CN105801258A - Bacillus subtilis fertilizer and preparation method and application thereof - Google Patents
Bacillus subtilis fertilizer and preparation method and application thereof Download PDFInfo
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
- C05D9/00—Other inorganic fertilisers
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- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract
The invention discloses a bacillus subtilis fertilizer and a preparation method and application thereof. The preservation number of bacillus subtilis JQ-001 is CGMCC No.11632, and the bacillus subtilis is obtained by being separated from soil of a JianSanjiang farm of Heilongjiang. The bacillus subtilis JQ-001 is subjected to seed activation, seed culture and fermented culture to be prepared into fermented liquid, then kieselguhr, a bonding agent, humic acid, cow dung and other components are added, and the bacillus subtilis fertilizer is prepared. As the bacillus subtilis has high biological activity, the inhibition capacity to potato scabs is high, environmental compatibility is good, a plant microorganism ecofauna can be adjusted, no hazard is caused to people and livestock, and use is convenient. The bacillus subtilis fertilizer prepared from the bacillus subtilis can inhibit the potato scabs, the yield of potatoes can be increased, the quality of the potatoes can be improved, the soil problem caused by potato continuous cropping all year round is solved, and intensive production development of modern agriculture is promoted.
Description
Technical field
The present invention relates to field of agricultural microorganism, be specifically related to a kind of bacillus subtilis bacterial manure and preparation side thereof
Method and application.
Background technology
Shot hole is considered the worldwide disease during Rhizoma Solani tuber osi produces, universal in China many Rhizoma Solani tuber osis main producing region
Exist, continuous cropping ground, meta-alkali ground and cultivation management improperly in the case of occurrence degree even more serious.In recent years,
Shot hole occurs in succession in a lot of Rhizoma Solani tuber osi production area of China, wherein Heilungkiang, the Inner Mongol, Hebei, mountain
The Rhizoma Solani tuber osi of multiple provinces and regions such as east, Shanxi, Shaanxi, Guizhou, Gansu, Sichuan, Hunan, Hubei, Yunnan
Main producing region is all related to the report of this disease, substantially covers northeast, North China, northwest and southwest.And at gas
The regional sickness rate waiting arid, soil meta-alkalescence, continuous cropping continuous cropping serious is higher.
The occurrence cause of potato scab:
1. potato seed is carried disease germs.In production of seed stock, the shot hole of micro potato is particularly acute, and the operating of potato seed is in spite of illness
One of important channel of disease spread.
2. soil meta-alkali.Northeast, northwest, some regional culture Rhizoma Solani tuber osis in North China usually use plant ash etc.
Alkaline fertilizer, soil gradually meta-alkali after accumulation, causes potato scab to increase the weight of year by year for many years.
3. pathogen accumulates in soil.South China, accumulates in a large number owing to continuous cropping for many years will also result in pathogenic bacteria.
4. attention degree is inadequate.Shot hole is the most little on yield and the meat impact of Rhizoma Solani tuber osi, although this disease is
Through becoming the big obstacle that Rhizoma Solani tuber osi produces, but also not causing the enough attention of people, a lot of areas are to this disease
The most all have little understanding, also become the reason that potato scab increases the weight of year by year.
Potato scab pathogenic bacteria is mainly caused harm tuber, invades from hole skin, and their early stage produces at tuber epidermis
Brown speckle, the most gradually expands, the tissue necrosis around infection court, and tuber surface is roughening, quality wood
Suberification.Simultaneously according to the difference of Species of Pathogens, ripe potato block often shows as raised or sunken table
Face scab, time serious, scab is in flakes, and the quality of potato block reduces.Simultaneously as after epidermal tissue is destroyed,
Easily by other pathogen infections, cause tuber rot, have a strong impact on the outward appearance of Rhizoma Solani tuber osi, reduce its quality and
The storage cycle.
Utilizing Biological control soil-borne disease is a kind of effective prophylactico-therapeutic measures, is must in plant protection
Few ingredient.Sick to target in particular with the plant rhizosphere beneficial microbe originally existed in nature
The antagonism of pathogenic microorganism, controls the harm of pathogenic microorganism, has less environmental risk, is one
Plant eco-friendly Prevention Technique.Along with food safety, ecological safety are paid attention to by people, Biological control is real
One of Critical policies of existing agricultural modernization sustainable development.
Summary of the invention
Problem to be solved by this invention is: provide a kind of can the hay bud of antagonism potato scab pathogenic bacteria
Spore bacillus, the bacterial manure thus prepared, thus reach the safe efficient pathogenetic purpose of inhibition of potato scab.
For solving above-mentioned technical problem, the technical scheme is that
Bacillus subtilis (Bacillus subtilis) JQ-001 that the present invention provides, is called for short JQ-001
Bacterial strain, is preserved in the common micro-life of China Committee for Culture Collection of Microorganisms on November 09th, 2015
CGMCC (is called for short, address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3) in thing center, deposit number
For CGMCC No.11632.
Bacillus subtilis (Bacillus subtilis) JQ-001 of the present invention is from Heilungkiang JianSanjiang farm even
Make what separation in more than 5 years Oryza sativa L. foundation soil, purification and screening step obtained, employing morphological characteristic,
On bio-chemical characteristics, molecular biology identification method and 16SrDNA homologous sequence comparison analysis finally determine
The bacterial strain stating deposit number is bacillus subtilis (Bacillus subtilis).
A kind of bacillus subtilis bacterial manure, by bacillus subtilis JQ-001 through seed activation, seed culture,
Make zymocyte liquid after fermentation culture, be subsequently adding humic acids, cattle manure is made；Wherein zymocyte liquid is in filter pressing
Before preferably add following composition: kieselguhr, adhesive aid；Described adhesive aid is preferably silicon dioxide.
The preparation method of a kind of bacillus subtilis bacterial manure, specifically includes:
A. actication of culture: by the bacillus subtilis JQ-001 inoculation of cryopreservation on solid medium,
Cultivating 34-38h in 35-38 DEG C, picking list bacterium colony is transferred on slant medium, in 35-38 DEG C of cultivation
16-24h, with media surface thalline under aseptic washing as inoculation liquid；
B. prepared by primary seed solution: accesses step a in seed culture medium according to the inoculum concentration of 5%-10% and prepares
Inoculation liquid, rotating speed 200-260rpm, temperature 35-38 DEG C, shaking table cultivate 16-24h, as first order seed
Liquid；
C. prepared by secondary seed solution: accesses step b in seed culture medium according to the inoculum concentration of 0.5%-3% and prepares
Primary seed solution, ventilation is 0.4-1.5vvm, and mixing speed is 180-260rpm, and incubation time is
18-24h, as secondary seed solution；
D. fermentation culture: access prepared by step c in fermentation medium according to the inoculum concentration of 5%-10% two grades
Seed liquor, ventilation is 0.4-1.5vvm, and mixing speed is 180rpm-260rpm, and incubation time is 24h-30h,
Obtain fermentation liquid；
E. the preparation of bacterial manure: by described zymocyte liquid through filter press and add humic acids, cattle manure makes bacterial manure.
Preferably, prepared by step e bacterial manure method particularly includes:
1. the zymocyte liquid obtained in above-mentioned fermentation culture is initially charged kieselguhr and adhesive aid, sheet frame after mixing
Filter；
2. 55-75% humic acid and the 20-35% cattle manure of gross weight, mixing is added according to filter cake bacteria containing amount；
3. by the above-mentioned material granulator granulation that mixes, spraying simultaneously is added the zymocyte liquid bonding of 10% and is made
Grain；
4. the granule oven drying at low temperature will made.
Preferably, above-mentioned steps 1. middle 3% kieselguhr adding gross weight and 1% adhesive aid.
Preferably, 3. above-mentioned steps adds 10% zymocyte liquid of gross weight.
Preferably, above-mentioned steps 4. oven drying at low temperature temperature is 70 DEG C.
Further, present invention also offers bacillus subtilis bacterial manure and prevent and treat potato scab medicine in preparation
Application in thing.
Owing to have employed technique scheme, the invention has the beneficial effects as follows: hay bud provided by the present invention
Spore bacillus has, to potato scab pathogen, the effect of significantly inhibiting.The bacterial manure thus prepared spraying or
After being manured into soil, bacillus subtilis can form dominant bacteria at crop rhizosphere Fast-propagation long term survival
Group, thus effectively prevent and treat potato scab, prevention effect reaches more than 85%, and potato growth is healthy and strong,
Produce without poisoning, improve yield and quality.Have safe and efficient simultaneously, noresidue, have no side effect, energy
Enough reducing the use of chemical pesticide, the soil issues making long-term continuous cropping Rhizoma Solani tuber osi produce has been resolved, thus
Having important application prospect in Biological control potato scab, the intensive manufacture contributing to modern agriculture is sent out
Exhibition.
Accompanying drawing explanation
The present invention is further described with embodiment below in conjunction with the accompanying drawings.
Fig. 1 is bacillus subtilis microscopic morphology and the colonial morphology of the present invention；
Fig. 2 is that dendrogram is analyzed in the 16SrDNA homologous sequence comparison of the bacillus subtilis of the present invention；
Fig. 3 is the bacillus subtilis antagonistic effect measurement result of the present invention.
In figure, Fig. 1: A is bacillus subtilis microscopic morphology；
B is bacillus subtilis colonial morphology.
Fig. 3: A be JQ-001 bacterial strain 0.1,0.5,1.0 hundred million/ml bacterium amount is to shot hole inhibition；
B be JQ-001 bacterial strain 1.5,2.0,2.5 hundred million/ml bacterium amount is to shot hole inhibition；
C be JQ-001 bacterial strain 3.0,3.5,4.0 hundred million/ml bacterium amount is to shot hole inhibition；
In figure, CK is comparison.
Detailed description of the invention
Below in conjunction with the accompanying drawings and embodiment, the present invention be expanded on further:
Experimental technique in following embodiment, if no special instructions, is conventional method.In following embodiment
Experiment material used, if no special instructions, is the routine biochemistry preparation that shop is bought.In following example
Quantitative test, bacterium arrange three times repeats test, results averaged.
Embodiment 1: acquisition, qualification and the preservation of bacillus subtilis JQ-001 bacterial strain
One, the acquisition of bacillus subtilis JQ-001 bacterial strain:
The bacillus subtilis of the present invention is to use soil dilute from Heilongjiang Reclamation Area build space The Rhizosphere of Rice soil
Releasing partition method and separate acquisition, separation method is:
1, soil sample collection: within the Oryza sativa L. normal growth phase, dig out rice root, takes related soil on root, one
Several sample is adopted in place, after mixing scalping, takes about 200g and is used as to separate；
2, prepared by culture medium flat plate: NA culture medium and LB culture medium is melted, pours sterilizing culture dish respectively into,
NA and LB flat board is made after cooled and solidified；
3,1g soil sample is weighed in band bead, equipped with in the 250ml conical flask of 100ml aquesterilisa, shaking table
Shaken well, makes 10—2Times Soil Slurry, then by 10 times of gradient dilutions to 10-7Times；
4, take respectively and above-mentioned be diluted to each gradient concentration Soil Slurry 0.1ml, join culture medium flat plate
On, uniform by coated with glass rod coating, each gradient is coated with three plates, is placed in 37 DEG C of constant incubators cultivation
1-2d；
5, single bacterium colony that picking cultivation obtains is rule in NA culture medium and is again obtained single bacterium colony, is transferred to NA
In test tube vamp culture medium, 37 DEG C of constant incubators are cultivated 12-24h；
6, with potato scab pathogen for target bacterium, use flat board list bacterium colony opposite culture method, screen
One strain has the bacterial strain of relatively strong inhibitory activity, numbered JQ 001 to Bacterial Spot Pathogen；
Described NA culture medium is the Nutrient agar finished product culture medium that Beijing overpass company produces；LB culture medium prescription
For peptone 10g, yeast extract 5g, NaCl10g, agar powder 16g, distilled water 1000ml, pH7.0,121 DEG C
Sterilizing 30min.
Two, the qualification of bacillus subtilis JQ-001 bacterial strain
1, colonial morphology and the microscopic morphology of bacillus subtilis JQ-001 bacterial strain is shown in Fig. 1.
2, Microbiological Characteristics: bacterial strain is under NA culture medium, 37 DEG C of environment, after cultivating 12-18h, bacterium colony
Rough surface is opaque, and dirty white or slightly yellow, flat dry tarnish, edge is irregular, does not produce color
Element.Bacterial strain is shaft-like, tool peritrichous, and Gram’s staining is positive, aerobic.
3, molecular biological characteristic: 16SrRNA gene sequencing result (see sequence table), by JQ-001 bacterium
16SrDNA extension increasing sequence comparison in ncbi database of strain, with Bacillus subtilis homology be
99.38%, in conjunction with morphological characteristic, the physiological and biochemical property of JQ-001 bacterial strain, determine that JQ-001 bacterial strain belongs to withered
Grass bacillus subtilis, the JQ-001 bacterial strain phylogeny based on 16SrDNA sequence
Tree is as shown in Fig. 2 (in figure, strain 1 is JQ-001 bacterial strain).
Three, the preservation of JQ-001 bacterial strain
Through identifying that this bacterial strain is bacillus subtilis (Bacillus subtilis).Bacillus subtilis
JQ-001, is called for short JQ-001 bacterial strain, is preserved in Chinese microorganism strain preservation on November 9th, 2015
(being called for short CGMCC, address is: BeiChen West Road, Chaoyang District, BeiJing City 1 at administration committee's common micro-organisms center
Institute 3), deposit number is CGMCC No.11632.
Embodiment 2:JQ 001 bacterial strain antagonistic effect measures
Solid medium: NA culture medium, Gause I culture medium: soluble starch 20g, KNO3 1g、NaCl
0.5g、K2HPO4 0.5g、MgSO4 0.5g、FeSO40.01g agar powder 16g, distilled water 1000ml, pH7.4,
During preparation, first use a small amount of cold water, by starch furnishing pasty state, pour in the water boiled, fire heats, limit
Stirring limit adds other composition, after thawing, supplies moisture to 1000ml, then average by 100ml conical flask
Subpackage 13 bottles, 121 DEG C of sterilizing 30min.
Fluid medium (hay fermentation medium): glucose 20g, Dried Corn Steep Liquor Powder 5g, peptone 1g,
Yeast powder 0.5g, MnSO420ml、MgSO410ml, tap water 500ml, pH7.5,121 DEG C of sterilizing 30min.
One, experimental technique: use flat board opposite culture method
1, potato scab pathogenicbacteria separation purification is cultivated
Sampling spot: in land for growing field crops, big whole village, Zhangbei County, Hebei province.
The disease taking morbidity potato tubers with blade is good for intersection, first carries out at sterilizing with mercuric chloride and ethanol
After reason, access in Gause I culture medium, access 4 pieces in each culture dish, in 25 DEG C of constant incubators
Take long pathogenic bacteria plate with card punch after cultivating 6 days and be purified cultivation, standby.
2, the preparation of bacillus subtilis JQ-001 fermentation liquid
The strain inoculating loop being saved on inclined-plane is accessed on nutrient agar panel streak culture, 37 DEG C of constant temperature
Cultivate 24h activation.The bacterial strain of activation is accessed equipped with in the 500ml triangular flask of 50ml fermentation medium,
34 DEG C, under the conditions of 230r/min, shaking table shaken cultivation 25h, prepare strain fermentating liquid, treat that it produces bud completely
After spore, measuring number of viable in fermentation liquid is 10,000,000,000/ml.
3, fermentation liquor treatment
The fermentation liquid measuring number of viable is diluted to 0.01,0.05,0.1,0.5,1.0,1.5,2.0
Hundred million/milliliter bacterium amount, takes each concentration bacterium solution 3ml and sterilizing respectively with 1ml liquid-transfering gun and is cooled to 45 DEG C and melts
The Gause I culture medium (this culture medium 100ml conical flask quantitative separating 75ml) of state is mixed in 1:25 ratio
Even, make the flat board that carries disease germs, average every plate contains 1ml bacterium solution；Not inoculate bacillus subtilis JQ-001's
The flat board that fermentation liquid changes into culture medium mixing is for compareing.The Rhizoma Solani tuber osi scab cultivated in advance is accessed in flat board
Sick pathogenic bacteria, gets truffle with the card punch of diameter 5mm, accesses in the culture dish of solidification, and every flat board uniformly connects
Plant two pieces, often process and be repeated 3 times.In 25 DEG C of calorstats, back-off is cultivated 6 days.
4, experimental data processing
In time compareing mycelia and cover with plate, Taking Pictures recording, observe thalline growing way situation and use decussation method to survey
Amount colony diameter, antibacterial circle diameter, calculate meansigma methods and relative bacteriostasis rate, record colony diameter size, than
The more different bacterium amounts inhibition to potato scab, calculates bacteriostasis rate according to the following formula.(see Fig. 3)
Bacteriostasis rate (%)=(comparison colony diameter-process colony diameter)/comparison colony diameter × 100%
5, result is shown
Table 1 bacillus subtilis JQ-001 antagonistic experiment result
Embodiment 3: the preparation of bacillus subtilis JQ 001 microbial-bacterial fertilizer and application
One, the preparation of bacterial manure
1, the activation of Bacillus subtilis strain
The strain being saved in-20 DEG C of refrigerators is inoculated in NA culture medium test tube, 37 DEG C of constant temperature culture 24h；
2, the preparation of first order seed
Prepare appropriate NA culture medium, with two eggplant-shape bottle subpackages, after autoclaving completes, take out tilting, system
Become slant medium, access the bacillus subtilis JQ-001 after activation, 37 DEG C of constant incubators are cultivated 2d；
3, the preparation of secondary seed
Strain after eggplant-shape bottle is cultivated is linked in the seed tank containing fermentation medium cultivation again, cultivates bar
Part is temperature 37 DEG C, ventilation 30m3/ h, seed tank rotating speed are 175r/min, cultivate 16h；Seed tank is trained
Support basis set conjunction as follows: yeast powder 0.3%, peptone 1%, sodium chloride 0.5%, glucose 1%, remaining is water.
By shaking table cultivate seed culture medium by 5% ratio access 500L seed tank, 37 DEG C, 180r/min, ventilation ratio
1:1.5, cultivate 24h.Above-mentioned percent is mass percent.
4, being inoculated in fermentation tank by 5% inoculum concentration by cultured seed, fermentation tank culture medium composition is as follows:
Semen Maydis powder 3%, bean cake 1%, glucose 0.5%, yeast extract 0.5%, glucose 0.5%, magnesium sulfate 0.02%,
Remaining is water.121 DEG C of sterilizings 30 minutes, are cooled to when 37 DEG C connect secondary seed, 96r/min, 37 DEG C of constant temperature
16-20h is to starting to produce spore in cultivation, and obtaining concentration is 10,000,000,000/milliliter bacterium solution.Bacterium solution is concentrated dry
Dry prepare dry powder.Above-mentioned percent is mass percent.
5, bacteria containing amount detection
Dilution-plate method is used to detect the bacteria containing amount fermented；
6, raw material mixture
The zymocyte liquid obtained in step 4 is added gross weight accounting 3% kieselguhr and 1% adhesive aid titanium dioxide
Silicon, is uniformly mixed.Use filter press after mixing, obtain moisture and be not higher than the filter cake of 30%.Root
Add 65% humic acid of gross weight accounting, 28% cattle manure according to filter cake bacteria containing amount, obtain every gram of bacteria containing amount more than 2
Hundred million mix material.
7, disc granulation
Granulating disc pelletize on material step 6 mixed, gross weight accounting 10% is added in spraying simultaneously
Zymocyte liquid bonded pelleting；
8, oven drying at low temperature
Use steam heating pelletizing drum chuck to 70 DEG C, by air-introduced machine, steam is taken out of, dry moisture extremely
Less than 20%.
9, subpackage
Finished product after up to standard for inspection is added automated packaging equipment, is distributed into bag by the market demand, is product.
Two, the field experiment of bacterial manure
1, the test of granular fertilizer: in big whole village, Zhangbei County, Hebei Hebei province, height above sea level 1600-1800 rice, soil
Earth is sand, organic 1.15%-1.35%.Continuous cropping 5 years and the serious Rhizoma Solani tuber osi plot of shot hole morbidity
Carry out field experiment.Test arranges three groups, and often group includes that three are repeated to process, and each repetition is processed as 20m2
Rhizoma Solani tuber osi plot.
First group (matched group): parallel control processes.
Second group (test group): on March 12nd, 2013, every square meter applies 150 grams of bacterial manure as base fertilizer one
Secondary property applies；
3rd group (test group): on March 12nd, 2013, every square meter applies 150 grams of common farm manure conducts
Base fertilizer disposably applies.
On June 3rd, 2013, gathering in the crops Rhizoma Solani tuber osi, investigation often group sickness rate also adds up yield.
Often organize and add up total potato block number and morbidity potato number of blocks respectively, calculate sickness rate, biological and ecological methods to prevent plant disease, pests, and erosion rate and yield.
Sickness rate=morbidity potato number of blocks/total potato number of blocks × 100%
2, result is shown:
Biological and ecological methods to prevent plant disease, pests, and erosion rate=(matched group sickness rate-test group sickness rate)/matched group sickness rate × 100%.
Sickness rate result in the preventing and treating experiment of table 2 bacterial manure
| Process group | Total potato block number | Morbidity block number | Sickness rate |
| Matched group | 289 | 167 | 57.79% |
| Process group one | 291 | 71 | 24.40% |
| Process group two | 274 | 53 | 19.34% |
| Process group three | 276 | 18 | 6.52% |
Table 3 bacterial manure Mean disease index and average prevention effect
| Process group | Mean disease index (%) | Average prevention effect (%) |
| First group | 24.40 | 57.78 |
| Second group | 19.34 | 66.53 |
| 3rd group | 6.52 | 88.72 |
Result shows, the bacillus subtilis JQ-001 bacterial manure that the present invention provides can significantly reduce Rhizoma Solani tuber osi
The sickness rate of shot hole, reaches more than 88% to the biological and ecological methods to prevent plant disease, pests, and erosion rate of potato scab, has finally played and significantly improved
The quality of Rhizoma Solani tuber osi and ensure its time stored.
Claims (9)
1. a bacillus subtilis bacterial manure, it is characterised in that: described bacterial manure is by bacillus subtilis JQ-001 warp
Make zymocyte liquid after seed activation, seed culture, fermentation culture, be subsequently adding humic acids, cattle manure system
Becoming, it is common that described bacillus subtilis JQ-001 is preserved in China Committee for Culture Collection of Microorganisms
Microorganism center, depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, the preservation time: 2015
In on November 9, in, preservation registration number is CGMCC No.11632.
2. bacillus subtilis bacterial manure as claimed in claim 1, it is characterised in that: described bacterial manure is additionally added following
Component: kieselguhr, adhesive aid.
3. bacillus subtilis bacterial manure as claimed in claim 2, it is characterised in that: described adhesive aid is dioxy
SiClx.
4. a preparation method for the bacillus subtilis bacterial manure described in claim 1, specifically includes:
A. actication of culture: by the bacillus subtilis JQ-001 inoculation of cryopreservation on solid medium,
Cultivating 34-38h in 35-38 DEG C, picking list bacterium colony is transferred on slant medium, in 35-38 DEG C of cultivation
16-24h, with media surface thalline under aseptic washing as inoculation liquid；
B. prepared by primary seed solution: access prepared by step a in seed culture medium according to the inoculum concentration of 5%-10%
Inoculation liquid, rotating speed 200-260rpm, temperature 35-38 DEG C, 16-24h cultivated by shaking table, as first order seed
Liquid；
C. prepared by secondary seed solution: accesses step b in seed culture medium according to the inoculum concentration of 0.5%-3% and prepares
Primary seed solution, ventilation is 0.4-1.5vvm, and mixing speed is 180-260rpm, incubation time
For 18-24h, as secondary seed solution；
D. fermentation culture: access two grades of kinds prepared by step c in fermentation medium according to the inoculum concentration of 5%-10%
Sub-liquid, ventilation is 0.4-1.5vvm, and mixing speed is 180rpm-260rpm, and incubation time is
24h-30h, obtains fermentation liquid；
E. the preparation of bacterial manure: by described zymocyte liquid through filter press and add humic acids, cattle manure makes bacterial manure.
5. the preparation method of bacillus subtilis bacterial manure as claimed in claim 4, it is characterised in that: described step
In e, the preparation of bacterial manure includes:
1. described zymocyte liquid is initially charged kieselguhr and adhesive aid, plate-and-frame filtration after mixing；
2. 55-75% humic acid and the 20-35% cattle manure of gross weight, mixing is added according to filter cake bacteria containing amount；
3. by the above-mentioned material granulator granulation that mixes, zymocyte liquid bonded pelleting is added in spraying simultaneously；
4. the granule oven drying at low temperature will made.
6. the preparation method of bacillus subtilis bacterial manure as claimed in claim 5, it is characterised in that: described step
1. 3% kieselguhr and 1% adhesive aid of gross weight accounting are added in.
7. the preparation method of bacillus subtilis bacterial manure as claimed in claim 5, it is characterised in that: described step
3. 10% zymocyte liquid of gross weight accounting is added.
8. the preparation method of bacillus subtilis bacterial manure as claimed in claim 5, it is characterised in that: described step
4. oven drying at low temperature temperature is 70 DEG C.
9. the application of bacillus subtilis bacterial manure as claimed in claim 1, it is characterised in that: described bacterial manure is in system
For preventing and treating the application in potato scab medicine.
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| CN106396864A (en) * | 2016-08-29 | 2017-02-15 | 佛山市艳晖生物科技有限公司 | Microbial fertilizer for control of potato scab and preparation method thereof |
| CN107721539A (en) * | 2017-09-12 | 2018-02-23 | 甘肃农业大学 | A kind of samphire is special to mix bacterial manure and preparation method |
| CN110278965A (en) * | 2019-08-06 | 2019-09-27 | 山东京青农业科技有限公司 | A kind of microbial bacterial agent and preparation method thereof for preventing and treating citrus bacterial canker disease |
| CN110845253A (en) * | 2019-11-20 | 2020-02-28 | 中国科学院成都生物研究所 | Preparation and application method of gamma-polyglutamic acid biological organic fertilizer |
| CN111647527A (en) * | 2020-05-21 | 2020-09-11 | 浙江工业大学 | Method for improving saline-alkali soil and preserving fertilizer by utilizing bacillus subtilis |
| CN112299917A (en) * | 2019-07-29 | 2021-02-02 | 中国科学院微生物研究所 | Preparation method of soil conditioning type disease-resistant yield-increasing water-soluble fertilizer additive |
| CN112409065A (en) * | 2020-11-19 | 2021-02-26 | 中国农业科学院草原研究所 | Soil finishing agent for overcoming continuous cropping obstacle of potatoes and application method thereof |
| CN113603525A (en) * | 2021-07-21 | 2021-11-05 | 山东京青农业科技有限公司 | Special organic bacterial fertilizer for araliaceae panax plants and preparation method thereof |
| CN114315482A (en) * | 2021-08-13 | 2022-04-12 | 北京市农林科学院 | Biological bacterial fertilizer for increasing roots and improving efficiency and preparation method and application thereof |
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| CN106396864A (en) * | 2016-08-29 | 2017-02-15 | 佛山市艳晖生物科技有限公司 | Microbial fertilizer for control of potato scab and preparation method thereof |
| CN107721539A (en) * | 2017-09-12 | 2018-02-23 | 甘肃农业大学 | A kind of samphire is special to mix bacterial manure and preparation method |
| CN112299917A (en) * | 2019-07-29 | 2021-02-02 | 中国科学院微生物研究所 | Preparation method of soil conditioning type disease-resistant yield-increasing water-soluble fertilizer additive |
| CN110278965A (en) * | 2019-08-06 | 2019-09-27 | 山东京青农业科技有限公司 | A kind of microbial bacterial agent and preparation method thereof for preventing and treating citrus bacterial canker disease |
| CN110845253A (en) * | 2019-11-20 | 2020-02-28 | 中国科学院成都生物研究所 | Preparation and application method of gamma-polyglutamic acid biological organic fertilizer |
| CN110845253B (en) * | 2019-11-20 | 2022-02-08 | 中国科学院成都生物研究所 | Preparation and application method of gamma-polyglutamic acid biological organic fertilizer |
| CN111647527A (en) * | 2020-05-21 | 2020-09-11 | 浙江工业大学 | Method for improving saline-alkali soil and preserving fertilizer by utilizing bacillus subtilis |
| CN112409065A (en) * | 2020-11-19 | 2021-02-26 | 中国农业科学院草原研究所 | Soil finishing agent for overcoming continuous cropping obstacle of potatoes and application method thereof |
| CN113603525A (en) * | 2021-07-21 | 2021-11-05 | 山东京青农业科技有限公司 | Special organic bacterial fertilizer for araliaceae panax plants and preparation method thereof |
| CN114315482A (en) * | 2021-08-13 | 2022-04-12 | 北京市农林科学院 | Biological bacterial fertilizer for increasing roots and improving efficiency and preparation method and application thereof |
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