Aspergillus niger spore powder obtained by culturing T8 culture medium in example 3, preparing into 10 concentration with sterile water 5 cfu/mL spore suspension to obtain Aspergillus niger bacterial liquid;
(3) the phosphate-solubilizing microbial agent of example 4 was prepared with sterile water to a concentration of 10 5 The spore suspension of cfu/mL is used for obtaining composite bacterial liquid;
(4) organic covering (drying pine bark and crushing to grain size 1-3 cm)
(5) Garden green soil (with phosphorite powder application history), soil pH value 7.56+/-0.04, organic matter 19.8+/-0.3 g/kg, total nitrogen 1.17+/-0.05 g/kg, total phosphorus 1.17+/-0.01 g/kg, total potassium 7.52+/-0.02 g/kg, hydrolyzed nitrogen 118.6+/-0.3 mg/kg, available phosphorus 9.82+/-0.14 mg/kg and quick-acting potassium 49.6+/-0.4 mg/kg.
(2) Test method
In order to attach the actual use environment of the phosphate-solubilizing microbial inoculum as much as possible, the whole test is carried out under outdoor and outdoor conditions. First, the bacterial liquids/sterile water of each group shown in Table 6 were mixed at a concentration of 1L/m 2 Uniformly spraying the organic covering on the surface of the soil to be tested (the soil to be tested is homogenized in advance and split-packed into seedling raising pots, in addition, the application method of the organic covering is that after bacterial liquid/sterile water is sprayed, 1 layer of organic covering with the thickness of 2cm is added on the surface of the soil), collecting soil samples after 2 months, collecting 1 mixed sample as a sample to be tested by a five-point sampling method in each seedling raising pot, detecting the effective phosphorus content of the soil by a phosphomolybdic blue colorimetric method, and each group contains 4 repetitions. Intra-group mean calculation and analysis of variance were performed using EXCEL 2016, and inter-group difference significance was multiple compared by LSD using SPSS 22.0.
Table 6 soil inoculation test design
(3) Test results and analysis
The effective phosphorus content of each group of soil is shown in table 7.
TABLE 7 soil available phosphorus content under different treatment conditions
Note that: the difference between the data represented by the different lower case letters was significant (P < 0.05).
As can be seen from table 7:
(1) the effective phosphorus content of the T3 group with the same bacterial liquid application amount is obviously higher than that of the T1 group and the T2 group, and the phosphorus dissolving effect of the composite bacterial agent is obviously better than that of single application effect of the two bacterial agents, so that the penicillium spore powder and the aspergillus niger spore powder obtained by adopting the specific culture medium for culture play a synergistic effect in the aspects of dissolving insoluble phosphorus sources in soil and the like, and the soil phosphorus utilization rate is obviously improved.
(2) The effective phosphorus content of the T3 group is 52.8% higher than that of CK1, and the effective phosphorus content of the T4 group is 65.8% higher than that of CK2, which shows that the effective phosphorus content of the soil can be obviously increased when the phosphate solubilizing microbial inoculum is applied to the soil surface.
(3) The effective phosphorus content of the T4 group is higher than that of the T3 group, which indicates that the phosphorus dissolving effect of the composite microbial inoculant can be further enhanced by adding an organic covering on the basis of the composite microbial inoculant.
EXAMPLE 6 petunia cultivation test
(1) Test materials
The petunia plug seedling, the phosphate-solubilizing microbial inoculum of the example 4, a culture medium (obtained by uniformly mixing the green land soil, the green waste compost and the like) and a seedling pot.
(2) Test method
Respectively diluting the phosphate-solubilizing bacteria agent into 10-concentration bacteria by using sterile water 8 cfu/mL、10 7 cfu/mL、10 6 cfu/mL、10 5 cfu/mL、10 4 cfu/mL、10 3 cfu/mL of the bacterial liquid was applied to T1 to T6 in the treatment manner shown in Table 8Around petunia root systems, CK groups were dosed with equal amounts of sterile water as a control, each group containing 5 replicates. Destructive sampling was performed after 2 months to measure dry weight.
Table 8 treatment method for petunia cultivation test
(3) Test results and analysis
The results of the dry weight statistics of petunia are shown in figure 7. It can be seen that: the T1 and T2 groups have no significant difference from the CK group; the T3-T5 groups can obviously increase the dry weight of petunia, and are respectively increased by 22.8%, 26.4% and 25.2% compared with CK groups; although the concentration of the T6 group is highest, the dry weight of petunia is not significantly different from that of the CK group and is inferior to that of the T3-T5 groups. Indicating that when the use amount of the phosphate solubilizing microbial inoculum is 10 5 ～10 7 cfu/mL, it is optimal for the pro-active effect of petunia.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The preparation method of the phosphate-solubilizing microbial inoculum is characterized by comprising the following steps:
s1, inoculating penicillium into a PDA solid culture medium, culturing until spores cover the surface of the culture medium, and collecting penicillium spore suspension after washing; inoculating the strain into a solid propagation culture medium, culturing until spores cover the surface of the culture medium, and collecting penicillium spore powder;
s2, inoculating aspergillus niger into a PDA solid culture medium, culturing until spores cover the surface of the culture medium, and collecting aspergillus niger spore suspension after washing; inoculating the strain into a solid propagation culture medium, culturing until spores cover the surface of the culture medium, and collecting Aspergillus niger spore powder;
s3, uniformly mixing the S1 penicillium spore powder and the S2 aspergillus niger spore powder to obtain the phosphate solubilizing microbial inoculum;
s1 and S2 are not limited in sequence;
the solid propagation culture medium comprises the following components in percentage by mass of 19.5-20.5: 9.5 to 10.5:4.5 to 5.5:14.5 to 15.5 percent of corn cob, rice husk, wheat bran and water.
2. The method according to claim 1, wherein the temperature of the culture is 25 to 30 ℃.
3. The preparation method according to claim 1, wherein the dosage ratio of the penicillium spore suspension to the solid propagation medium is 800-1200 cfu/g; the dosage ratio of the aspergillus niger spore suspension to the solid propagation culture medium is 800-1200 cfu/g.
4. The preparation method according to claim 1, wherein the mass ratio of the S1 penicillium spore powder to the S2 aspergillus niger spore powder is 0.8-1.2: 0.8 to 1.2.
5. The method of claim 1, wherein the solid propagation medium further comprises agar.
6. The phosphate solubilizing microbial inoculum prepared by the method of any one of claims 1 to 5.
7. The use of the phosphate solubilizing microbial inoculum of claim 6 in improving the utilization rate of phosphorus in soil.
8. The method for preserving a phosphate solubilizing microbial agent according to claim 6, wherein the phosphate solubilizing microbial agent is dried to a water content of 9 to 39% and preserved at 3 to 5 ℃.
9. The solid propagation culture medium is characterized by comprising the following components in percentage by mass of 19.5-20.5: 9.5 to 10.5:4.5 to 5.5:14.5 to 15.5 percent of corn cob, rice husk, wheat bran and water.
10. The use of the solid propagation medium according to claim 9 for preparing a phosphate solubilizing bacterial agent.
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