CN109320363A - A kind of bacillus liquid bluk recombination microbial-bacterial fertilizer and preparation method thereof - Google Patents
A kind of bacillus liquid bluk recombination microbial-bacterial fertilizer and preparation method thereof Download PDFInfo
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- CN109320363A CN109320363A CN201811493305.5A CN201811493305A CN109320363A CN 109320363 A CN109320363 A CN 109320363A CN 201811493305 A CN201811493305 A CN 201811493305A CN 109320363 A CN109320363 A CN 109320363A
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C21/00—Methods of fertilising, sowing or planting
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
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- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention discloses a kind of bacillus liquid bluk recombination microbial-bacterial fertilizers and preparation method thereof, belong to field of agricultural microbial technology, by weight, its component proportion is as follows: bacillus subtilis Bacillus subtilis SEM-9 bacterium solution 40-60 weight %, nutrition complex 20-40 weight %, manganese sulfate are 1-3 weight %, sodium methylene bis-naphthalene sulfonate is 4-8 weight %, and surplus is water.Bacillus subtilis Bacillus subtilis SEM-9 bacterium source is in silkworm excrement in bacillus liquid bluk recombination microbial-bacterial fertilizer of the invention, its fermenting and producing silkworm excrement liquid bio bacterial manure is utilized simultaneously, one growing environment close to ecosystem can either be provided to Bacillus subtilis SEM-9 bacterial strain, living spores quantity is improved, the release of various nutriments in silkworm excrement can be also more advantageous to.Composite microbic bacterial fertilizer of the invention can significantly improve the yield of crop and the resistivity to wilt disease for crops such as green cucumber, pakchois.
Description
Technical field
The present invention relates to a kind of bacillus liquid bluk recombination microbial-bacterial fertilizer and preparation method thereof, in particular to a kind of gemma
Bacillus Bacillus subtilis SEM-9 liquid composite microbial bacterial manure and preparation method thereof, belongs to composite microbic bacterial fertilizer
Field.
Background technique
Wilt disease is a kind of global plant epiphyte soil-borne disease, is the major microorganisms disease for causing crop failure
One of.Fusarium oxysporum (Fusarium oxysporum Schl) is one of its most important pathogen, and host range is wide.Change
Learning drug is the effective efficiently method for preventing and treating wilt disease, but chemicals use leads to soil degradation, destroys the ecological balance, very
To endangering human health.
With social development and government guidance, the ecological agriculture of sustainable development has become main trend, the disease-resistant product of breeding
Kind, crop rotation and work and biological control etc. are at the main means for preventing and treating wilt disease.Shortage resistant gene, time-consuming, stability
Difference is the major defect of disease-resistant variety breeding；Crop rotation and a work can effectively prevent wilt disease, but economic benefit is low.
Therefore, it is current wilt disease prevention and treatment that exploitation, which not only can guarantee continuous cropping but also can prevent and treat the biological control method of wilt disease,
Research hotspot.
Existing research at present is screened from silkworm excrement with inorganic elements such as phosphorus decomposing, potassium, and has antagonism Fusarium oxysporum
Microorganism, such as the Chinese invention patent of Patent No. 2017104163586 of September 12 announcements in 2017 discloses one plant
From the bacillus subtilis strain (Bacillus subtilis SEM-9) of silkworm excrement.
Composite microbic bacterial fertilizer refers to the bio-feritlizer containing a large amount of plant growth-promoting rhizobacterias (PGPR), plant rhizosphere growth-promoting
Bacterium (PGPR) can synthesize plant nutrient substance or hormone, inhibit phytopathogen growth, release environment-stress, improve microelement
Existence form, promote fertilizer absorption；Plant growth can thus be promoted, improve crop yield.
Padding of the silkworm faeces just, in mulberry leaf residue and silkworm rearing bed is referred to as silkworm excrement, is the principal by product of silkworm quotient's industry, because of its thick egg
The inorganic element contents such as the organic nutrient substances such as white, carbohydrate and phosphorus, potassium are abundant, are the first choices of base fertilizer to agricultural crops, but straight
Connect will lead to using silkworm excrement as base manure it is insufficient to its nutritional utilization, such as the inorganic elements such as phosphorus, potassium using insufficient, cause
Waste.
Therefore it provides it is a kind of have both efficiently prevention and treatment crop wilt disease and growth promoting volume increase liquid composite microbial bacterial manure just at
For technical field technical problem urgently to be solved.
Summary of the invention
An object of the present invention is using silkworm excrement as fermentation substrate and to utilize bacillus subtilis strain (Bacillus
Subtilis SEM-9) develop a kind of liquid composite microbial bacterial manure for efficiently preventing and treating crop wilt disease, growth promoting volume increase.
Above-mentioned purpose of the invention reaches by the following technical programs:
A kind of bacillus liquid bluk recombination microbial-bacterial fertilizer, by weight, component proportion are as follows:
Bacillus subtilis Bacillus subtilis SEM-9 bacterium solution 40%-60%, nutrition complex 20%-40%,
Manganese sulfate is 1%-3%, sodium methylene bis-naphthalene sulfonate 4%-8%, and surplus is water；
The bacillus subtilis Bacillus subtilis SEM-9 bacterium solution is obtained by fermentation；Its fermentating formula is such as
Under, in terms of mass concentration proportion, 10~90g/L of silkworm excrement, 20~30g/L of cornstarch, 15~25g/L of bean cake powder, (NH4)2SO4
For 0.5~1.5g/L, CaCO3For 3~6g/L, K2HPO4For 0.2~0.3g/L, MgSO4·7H2O be 0.15~0.25g/L,
MnSO4·H2O is 0.15~0.25g/L, NaCl2It is suitable for 10-30g/L, pH=6.5~7.5, polysiloxane defoaming agent SXP-101
Amount, surplus is water；
The nutrition complex is amino acid, fulvic acid, alginic acid, humic acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, urine
The mixing of one or more of plain amino acid chelated calcium or sodium ethylene diamine tetracetate magnesium arbitrary proportion.
Preferably, the content of the silkworm excrement is 30~70g/L.
Preferably, the content of the silkworm excrement is 50~70g/L.
Preferably, in the nutrition complex, 7 weight %, the humic acid that fulvic acid accounts for total bacterial manure amount account for total bacterial manure amount
3 weight %, the dipotassium hydrogen phosphate that 12 weight %, ethylenediamine tetraacetic acid disodium magnesium salt account for total bacterial manure amount account for total bacterial manure amount 4 weight %,
Potassium dihydrogen phosphate accounts for 4 weight % of total bacterial manure amount.
It is a further object of the present invention to provide the preparations of above-mentioned bacillus liquid bluk recombination microbial-bacterial fertilizer.
Above-mentioned purpose of the invention reaches by the following technical programs:
A kind of preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer, its step are as follows:
(1) actication of culture:
(deposit number is GDMCC NO.60619 to the Bacillus subtilis SEM-9 strain that 4 DEG C of inclined-planes are saved, extensively
East saves Culture Collection preservation) it transfers in LB culture medium flat plate, 32~37 DEG C of 60~72h of culture；
(2) prepared by first order seed:
Selecting, there is the single bacterium of fixed form to fall in the LB liquid medium containing 50~100mL (250mL tri- on plate
Angle bottle), 14~20h of constant temperature incubation under the conditions of 32~37 DEG C, 150~200rpm/min obtains primary seed solution；
(3) prepared by secondary seed solution:
Primary seed solution is accessed into seeding tank by 1~4% inoculum concentration, fermentation temperature is 32~37 DEG C, 400rpm/min,
Ventilatory capacity is pressed ventilates with the ratio of fermenter volume 1:1, and tank presses 0.06~0.07Mpa, cultivates 7~10h, obtains secondary seed
Liquid；
(4) prepared by Bacillus subtilis SEM-9 zymocyte liquid:
1) prepared by fermentation liquid: as follows using fermentating formula: 10~90g/L of silkworm excrement, 20~30g/L of cornstarch, bean cake powder
15~25g/L, (NH4)2SO4For 0.5~1.5g/L, CaCO3For 3~6g/L, K2HPO4For 0.2~0.3g/L, MgSO4·7H2O
For 0.15~0.25g/L, MnSO4·H2O is 0.15~0.25g/L, NaCL2For 10-30g/L, pH=6.5~7.5, poly- silicon oxygen
Alkane defoaming agent SXP-101 is appropriate, and surplus is water；
2) by step (3) preparation secondary seed solution by 3~5% inoculum concentration access fermentor, tank pressure 0.06~
0.07Mpa, ventilatory capacity is pressed ventilates with the ratio of fermentation volume 1:1, and 400rpm/min, 32~37 DEG C of 18~22h of ferment at constant temperature are obtained
To Bacillus subtilis SEM-9 zymocyte liquid；
(5) preparation of nutrition complex
Amino acid, fulvic acid, alginic acid, humic acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, urea amino acid chelated calcium or
The mixing of one or more of sodium ethylene diamine tetracetate magnesium arbitrary proportion, obtains nutrition complex；
(6) preparation of silkworm excrement composite microbic bacterial fertilizer
The Bacillus subtilis SEM-9 zymocyte liquid 40-60 weight % that step (4) is obtained and step (5) are made
Standby nutrition complex 20-40 weight % mixing, adds the synergist manganese sulfate and 4-8% weight % of weight 1-3%%
Auxiliary agent sodium methylene bis-naphthalene sulfonate or polycondensation naphthalenesulfonatedispersant dispersant D-425, surplus be water, obtain silkworm excrement composite microbial bacteria
Fertilizer.
Preferably, the Bacillus subtilis SEM-9 zymocyte liquid is 50 weight %, 30 weight of nutrition complex
Amount %, manganese sulfate are 2.5 weight %, sodium methylene bis-naphthalene sulfonate is 5 weight %, and surplus is water.
The nutrition complex of the 30 weight % includes: the fulvic acid for accounting for total 7 weight % of bacterial manure amount, accounts for total 12 weight of bacterial manure amount
The humic acid for measuring %, the ethylenediamine tetraacetic acid disodium magnesium salt for accounting for total 3 weight % of bacterial manure amount, the phosphorus for accounting for total 4 weight % of bacterial manure amount respectively
Sour hydrogen dipotassium and potassium dihydrogen phosphate.
Preferably, in step 1) the fermentation liquid preparation of the step (4), the content of silkworm excrement is 30~70g/L.
Preferably, in step 1) the fermentation liquid preparation of the step (4), the content of silkworm excrement is 30~50g/L.
It is a further object of the present invention to provide the applications of above-mentioned bacillus liquid bluk recombination microbial-bacterial fertilizer.
Above-mentioned purpose of the invention reaches by the following technical programs:
The application of above-mentioned bacillus liquid bluk recombination microbial-bacterial fertilizer, direct pouring root.
Specific method of administration: after bacillus liquid bluk recombination microbial-bacterial fertilizer of the invention is diluted 500~1000 times,
For pouring root, amount of application are as follows: 1000~2000g/ mus.
The beneficial effects of the present invention are:
Bacillus subtilis SEM-9 living spores quantity in fermentation liquid is increased to 9~15 by the present invention ×
109cfu/mL。
The bioactive substances such as crude protein, carbohydrate and water soluble vitamin rich in silkworm excrement, while silkworm
The inorganic elements such as husky P, K also rich in, frequently as Chinese medicine, feed and agricultural base manure come using；The present invention makees silkworm excrement
It ferments for liquid fermentation substrate, produces liquid composite microbial bacterial manure, sufficiently release organic and inorganic battalion in silkworm excrement
Support substance.
Bacillus subtilis SEM-9 bacterial strain is used to produce silkworm excrement liquid bio bacterial manure by the present invention, which comes
Derived from silkworm excrement, while its fermenting and producing silkworm excrement liquid bio bacterial manure is utilized, Bacillus subtilis SEM-9 bacterium can either be given
Strain provides a growing environment close to ecosystem, improves living spores quantity, can also be more advantageous to various nutriments in silkworm excrement
Release.
Bacillus subtilis SEM-9 liquid composite microbial bacterial manure of the invention is for agricultures such as green cucumber, pakchois
Crop can significantly improve the yield of crop and the resistivity to wilt disease.
Below by the drawings and specific embodiments, the present invention is described in detail.It should be understood that the reality
Apply example and merely relate to the preferred embodiments of the invention, when not departing from the spirit and scope of the present invention, various composition and
The changes and improvements of content are all possible.
Detailed description of the invention
In Fig. 1 in Application Example 1 of the present invention control experiment photo.
Fig. 2 is photo when Application Example 2 of the present invention starts with fertilizer.
Fig. 3 is to apply SEM-9 bacterial manure in Application Example 2 of the present invention and compare the cucumber seedling of bacterial manure April 10 in 2018
Growing way photo when day checks and accepts.
Fig. 4-1 is that the cucumber seedling of SEM-9 bacterial manure is applied in Application Example 2 of the present invention in examination on April 10th, 2018
Partial enlargement growing way photo.
Fig. 4-2 is the cucumber seedling of application control bacterial manure in Application Example 2 of the present invention when checking and accepting on April 10th, 2018
Partial enlargement growing way photo.
Fig. 4-3 is part of the cucumber seedling of blank control in Application Example 2 of the present invention when checking and accepting on April 10th, 2018
Amplify growing way photo.
Fig. 5-1 is that the cucumber seedling of SEM-9 bacterial manure is applied in Application Example 2 of the present invention in examination on April 10th, 2018
Partial enlargement growing way photo.
Fig. 5-2 is the cucumber seedling of application control bacterial manure in Application Example 2 of the present invention when checking and accepting on April 10th, 2018
Partial enlargement growing way photo.
Specific embodiment
Embodiment 1
A kind of preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer, its step are as follows:
(1) actication of culture: the Bacillus subtilis SEM-9 strain transfer that 4 DEG C of inclined-planes are saved is in LB culture medium
Plate, 35 DEG C of culture 66h；
(2) prepared by first order seed: selecting the single colonie on plate with fixed form, is added and trains containing 75mL LB liquid
In the 250mL triangular flask for supporting base, under the conditions of 35 DEG C, 175rpm/min, constant temperature incubation 17h obtains primary seed solution；
(3) prepared by secondary seed solution: the primary seed solution that step (2) is obtained, and accesses seed by 2.5% inoculum concentration
Tank, fermentation temperature are 35 DEG C, 400rpm/min, and ventilatory capacity is pressed ventilates with the ratio of seed tank volume 1:1, and tank presses 0.065Mpa,
8.5h is cultivated, secondary seed solution is obtained；
(4) prepared by Bacillus subtilis SEM-9 zymocyte liquid:
1) fermentation liquid prepare: using fermentating formula it is as follows: silkworm excrement 50g/L, bean cake powder 20g/L, cornstarch 27g/L,
(NH4)2SO4For 1g/L, CaCO3For 4g/L, K2HPO4For 0.3g/L, MgSO4·7H2O is 0.25g/L, MnSO4·H2O is
0.25g/L、NaCl2Appropriate for 20g/L, pH=7.0, polysiloxane defoaming agent SXP-101, surplus is water.
2) secondary seed solution for preparing step (3) accesses fermentor by 4% inoculum concentration, and tank presses 0.065Mpa, ventilation
Amount is ventilated by with the ratio of fermentation volume 1:1, and 400rpm/min, 37 DEG C of ferment at constant temperature 22h obtain Bacillus subtilis
SEM-9 zymocyte liquid；
(5) preparation of nutrition complex
By amino acid, fulvic acid, alginic acid, humic acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, urea amino acid chelated calcium
It is mixed with sodium ethylene diamine tetracetate magnesium, obtains nutrition complex, including: it accounts for the fulvic acid of total 7 weight % of bacterial manure amount, account for always
The humic acid of 12 weight % of bacterial manure amount, accounts for total bacterial manure amount 4 at the ethylenediamine tetraacetic acid disodium magnesium salt for accounting for total 3 weight % of bacterial manure amount respectively
The dipotassium hydrogen phosphate and potassium dihydrogen phosphate of weight %；
(6) preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer
Prepared by the 50 weight % of Bacillus subtilis SEM-9 zymocyte liquid that step (4) is obtained and step (5)
30 weight % of nutrition complex, 2.5 weight % of synergist manganese sulfate and auxiliary agent sodium methylene bis-naphthalene sulfonate are 5 weight %, are mixed
It closes, surplus is water, obtains bacillus liquid bluk recombination microbial-bacterial fertilizer 1.
Embodiment 2
It is other same as Example 1, the difference is that: the 1 of step (4)) in, silkworm excrement content is 10g/L；Obtain gemma
Bacillus liquid composite microbial bacterial manure 2.
Embodiment 3
It is other same as Example 1, the difference is that: the 1 of step (4)) in, silkworm excrement content is 30g/L；Obtain gemma
Bacillus liquid composite microbial bacterial manure 3.
Embodiment 4
It is other same as Example 1, the difference is that: the 1 of step (4)) in, silkworm excrement content is 70g/L；Obtain gemma
Bacillus liquid composite microbial bacterial manure 4.
Embodiment 5
It is other same as Example 1, the difference is that: the 1 of step (4)) in, silkworm excrement content is 90g/L；Obtain gemma
Bacillus liquid composite microbial bacterial manure 5.
Embodiment 6
A kind of preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer, its step are as follows:
(1) actication of culture: the Bacillus subtilis SEM-9 strain transfer that 4 DEG C of inclined-planes are saved is in LB culture medium
Plate, 32 DEG C of culture 60h；
(2) prepared by first order seed: selecting the single colonie on plate with fixed form, the LB liquid training containing 50mL is added
In the 250mL triangular flask for supporting base, under the conditions of 32 DEG C, 150rpm/min, constant temperature incubation 14h obtains primary seed solution；
(3) prepared by secondary seed solution: the primary seed solution that step (2) is obtained, and accesses seeding tank by 1% inoculum concentration,
Fermentation temperature is 32 DEG C, 400rpm/min, and ventilatory capacity is pressed ventilates with the ratio of seed tank volume 1:1, and tank presses 0.06Mpa, culture
7h obtains secondary seed solution；
(4) prepared by Bacillus subtilis SEM-9 zymocyte liquid:
1) fermentation liquid prepare: using fermentating formula it is as follows: silkworm excrement 10g/L, cornstarch 20g/L, bean cake powder 15g/L,
(NH4)2SO4For 0.5g/L, CaCO3For 3g/L, K2HPO4For 0.2g/L, MgSO4·7H2O is 0.15g/L, MnSO4·H2O is
0.15g/L、NaCl2Appropriate for 10g/L, pH=6.5, polysiloxane defoaming agent SXP-101, surplus is water.
2) secondary seed solution for preparing step (3) accesses fermentor by 3% inoculum concentration, and tank presses 0.06Mpa, ventilation
Amount is ventilated by with the ratio of fermentation volume 1:1, and 400rpm/min, 37 DEG C of ferment at constant temperature 22h obtain Bacillus subtilis
SEM-9 zymocyte liquid；
(5) preparation of nutrition complex
By amino acid, fulvic acid, alginic acid, humic acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, urea amino acid chelated calcium
It is mixed with sodium ethylene diamine tetracetate magnesium, obtains nutrition complex, including: it accounts for the fulvic acid of total 7 weight % of bacterial manure amount, account for always
The humic acid of 12 weight % of bacterial manure amount, accounts for total bacterial manure amount 4 at the ethylenediamine tetraacetic acid disodium magnesium salt for accounting for total 3 weight % of bacterial manure amount respectively
The dipotassium hydrogen phosphate and potassium dihydrogen phosphate of weight %；
(6) preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer
Prepared by the 40 weight % of Bacillus subtilis SEM-9 zymocyte liquid that step (4) is obtained and step (5)
20 weight % of nutrition complex, synergist manganese sulfate 1.5 weight % and auxiliary agent polycondensation naphthalenesulfonatedispersant dispersant D-425 are 4 weights
% is measured, mixing, surplus is water, obtains bacillus liquid bluk recombination microbial-bacterial fertilizer 6.
Embodiment 7
A kind of preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer, its step are as follows:
(1) actication of culture: the Bacillus subtilis SEM-9 strain transfer that 4 DEG C of inclined-planes are saved is in LB culture medium
Plate, 37 DEG C of culture 72h；
(2) prepared by first order seed: selecting the single colonie on plate with fixed form, the LB liquid containing 100mL is added
In the 250mL triangular flask of culture medium, under the conditions of 37 DEG C, 200rpm/min, constant temperature incubation 20h obtains primary seed solution；
(3) prepared by secondary seed solution: the primary seed solution that step (2) is obtained, and accesses seeding tank by 4% inoculum concentration,
Fermentation temperature is 37 DEG C, 400rpm/min, and ventilatory capacity is pressed ventilates with the ratio of seed tank volume 1:1, and tank presses 0.07Mpa, culture
10h obtains secondary seed solution；
(4) prepared by Bacillus subtilis SEM-9 zymocyte liquid:
1) fermentation liquid prepare: using fermentating formula it is as follows: silkworm excrement 90g/L, cornstarch 30g/L, bean cake powder 25g/L,
(NH4)2SO4For 1.5g/L, CaCO3For 6g/L, K2HPO4For 0.3g/L, MgSO4·7H2O is 0.25g/L, MnSO4·H2O is
0.25g/L、NaCl2Appropriate for 30g/L, pH=7.5, polysiloxane defoaming agent SXP-101, surplus is water；
2) secondary seed solution for preparing step (3) accesses fermentor by 5% inoculum concentration, and tank presses 0.07Mpa, ventilation
Amount is ventilated by with the ratio of fermentation volume 1:1, and 400rpm/min, 37 DEG C of ferment at constant temperature 22h obtain Bacillus subtilis
SEM-9 zymocyte liquid；
(5) preparation of nutrition complex
By amino acid, fulvic acid, alginic acid, humic acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, urea amino acid chelated calcium
It is mixed with sodium ethylene diamine tetracetate magnesium, obtains nutrition complex, including: it accounts for the fulvic acid of total 7 weight % of bacterial manure amount, account for always
The humic acid of 12 weight % of bacterial manure amount, accounts for total bacterial manure amount 4 at the ethylenediamine tetraacetic acid disodium magnesium salt for accounting for total 3 weight % of bacterial manure amount respectively
The dipotassium hydrogen phosphate and potassium dihydrogen phosphate of weight %；
(6) preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer
Prepared by the 60 weight % of Bacillus subtilis SEM-9 zymocyte liquid that step (4) is obtained and step (5)
40 weight % of nutrition complex, 3.0 weight % of synergist manganese sulfate and auxiliary agent sodium methylene bis-naphthalene sulfonate are 6 weight %, are mixed
It closes, surplus is water, obtains bacillus liquid bluk recombination microbial-bacterial fertilizer 7.
In bacillus liquid bluk recombination microbial-bacterial fertilizer with the detection embodiment 1-5 preparation of viable plate count detection method
Bacillus subtilis SEM-9 gemma number, as a result see the table below 1:
Influence of the 1 silkworm excrement content of table to Bacillus subtilis SEM-9 gemma number
As can be seen from Table 1: adding a certain amount of silkworm excrement compared with the control group, in fermentation medium is beneficial to Bacillus
The high density fermentation of subtilis SEM-9；Also, when silkworm excrement content is in 50~70g/L, living spores quantity can reach
To 145~152 × 109cfu/mL。
Application Example 1
The promotion growth experiment of bacillus liquid bluk recombination microbial-bacterial fertilizer of the invention to green cucumber
Experimental material:
Green cucumber seed passes through local market of farm produce purchase；Bacillus subtilis SEM-9 liquid composite microbial bacterium
Fertilizer is prepared according to the embodiment of the present invention 1；Giant dragon bacterial manure of taking root is manufactured by Guangdong Plants dragon Biotechnology Ltd. to be sold；
More spore bacterium solution products that control bacterial manure is sold by Dezhou Yuan Henong Science and Technology Ltd..
Experimental site: Guangdong Province plant dragon Biotechnology Ltd. arable farming room；
Experimental method: it when the true leaf of melon seedling is fully deployed, applies fertilizer for the first time；It carries out applying fertilizer for second after a week；Each place
Reason set 12 caves (parallel), one, every cave seedling, apply fertilizer encapsulating amount for every 50~60mL/ times；The specific extension rate of every kind of fertilizer is such as
The following table 2；After second is applied fertilizer, every two days, first time examination is carried out to experiment, statistical test group relative comparison group is distinguished after seven days
Dry weight and weight in wet base growth rate；Specific growing state is shown in attached drawing 1.
Table 1
Experimental result is as shown in Figure 1, apply the green cucumber of Bacillus subtilis SEM-9 liquid composite microbial bacterial manure
Seedling shoot thickness is dark green greatly, and applies other bacterial manure and clear water control group, all there is obvious nutritional deficiency symptom, and leaf is partially yellow, under cotyledon hair wrinkle
It hangs down.
It is following table 3 and table 4 show use different bio-bacterial manures to the growth promoting degree of green cucumber seedling, as the result is shown: using
Bacterial manure can promote the growth of green cucumber seedling in varying degrees, and application control bacterial manure and the effect gap for giant dragon bacterial manure of taking root are little,
But the weight in wet base and dry weight of the green cucumber seedling of application Bacillus subtilis SEM-9 liquid composite microbial bacterial manure increase separately
6.17 times and 11.2 times.
2 fresh weight growth rate of table
3 dry weight growth rate of table
Application Example 2
Bacillus liquid bluk recombination microbial-bacterial fertilizer of the invention compares growing way difference between each bacterial manure, while observing to crop
The preventive effect of soil-borne disease.
Test period: on April 10,23 days to 2018 March in 2018.
Test material: Bacillus subtilis SEM-9 liquid composite microbial bacterial manure, according to the embodiment of the present invention 1
It prepares；Giant dragon bacterial manure of taking root is manufactured by Guangdong Plants dragon Biotechnology Ltd. to be sold；Compare bacterial manure by Dezhou member and
More spore bacterium solution products of agriculture Science and Technology Ltd. sale.
Test site: test site is located at Zhuhai City lantern crossing.
Experimental field overview: experimental field the poor retain water and nutrients ability of soil property is weak for this, and it is withered to have part cucumber seedling before early period fertilizer
It withers.Whole management level is general.
Trial crops: cucumber.Without insect pest, disease generation is generally mainly based on downy mildew.
Cell arrangement: 4 processing, 2 repetitions of every processing, totally 8 cells, about 15 square meter of every cell, cell processing use
Random alignment.At least isolation strip of 50cm is set between different disposal.
Handle time and number
This, which is tested, starts with fertilizer when selected cucumber seedling height is about 15cm (as shown in Fig. 2, real for present invention application
Apply photo when example 2 starts with fertilizer), every 7 days with primary, i.e. on March 23rd, 2018 and on March 30th, 2018, leaching is twice altogether
Fertilizer is carried out an acceptance inspection in second with 10 days (i.e. on April 9th, 2018) after fertilizer, main to measure plant height and statistics dead seedling situation.
Weather condition during test: 13~27 DEG C of temperature during test, 21 degree of mean daily temperature；Relative humidity is 34%
~94%, per day relative humidity 75%.Leaching fertilizer same day weather condition: on March 23rd, 2018 is fine, and on March 30th, 2018 is cloudy.
Use instrument and fertilization mode: using irrigation method after clear water dilution.Every seedling about 400 milliliters/time of watering, bacterial manure
Average application.
Plant height investigation: fertilising prefixed point investigates 20 plants of cucumber seedling height, after using fertilizer twice, in latter 10 days (i.e. 2018
On April 9, in) plant height is measured.
Dead seedling investigation: dead seedling situation is counted with 10 days (i.e. on April 9th, 2018) after fertilizer for the second time.
Plant height Growth Rate Calculation method:
Death rate calculation method:
As a result with analysis:
Cucumber seedling plant height growth rate is shown in Table 5:
Table 5
Death rate and preventive effect are shown in Table 6:
Table 6
As shown in figure 3, for application SEM-9 bacterial manure in Application Example 2 of the present invention and compareing the cucumber seedling of bacterial manure 2018
Growing way photo when on April 10, in checks and accepts；
As shown in Fig. 4-1,4-2,4-3, in Application Example 2 respectively of the present invention apply SEM-9 bacterial manure, control bacterial manure and
Partial enlargement growing way photo of the cucumber seedling of blank control when checking and accepting on April 10th, 2018；
Fig. 5-1 and 5-2 is respectively to apply SEM-9 bacterial manure in Application Example 2 of the present invention to exist with the cucumber seedling for compareing bacterial manure
Partial enlargement growing way photo when on April 10th, 2018 checks and accepts.
Growing way interpretation of result is tested from this, bacillus liquid bluk recombination microbial-bacterial fertilizer SEM-9 bacterial manure of the invention
Effect is best, and the cucumber seedling growing way handled using SEM-9 bacterial manure is obvious, and for 10 days growth rates up to 238.96%, blade is thick after fertilising
Big dark green, stem is sturdy, prematurity result.
And from Disease-resistance Analysis, due to having existed part dead seedling before applying bacterial manure, and with SEM-9 bacterial manure after fertilizer in certain journey
Degree is upper can to slow down dead seedling degree, death rate 12.38%, this than compare bacterial manure with take root that giant dragon death rate is low, and disease resistance is stronger.
Application Example 3
The growth promoting production-increasing function of bacillus liquid bluk recombination microbial-bacterial fertilizer of the invention to pakchoi
Experimental material: it pakchoi seedling: is provided by Huidong County's town Liang Hua agrotechnical station；
Experimental site: Huidong County of Huizhou City town Liang Hua village Guang Chang；
Experimental design: as shown in table 7:
Table 7SEM-9 composite bacterial fertilizer is to black leaf pakchoi growth promoting experimental design
A: Ba Tian compound fertilizer (N:P:K=15:6:8) is respectively 15kg/ mus of top dressing after seedling the 4th, 11,18 day；
B:SEM-9 biological composite bacterial manure irrigation time is identical as dressing time, extension rate 400；In addition, in pakchoi
The time of infertility separately irrigates twice.
Experimental result: as shown in table 8, influence of the different disposal to pakchoi Main Agronomic shape, for processing one,
After leaching applies " SEM-9 composite microbic bacterial fertilizer " three times, the plant height of pakchoi has increased separately 85.4%, 97.8% and 36.9%,
Leaf width has increased separately 46.9%, 42.6% and 29.9%, and leaf length has increased separately 88.9%, 82.4% and 37.4%.
For processing two, leaching apply " the SEM-9 composite microbic bacterial fertilizer of inactivation " three times after, the plant height of pakchoi point
Not Zeng Jia by 91.3%, 96.6% and 35.5%, leaf width has increased separately 45.2%, 42.2% and 31.3%, and leaf length increases separately
95.3%, 81.0% and 36.8%.
For processing three, the plant height amplification of the pakchoi same period is respectively 73.5%, 101.2% and 33.3%, and leaf width increases
Width is respectively 41.9%, 38.6% and 27.9%, and the long amplification of leaf is respectively 88.6%, 81.9% and 34.4%.
For no processing four for applying any fertilizer, the same period plant height amplification of pakchoi is only 49.0% respectively,
100.0% and 12.5%, leaf width amplification is respectively 18.2%, 41.0% and 7.3%, the long amplification of leaf is respectively 57.4%,
59.5% and 15.3%.
When harvesting, processing one is compared to processing two, and plant height, leaf width, leaf are long and the number of sheets increases separately 3.0%, 3.6%, 2.4%
With 3.6%；Compared to processing three, plant height, leaf width, the leaf length and the number of sheets for handling one have increased separately 7.7%, 11.5%, 4.9% and
9.6%；Compared to processing four, plant height, leaf width, the leaf length and the number of sheets for handling one have increased separately 40.9%, 47.5%, 56.6% and
33.9%.
Influence of 8 different disposal of table to pakchoi Main Agronomic shape
As shown in table 9, influence of the different disposal to yield of pakchoi passes through single factor test significance analysis (p < 0.05, SPSS
22) find, there are significant differences for the yield of pakchoi between four processing, wherein handle a yield highest, respectively relatively processing two,
Three and four amplification 5.04%, 10.30% and 190.94%.The yield of followed by processing two is higher, and relatively processing three and four increases respectively
5.01% and 176.98%, and handle that four yield are minimum, and per mu yield is only 294.44 ± 17.60Kg.
Influence of 9 different disposal of table to yield of pakchoi
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (10)
1. a kind of bacillus liquid bluk recombination microbial-bacterial fertilizer, by weight, component proportion are as follows:
Bacillus subtilis Bacillus subtilis SEM-9 bacterium solution 40-60 weight %, nutrition complex 20-40 weight %,
Manganese sulfate is 1-3 weight %, sodium methylene bis-naphthalene sulfonate is 4-8 weight %, and surplus is water；
The bacillus subtilis Bacillus subtilis SEM-9 bacterium solution is obtained by fermentation；Its fermentating formula is as follows, with
Mass concentration proportion meter, 10~90g/L of silkworm excrement, 20~30g/L of cornstarch, 15~25g/L of bean cake powder, (NH4)2SO40.5~
1.5g/L、CaCO33~6g/L, K2HPO40.2~0.3g/L, MgSO4·7H20.15~0.25g/L of O, MnSO4·H2O
0.15~0.25g/L, NaCl210~30g/L, pH=6.5~7.5, polysiloxane defoaming agent SXP-101 are appropriate, and surplus is
Water；
The nutrition complex is amino acid, fulvic acid, alginic acid, humic acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, urea ammonia
The mixing of base acid chelating one or more of calcium or sodium ethylene diamine tetracetate magnesium arbitrary proportion.
2. bacillus liquid bluk recombination microbial-bacterial fertilizer according to claim 1, it is characterised in that: the content of the silkworm excrement is
30~70g/L.
3. bacillus liquid bluk recombination microbial-bacterial fertilizer according to claim 1, it is characterised in that: the content of the silkworm excrement is
50~70g/L.
4. bacillus liquid bluk recombination microbial-bacterial fertilizer according to claim 1, it is characterised in that: the nutrition complex
In, 12 weight %, the ethylenediamine tetraacetic acid disodium magnesium salt that 7 weight %, the humic acid that fulvic acid accounts for total bacterial manure amount account for total bacterial manure amount account for
4 weight %, the potassium dihydrogen phosphate that 3 weight %, the dipotassium hydrogen phosphate of total bacterial manure amount account for total bacterial manure amount account for 4 weights of total bacterial manure amount
Measure %.
5. the preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer described in any one of claim 1-4, its step are as follows:
(1) actication of culture:
The Bacillus subtilis SEM-9 strain transfer that 4 DEG C of inclined-planes are saved is in LB culture medium flat plate, 32~37 DEG C of cultures
60~72h；
(2) prepared by first order seed:
Selecting, there is the single bacterium of fixed form fallen on plate in the LB liquid medium containing 50~100mL, 32~37 DEG C,
14~20h of constant temperature incubation under the conditions of 150~200rpm/min, obtains primary seed solution；
(3) prepared by secondary seed solution:
Primary seed solution is accessed into seeding tank by 1~4% inoculum concentration, fermentation temperature is 32~37 DEG C, ventilation, tank pressure 0.06~
0.07Mpa cultivates 7~10h, obtains secondary seed solution；
(4) prepared by Bacillus subtilis SEM-9 zymocyte liquid:
1) prepared by fermentation liquid: as follows using fermentating formula: 10~90g/L of silkworm excrement, 20~30g/L of cornstarch, bean cake powder 15~
25g/L、(NH4)2SO4For 0.5~1.5g/L, CaCO3For 3~6g/L, K2HPO4For 0.2~0.3g/L, MgSO4·7H2O 0.15
~0.25g/L, MnSO4·H2O is 0.15~0.25g/L, NaCL2For 10-30g/L, pH=6.5~7.5, silicone antifoam
Agent SXP-101 is appropriate, and surplus is water；
2) secondary seed solution of step (3) preparation being accessed into fermentor by 3~5% inoculum concentration, tank presses 0.06~0.07Mpa,
Ventilation, 32~37 DEG C of 18~22h of ferment at constant temperature obtain Bacillus subtilis SEM-9 zymocyte liquid；
(5) preparation of nutrition complex
Amino acid, fulvic acid, alginic acid, humic acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, urea amino acid chelated calcium or second two
The mixing of one or more of amine tetraacethyl sodium magnesium arbitrary proportion, obtains nutrition complex；
(6) preparation of silkworm excrement composite microbic bacterial fertilizer
The nutrition of the Bacillus subtilis SEM-9 zymocyte liquid 40-60% that step (4) is obtained and step (5) preparation
Complex 20-40 weight % mixing, adds the synergist manganese sulfate of 1-3 weight % and the auxiliary agent methylene of 4-8 weight %
Double sodium naphthalene sulfonates or polycondensation naphthalenesulfonatedispersant dispersant D-425, surplus are water, obtain silkworm excrement composite microbic bacterial fertilizer.
6. the preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer according to claim 5, it is characterised in that: the step
(6) the SEM-9 zymocyte liquid of Bacillus subtilis described in is 50 weight %, 30 weight % of nutrition complex, manganese sulfate
It is 5 weight % for 2.5 weight %, sodium methylene bis-naphthalene sulfonate, surplus is water.
7. the preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer according to claim 6, it is characterised in that: 30 weight
The nutrition complex of amount % includes: the fulvic acid for accounting for total 7 weight % of bacterial manure amount, the humic acid for accounting for total 12 weight % of bacterial manure amount, accounts for
The ethylenediamine tetraacetic acid disodium magnesium salt of total 3 weight % of bacterial manure amount, the dipotassium hydrogen phosphate and di(2-ethylhexyl)phosphate for accounting for total 4 weight % of bacterial manure amount respectively
Hydrogen potassium.
8. the preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer according to claim 5, it is characterised in that: the step
(4) in step 1) fermentation liquid preparation, the content of silkworm excrement is 30~70g/L.
9. the preparation of bacillus liquid bluk recombination microbial-bacterial fertilizer according to claim 5, it is characterised in that: the step
(4) in step 1) fermentation liquid preparation, the content of silkworm excrement is 30~50g/L.
10. the application of bacillus liquid bluk recombination microbial-bacterial fertilizer described in any one of claim 1-4.
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