CN106929443A - A kind of vibrios LX6 2 and its purposes in biological alga fertilizer is prepared - Google Patents
A kind of vibrios LX6 2 and its purposes in biological alga fertilizer is prepared Download PDFInfo
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Abstract
The present invention relates to a kind of vibrios LX6 2 and its purposes in biological alga fertilizer is prepared.The biological alga fertilizer preparation process includes the step such as the inclined-plane cultures of vibrios LX6 2, seed culture, the fermentation for preparing marine alga digestive juice and biological marine alga liquid.More than the GA content 5000ppm of biology alga fertilizer of the invention, 12 times are improve compared with conventional chemical processing method and other biological method, are effectively facilitated growth and development of plants and are improved crop yield.Compound bio alga fertilizer of the invention also has promotion Zinc fractions crumb structure, strengthen the soil improvement action of soil permeability, with significantly improving plant drought anti-adversity ability, the function of growth-promoting effect is significant, there is good preventive effect to take-all and pepper ralstonia solanacearum, have good prevention effect to Rhizoctonia cerealis, Rhizoctonia solani Kuhn etc..
Description
【Technical field】
The invention belongs to technical field of biological fertilizer.More particularly it relates to a kind of vibrios LX6-2, further relates to institute
State purposes of the vibrios LX6-2 in biological alga fertilizer is prepared.
【Background technology】
Marine alga is a kind of marine organisms of abundance, is widely used in food, the raw material of industry, botanical.By sea
Algae is applied to agricultural and the existing centuries history of Animal husbandry production.The new fertilizer that alga fertilizer is that a class is pure natural, has no toxic side effect
Material, it contains abundant amino acid, mineral matter, polysaccharide, vitamin and physiological activator, with raising crop yield, improves
The effect such as quality and enhancing crop drought resistance, cold-resistant, disease resistance.Therefore, current new fertilizer is turned into using marine alga production organic fertilizer
Expect one of important focus of research.
It with the large-scale fast-growing brown algae being grown in ocean is raw material that alga fertilizer is, using the side of chemistry, physics or biology
The active ingredient that method is extracted in marine alga is made fertilizer, and bestowing plant can promote plant growth, improve yield, improve agricultural product product
Matter.Alga fertilizer is natural organic fertilizer, and its fertilizer efficiency depends on contained cytokinin in alga fertilizer, auximone, red
The main active substances such as mycin, abscisic acid, ethene, glycine betaine, polyamines.Alga fertilizer is removed and contains substantial amounts of non-nitrogen containing organic matter and one
Outside the amino acid of fixed number amount, protein and micronutrient element, also contain the distinctive algal polysaccharides of marine organisms, alginic acid, height
The trace quantity mineral substance elements such as zinc, bromine, the iodine that degree unrighted acid and terrestrial plant growth need.
At present, domestic and international marine alga fertilizer production method is usually chemical hydrolysis (i.e. potassium hydroxide Hydrolyze method), physics extraction
Method, biological fermentation process (i.e. enzyme edman degradation Edman) etc..In addition, bioactivator is extracted from seaweed industry discarded object, then through science
Fertilizer obtained from preparation.Extracting method for Product Activity material and nutrient composition content influence greatly, same amount but
Difference is also very big if active difference.
Overwhelming majority producer mainly produces alga fertilizer, the greatest drawback of this method using chemical hydrolysis both at home and abroad now
It is activity that highly basic high temperature can destroy marine alga endogenous substance.Physics extraction method uses high pressure low temperature cooling technique to reach marine alga
The broken purpose of cell membrane, the extracting method is better than chemical leaching test, but physical purification technical difficulty is big, industrial exploitation difficult weight
Weight.
Biological fermentation process is the various enzymes produced in the metabolic process with marine alga etc. as nutrient using microorganism, will be constituted
Marine alga macromolecular substances are degraded into small molecule, water miscible material, because fermentation process does not have the highly basic of chemical method and high temperature,
There is no the high pressure of Physical and low temperature, therefore intactly remain bioactivator and nutrients in marine alga to greatest extent
Matter, and can be absorbed and used by plants.Laboratory research is at home and abroad only limitted to using biological fermentation process production alga fertilizer at present,
Deep system research is lacked to its development technology and application mechanism, blank is still belonged in terms of industrialization technology.
The application method of alga fertilizer mainly has three kinds：(1) soil application：The native compound such as marine alga that alga fertilizer contains are more
Sugar is Natural soil conditioner, can not only chelate heavy metal ion, increases the persistence and validity of active ingredient in soil, and
And the formation of soil granular structure can be promoted, and improve soil internal void space, coordinate solid, liquid, gas proportions in soil, carry
The aeration of high-moisture conservation rate and soil, recovers the lac balance of soil, increases the soil organism.(2) seed immersion：
Seed Treatment is had been demonstrated for promoting germination ahead of time and raising early growth period anti-adversity ability largely effective.(3) foliage-spray：Leaf
It is most widely used fertilization mode that face sprays, and the edge that can quickly supplement Plant To Nutrient composition lacks, and stimulates the hair of rhizome
Educate and the absorption to nutritional ingredient.Alginic acid in alga fertilizer can reduce the surface tension of water, and one is formed in leaf surface of plant
Layer film, increases contact area, makes water or water-soluble substances be easier to enter plant cell through leaf surface cell membrane, makes plant
Thing most effectively absorbs the nutritional ingredient in seaweed extracted liquor.
Therefore alga fertilizer have application process various, it is easy to use, using it is in extensive range the advantages of, so, with alga fertilizer
Based on develop more diversified seaweed products according to different demands, will more adapt to the demand in market.
【The content of the invention】
[technical problem to be solved]
It is an object of the invention to provide a kind of vibrios LX6-2.
It is a further object to provide purposes of the vibrios LX6-2 in biological alga fertilizer is prepared.
[technical scheme]
The present invention is achieved through the following technical solutions.
The present invention relates to a kind of vibrios (Vibrio sp.) LX6-2, the bacterial strain is on March 23rd, 2015 in court of Beijing
No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica of institute of positive area's North Star West Road 1 are commonly micro-
Bio-Centers preservation, its preserving number is CGMCC No.10659.
The present invention relates to purposes of the described vibrios LX6-2 in biological alga fertilizer is prepared.
A preferred embodiment of the invention, the preparation process of the biological alga fertilizer is as follows：
A. vibrios LX6-2 inclined-plane cultures
Prepare slant medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium, 2g/l dipotassium hydrogen phosphates and 20g/l agar powders, then sterilize 30min under conditions of 121 DEG C of temperature,
Obtain described slant medium；
Vibrios LX6-2 bacterial strain glycerol tube strains are inoculated on the inclined-plane of test tube slant solid medium, then in constant temperature
In incubator under conditions of 28~30 DEG C of temperature 2~3d of inclined-plane culture, obtain slant culture；
B. seed culture
Prepare seed culture medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium and 2g/l dipotassium hydrogen phosphates, then sterilize 30min under conditions of 121 DEG C of temperature, obtains described kind
Sub- culture medium；
The slant culture that a ring step A is obtained is taken using oese, it is inoculated into described seed culture medium, so
1~2d is cultivated under conditions of 28~30 DEG C of temperature with rotating speed 180rpm in the constant-temperature table afterwards, obtains inoculum；
C. marine alga digestive juice is prepared
The marine alga raw material that dries crushed is well mixed with weak base aqueous solution, then in 50~60 DEG C of temperature and the condition for stirring
Under carry out 1~2h of digestion, obtain a kind of marine alga digestive juice；
D. the fermentation of biological marine alga liquid
In the fermentation tank equipped with marine alga digestive juice, according to 0.1~10g/l peptones, 0.1~10g/l sodium chloride, 0.1
~10g/l magnesium sulfate and 0.1~10g/l dipotassium hydrogen phosphates, peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate culture medium
Composition is added in the marine alga digestive juice that step C is obtained；
Then according to the 1.5~2.5% of marine alga digestive juice volume, the inoculum that step B is obtained is linked into fermentation
In tank, in 28~30 DEG C of temperature, throughput 1:1.0~1.2, the condition of 100~200rpm of 0.04~0.06MPa of pressure and rotating speed
Lower constant temperature stirs 24h, and marine alga digestive juice complete fermentation obtains described biological alga fertilizer.
Another preferred embodiment of the invention, described biological alga fertilizer is also containing a kind of poly- selected from macromolecular
Glutami acid fermentation liquor, small molecule polyglutamic acid zymotic fluid, P. fluorescens fermentation liquid or bacillus amyloliquefaciens zymotic fluid.
Another preferred embodiment of the invention, described macromolecular polyglutamic acid zymotic fluid is basis
The method of CN101109010B descriptions is the bacillus subtilis (Bacillus of CGMCC NO.2108 by deposit number
Subtillis) the zymotic fluid that fermented and cultured is obtained.
Another preferred embodiment of the invention, described small molecule polyglutamic acid zymotic fluid is basis
The method of CN104694437A descriptions is the bacillus licheniformis (Bacillus of CGMCC NO.8821 by deposit number
Licheniformis, B.licheniformis) zymotic fluid that obtains of LW fermented and cultureds.
Another preferred embodiment of the invention, described P. fluorescens fermentation liquid is basis
The method of CN104152380A descriptions is the ultraviolet mutagenesis type Pseudomonas fluorescens of CGMCC No.8820 by deposit number
The zymotic fluid that (Pseudomonasflorescens, P.florescens) F1 fermented and cultureds are obtained.
Another preferred embodiment of the invention, described bacillus amyloliquefaciens zymotic fluid is basis
The method of CN105316266A descriptions is the bacillus amyloliquefaciens of CGMCC No.11263 by deposit number
(Bacillusamyloliquefaciens) zymotic fluid that LX-J1 fermented and cultureds are obtained.
Another preferred embodiment of the invention, in the biological alga fertilizer containing the zymotic fluid, withered grass bud
Spore bacillus CGMCC NO.2108, bacillus licheniformis LW, ultraviolet mutagenesis type Pseudomonas fluorescens F1 and bacillus amyloliquefaciens
The living bacteria count of LX-J1 is respectively 500,000,000/more than ml.
Another preferred embodiment of the invention, in the biological alga fertilizer containing the zymotic fluid, macromolecular
Polyglutamic acid zymotic fluid, small molecule polyglutamic acid zymotic fluid, P. fluorescens fermentation liquid and bacillus amyloliquefaciens zymotic fluid
Content be respectively with the biological alga fertilizer total volume meter 10%~50%.
The present invention is described in more detail below.
The present invention relates to a kind of vibrios (Vibrio sp.) LX6-2, the bacterial strain is on March 23rd, 2015 in court of Beijing
No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica of institute of positive area's North Star West Road 1 are commonly micro-
Bio-Centers preservation, its preserving number is CGMCC No.10659.
Below by the more specific screening process for describing the bacterial strain.
The present inventor isolates 42 plants of bacteriums from rotten sea-tangle, is tested by following plate screenings and determined, wherein 19 plants
Bacterial strain can to some extent degrade alginic acid under conditions of flat board culture, at the beginning of then carrying out marine alga acid degradation to 19 plants of bacterial strains
Sieve, wherein 7 plants of strains for degrading alginic acid effects are more preferably, 7 plants of bacterium 16S rDNA sequencings determine that they are vibrios, its strain number
It is listed in Table 1 below respectively.
Detailed screening technique is described as follows：
The sea-tangle picked up by the sea is steeped in seawater, place at room temperature 7 days, 15 days, 30 days when difference picking sea-tangle it is rotten
Rotten part, move to by 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l magnesium sulfate, 2g/l dipotassium hydrogen phosphates with
On the pH7.0 plating mediums of 20g/l agar powders composition.Incubated 2-3 days, the microorganism species on sea-tangle were enriched with.
The ring of picking one is enriched with lawn in culture of being rule on above-mentioned plating medium, purifying, pure by repeated multiple times separation
Change, finally isolate 42 plants of bacteriums in the sea-tangle of different times difference rotten level.
Transparent circle degradation experiment is carried out on flat board, bacterium is rule on above-mentioned plating medium respectively and is cultivated 3-
4 days, there are 19 plants of bacterium to degrade to some extent alginic acid, through primary dcreening operation, wherein 7 plants of bacterium degradation effects are preferably, then to this 7 plants
Bacterium carries out 16S rDNA sequencings, determines that they are vibrios.
16S rDNA sequencing conditions are as follows：
The preparation of sample total DNA：DNA of bacteria extracting method routinely is carried out.
PCR primer：Forward primer 27F, reverse primer 1492R；
PCR reaction systems：Reaction system volume：20 μ L, reaction solution composition：1 μ L DNA profilings, 10 μ L 2 ×
MastarMix, 0.4 μ L 27F, 0.4 μ L 1492R, ultra-pure water complement to 20 μ L.
PCR amplification programs：95℃2min；30 circulations (84 DEG C of 1min, 52 DEG C of 1min, 65 DEG C of 8min)；65℃18min.
Amplified production through 0.7% agarose gel electrophoresis detection it is qualified after send Beijing three win polygala company be sequenced.To measure
DNA sequence dna manually corrected after, compared online using the BIAST softwares in NCBI, through comparison, 9 bacterium identified
Strain is 99% with the similarity of vibrios, so concluding that this 9 bacterial strains are vibrio.
To pick out the bacterial strain for being more suitable for processing algae leaching liquor, the comparative experiments of algae leaching liquor treatment is carried out, it is real
Proved recipe method is as follows：
Algae leaching liquor is prepared first.Appropriate sargassum is weighed, the water of about 10~20 times of sargassum volume is incorporated as, then add
Enter a small amount of carbonic acid soda and be processed into thin pulp pasty state slurries, then according to 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
Magnesium and 2g/l dipotassium hydrogen phosphates, peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate are added toward this slurries, obtain described
Algae leaching liquor.
Next prepares seed culture fluid.7 plants of bacterial strains that primary dcreening operation is obtained are inoculated into respectively and contain 5g/l sodium alginates, 3g/l
In the pH7.0 shake-flask seed culture mediums of peptone, 3g/l sodium chloride, 0.1g/l magnesium sulfate and 2g/l dipotassium hydrogen phosphates, then put
1~2d is cultivated under conditions of 28~30 DEG C of temperature with rotating speed 180rpm in constant-temperature table, inoculum is obtained；
3rd, according to the 2% of seaweed extracted liquor volume, above-mentioned bacterial strains inoculum is respectively connected to above-mentioned marine alga leaching
Extract, constant temperature is at the uniform velocity stirred at 28~30 DEG C of temperature, in different time sampling, its viscosity is determined using Ubbelohde viscometer method,
The results are shown in Table 1 for its experiment：
Table 1：7 plants of algae leaching liquor degradation effects of cholerae strain
The experimental result of table 1 clearly shows that vibrios LX6-2 bacterial strains (6. -2) are with the obvious advantage, just can be by extract solution in 24h
Viscosity can be reduced to less than 60 seconds.
Vibrios LX6-2 has following biological characteristicses：
Morphological feature：
Observed by microscope and flat-plate bacterial colony, the Pseudomonas negative bacillus, the raw flagellum in end can be moved, without gemma.In liquid
Under condition of culture, the bacterium into tiny shaft-like, without obvious crooked radian.Under the conditions of solid culture flat board, when cultivating 2-3 days, bacterium
The circular projection of completion, diameter 0.5-1.0 millimeters, neat in edge, bacterium colony is into faint yellow translucent, surface wettability.
It is vibrios (vibrio sp.) by the Strain Designation with reference to the morphological feature and 16SrDNA sequencing results of the bacterial strain
LX6-2, the bacterial strain grinds on March 23rd, 2015 in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism
Jiu Suo China Committee for Culture Collection of Microorganisms's common micro-organisms centers preservation, its preserving number is CGMCC No.10659.
The invention further relates to purposes of the described vibrios LX6-2 in biological alga fertilizer is prepared.
According to the present invention, the preparation process of the biological alga fertilizer is as follows：
A. vibrios LX6-2 inclined-plane cultures
Prepare slant medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium, 2g/l dipotassium hydrogen phosphates and 20g/l agar powders, then sterilize 30min under conditions of 121 DEG C of temperature,
Obtain described slant medium；
Vibrios LX6-2 bacterial strain glycerol tube strains are inoculated on the inclined-plane of test tube slant solid medium, then in constant temperature
In incubator under conditions of 28~30 DEG C of temperature 2~3d of inclined-plane culture, obtain slant culture；
The peptone used when slant culture is prepared is, for example, to be produced by the extensive and profound in meaning star Bioisystech Co., Ltd in Beijing
Trade name tryptone product；Agar powder is, for example, the trade name produced by the extensive and profound in meaning star Bioisystech Co., Ltd in Beijing
The product of agar powder.
In this step, the inclined-plane culture equipment for using all is the product sold in the market, such as by Shanghai reality
Test the constant incubator of instrument Co., Ltd., Factory production；The used equipment of sterilizing is the product sold in the market, for example
The normal limited public affairs of instrument instrument and equipment of Portable pressure steam sterilizing device or Xi'an produced by Jiangyin Binjiang Medical Equipment Co., Ltd.
The high-pressure sterilizing pot product of department.
B. seed culture
Prepare seed culture medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium and 2g/l dipotassium hydrogen phosphates, then sterilize 30min under conditions of 121 DEG C of temperature, obtains described kind
Sub- culture medium；
The slant culture that a ring step A is obtained is taken using oese, it is inoculated into described seed culture medium, so
1~2d is cultivated under conditions of 28~30 DEG C of temperature with rotating speed 180rpm in the constant-temperature table afterwards, obtains inoculum；
The sodium alginate that the step is used is the product sold in the market, such as by extensive and profound in meaning star company with trade name
The product of sodium alginate sale, other reagents reagent usually used as previously described or in chemical technology field.This step
The rapid equipment for using as previously described, will not be repeated here.
C. marine alga digestive juice is prepared
The marine alga raw material that dries crushed is well mixed with weak base aqueous solution, then in 50~60 DEG C of temperature and the condition for stirring
Under carry out 1~2h of digestion, obtain a kind of marine alga digestive juice；
Marine alga digestion liquid and preparation method thereof of the present invention is according to CN 201510287164.1, denomination of invention《A kind of active alga
Fertilizer and its production method》Method described by 0029-0039 sections of patent application specification.
In the present invention, described marine alga is one or more marine alga selected from sargassum, sea-tangle or yellow tang.Horse hair
Algae, sea-tangle and yellow tang are all the products sold in the market.
Drying marine alga raw material is crushed using equipment usually used in current food processing technology field, for example
The disintegrating apparatus sold with trade name SCF twin shafts atomizer by the company of powder equipment Co., Ltd of Shanghaiization three.Dry marine alga
Raw material grinding particle size is not particularly critical, but suitable granularity is then conducive to follow-up digestion process.Usually, marine alga is dried
Granularity is 50~200 mesh.
According to the present invention, described weak base is potassium carbonate, saleratus, sodium carbonate or sodium acid carbonate, preferably carbonic acid
Potassium, saleratus or sodium carbonate, more preferably potassium carbonate or saleratus.
The concentration of the weak base aqueous solution that the present invention is used is 0.001~0.004g/ml.If the weak base aqueous solution concentration
It is too high or too low be all it is inappropriate, because if weak base aqueous solution concentration be higher than 0.004g/ml, then pH value of solution is higher, can break
The activity of natural nutrient substance in bad marine alga；If weak base aqueous solution concentration is less than 0.001g/ml, weak base is to the swelling of marine alga
Effect is limited, and influence enzymolysis process below, active material recovery rate declines.
According to the present invention, the marine alga is 5~8 with the mass ratio of the weak base:1.Preferably, the marine alga is weak with described
The mass ratio of alkali is 6~8:1.It is highly preferred that the marine alga is 7~8 with the mass ratio of the weak base:1.
Need to be stirred continuously when being digested using weak base, so that marine alga powder particles can be fully contacted with weak base aqueous solution,
Digestion reaction is complete.
In this step, if digestion reaction temperature is less than 50 DEG C, digestion reaction is carried out not exclusively, alginic cell wall
Swelling action be limited, be unfavorable for the dissolution of nutriment in marine alga；If digestion reaction temperature is higher than 60 DEG C, have in marine alga
The activity for imitating composition is damaged, and influences the using effect of alga fertilizer.Therefore, the temperature of digestion reaction is 50~60 DEG C, preferably
It is 52~58 DEG C, more preferably 54~56 DEG C.
Under conditions of 50~60 DEG C of digestion reaction temperature, if digestion time is less than 1h, digestion reaction cannot not carried out completely
Entirely, the swelling action of alginic cell wall is limited, and is unfavorable for the dissolution of nutriment in marine alga；If digestion time is more than 2h,
Digestion reaction tends towards stability, and causes the significant wastage of the energy, and cost increases.Therefore, the time of digestion reaction for 1~2h be reasonable
, preferably 1.2~1.8h, more preferably 1.4~1.6h.
D. the fermentation of biological marine alga liquid
In the fermentation tank equipped with marine alga digestive juice, according to 0.1~10g/l peptones, 0.1~10g/l sodium chloride, 0.1
~10g/l magnesium sulfate and 0.1~10g/l dipotassium hydrogen phosphates, peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate culture medium
Composition is added in the marine alga digestive juice that step C is obtained；
Then according to the 1.5~2.5% of marine alga digestive juice volume, the inoculum that step B is obtained is linked into fermentation
In tank, in 28~30 DEG C of temperature, throughput 1:1.0~1.2, the condition of 100~200rpm of 0.04~0.06MPa of pressure and rotating speed
Lower constant temperature stirs 24h, and marine alga digestive juice complete fermentation obtains described biological alga fertilizer.
The gibberellin of the biological alga fertilizer prepared by LX6-2 strain fermentations using high performance liquid chromatography detection, the present invention is contained
Amount more than 5000ppm, compared with conventional chemical processing method and other biological method, GA content improves 1-2 times, maximum journey
Degree protects the active material in marine alga Central Plains, more efficiently promotes growing and improving crop yield for plant.
According to the present invention, described biological alga fertilizer is also selected from macromolecular polyglutamic acid zymotic fluid, small molecule containing a kind of
The zymotic fluid of polyglutamic acid zymotic fluid, P. fluorescens fermentation liquid or bacillus amyloliquefaciens zymotic fluid.
In the present invention, macromolecular polyglutamic acid zymotic fluid, small molecule polyglutamic acid zymotic fluid, Pseudomonas fluorescens are contained
The biological alga fertilizer of zymotic fluid or bacillus amyloliquefaciens zymotic fluid, also referred to as compound bio alga fertilizer.
Described macromolecular polyglutamic acid zymotic fluid is that the method described according to CN101109010B is by deposit number
The zymotic fluid that bacillus subtilis (Bacillus subtillis) fermented and cultured of CGMCC NO.2108 is obtained.
Described small molecule polyglutamic acid zymotic fluid is that the method described according to CN104694437A is by deposit number
Bacillus licheniformis (Bacillus licheniformis, B.licheniformis) LW fermented and cultureds of CGMCC NO.8821
The zymotic fluid for obtaining.
Described P. fluorescens fermentation liquid is that the method described according to CN104152380A is CGMCC by deposit number
Ultraviolet mutagenesis type Pseudomonas fluorescens (Pseudomonas florescens, P.florescens) F1 fermentation trainings of No.8820
Support the zymotic fluid for obtaining.
Described bacillus amyloliquefaciens zymotic fluid is that the method described according to CN105316266A is by deposit number
Bacillus amyloliquefaciens (Bacillusamyloliquefaciens) LX-J1 fermented and cultureds of CGMCC No.11263 are obtained
Zymotic fluid.
In the present invention, detected using viable plate count method known to those skilled in the art, containing
State in the biological alga fertilizer of zymotic fluid, bacillus subtilis CGMCC NO.2108, bacillus licheniformis LW, ultraviolet mutagenesis type are glimmering
The living bacteria count of light pseudomonad F1 and bacillus amyloliquefaciens LX-J1 is respectively 500,000,000/more than ml.
According to the present invention, in the biological alga fertilizer containing the zymotic fluid, macromolecular polyglutamic acid zymotic fluid, small molecule
The content of polyglutamic acid zymotic fluid, P. fluorescens fermentation liquid and bacillus amyloliquefaciens zymotic fluid is respectively with the biology
Alga fertilizer total volume meter 10%~50%.
In addition, present invention biology alga fertilizer is combined life obtained from mother liquor is equipped with above-mentioned other functional form microbial inoculums
Thing alga fertilizer, it also has functions that other functional form microorganism fertilizers in addition to biology alga fertilizer of the invention in itself effect,
Function is more enriched and is embodied, and can meet the crop for having different demands, effectively raises fertilising efficiency.
Biology alga fertilizer of the invention is sent out according to the 10%~50% of its cumulative volume with bacillus subtilis CGMCC No.2108
The compound bio alga fertilizer that zymotic fluid is mixed to get, uses CTAB methods to detect that its polyglutamic acid content is 3g/l~15g/l, uses
Viable plate count method detection known to those skilled in the art, its bacillus subtilis living bacteria count >=500,000,000/
Ml, this compound bio alga fertilizer in addition to the function effect with biology alga fertilizer of the invention, also with promoting Zinc fractions group
Kernel structure, strengthens the soil improvement action of soil permeability.
Biology alga fertilizer of the invention is sent out according to the 10%~50% of its cumulative volume with bacillus licheniformis CGMCC No.8821
The compound bio alga fertilizer that zymotic fluid is mixed to get, uses CTAB methods to detect that its small molecule polyglutamic acid content is 4g/l~20g/
L, and detected using viable plate count method known to those skilled in the art, its bacillus licheniformis effectively lives
Bacterium number >=500,000,000/ml, this compound bio alga fertilizer in addition to the function effect with biology alga fertilizer of the invention, also with obvious
Improve plant drought anti-adversity ability, the function of growth-promoting effect is significant.
Biology alga fertilizer of the invention is sent out according to the 10%~50% of its cumulative volume with Pseudomonas fluorescens CGMCC No.8820
The compound bio alga fertilizer that zymotic fluid is mixed to get, is examined using viable plate count method known to those skilled in the art
Survey, its Pseudomonas fluorescens living bacteria count >=500,000,000/ml, this compound bio alga fertilizer is except with present invention biology marine alga
Outside function effect of fertilizer, also there is good preventive effect to take-all and pepper ralstonia solanacearum；
Biology alga fertilizer of the invention according to its cumulative volume 10%~50% with bacillus amyloliquefaciens CGMCC
The compound bio alga fertilizer that No.11263 zymotic fluids are mixed to get, using flat board viable bacteria known to those skilled in the art
Counting method detects that its solution starch living bacteria count >=500,000,000/ml, this compound bio alga fertilizer is removed with the biological sea of the present invention
Outside function effect of algae fertilizer, also to Rhizoctonia cerealis, Rhizoctonia solani Kuhn, Pythium ultimum, sclerotium rolfsii, the black hemorrhoid disease of potato, flower
Raw southern blight has good prevention effect.
[beneficial effect]
The beneficial effects of the invention are as follows：The GA content of the biological alga fertilizer that the present invention is prepared by LX6-2 strain fermentations
More than 5000ppm, compared with conventional chemical processing method and other biological method, GA content improves 1-2 times, effectively promotees
Enter growth and development of plants and improve crop yield.
Biology alga fertilizer of the invention for mother liquor be equipped with macromolecular polyglutamic acid zymotic fluid, small molecule polyglutamic acid zymotic fluid,
P. fluorescens fermentation liquid or bacillus amyloliquefaciens zymotic fluid, the compound bio alga fertilizer for obtaining is except with above-mentioned functions work(
Effect is outer, also with Zinc fractions crumb structure is promoted, strengthens the soil improvement action of soil permeability, with significantly improving plant
Drought resisting anti-adversity ability, the function of growth-promoting effect is significant has good preventive effect to take-all and pepper ralstonia solanacearum, to cereal silk
The black hemorrhoid disease of pyrenomycetes, Rhizoctonia solani Kuhn, Pythium ultimum, sclerotium rolfsii, potato, the good prevention effect of peanut sclerotium rolfsii.
【Specific embodiment】
The present invention is will be better understood that by following embodiments.
Embodiment 1：The preparation of biology alga fertilizer of the invention
The implementation steps of the embodiment are as follows：
A. vibrios LX6-2 inclined-plane cultures
Prepare slant medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium, 2g/l dipotassium hydrogen phosphates and 20g/l agar powders, then sterilize 30min under conditions of 121 DEG C of temperature,
Obtain described slant medium；
Vibrios LX6-2 bacterial strain glycerol tube strains are inoculated on the inclined-plane of test tube slant solid medium, then in constant temperature
In incubator under conditions of 28 DEG C of temperature inclined-plane culture 2d, obtain slant culture；
B. seed culture
Prepare seed culture medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium and 2g/l dipotassium hydrogen phosphates, then sterilize 30min under conditions of 121 DEG C of temperature, obtains described kind
Sub- culture medium；
The slant culture that a ring step A is obtained is taken using oese, it is inoculated into described seed culture medium, so
1d is cultivated under conditions of 29 DEG C of temperature with rotating speed 180rpm in the constant-temperature table afterwards, obtains inoculum；
C. marine alga digestive juice is prepared
Mass ratio according to marine alga and weak base is 6:1, the sargassum raw material that dries of crushing is 0.0034g/ml's with concentration
Aqueous sodium carbonate is well mixed, and then carries out digestion 1.8h under conditions of 58 DEG C of temperature and stirring, obtains a kind of marine alga and disappears
Change liquid；
D. the fermentation of biological marine alga liquid
In the fermentation tank equipped with marine alga digestive juice, according to 0.1g/l peptones, 3g/l sodium chloride, 6g/l magnesium sulfate with
Peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate medium component, are added to what step C was obtained by 0.1g/l dipotassium hydrogen phosphates
In marine alga digestive juice；Then
According to the 2.2% of marine alga digestive juice volume, the inoculum that step B is obtained is linked into fermentation tank, in temperature
Degree 29 DEG C, throughput 1:1.0th, constant temperature stirring 24h under conditions of pressure 0.04MPa and rotating speed 100rpm, marine alga digestive juice is complete
Fermentation, obtains described biological alga fertilizer.
Embodiment 2：The preparation of biology alga fertilizer of the invention
The implementation steps of the embodiment are as follows：
A. vibrios LX6-2 inclined-plane cultures
Prepare slant medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium, 2g/l dipotassium hydrogen phosphates and 20g/l agar powders, then sterilize 30min under conditions of 121 DEG C of temperature,
Obtain described slant medium；
Vibrios LX6-2 bacterial strain glycerol tube strains are inoculated on the inclined-plane of test tube slant solid medium, then in constant temperature
In incubator under conditions of 29 DEG C of temperature inclined-plane culture 3d, obtain slant culture；
B. seed culture
Prepare seed culture medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium and 2g/l dipotassium hydrogen phosphates, then sterilize 30min under conditions of 121 DEG C of temperature, obtains described kind
Sub- culture medium；
The slant culture that a ring step A is obtained is taken using oese, it is inoculated into described seed culture medium, so
2d is cultivated under conditions of 28 DEG C of temperature with rotating speed 180rpm in the constant-temperature table afterwards, obtains inoculum；
C. marine alga digestive juice is prepared
Mass ratio according to marine alga and weak base is 7:1, the sargassum raw material that dries of crushing is the carbon of 0.002g/ml with concentration
Sour hydrogen sodium water solution is well mixed, and then carries out digestion 2.0h under conditions of 52 DEG C of temperature and stirring, obtains a kind of marine alga and disappears
Change liquid；
D. the fermentation of biological marine alga liquid
In the fermentation tank equipped with marine alga digestive juice, according to 2g/l peptones, 0.1g/l sodium chloride, 4g/l magnesium sulfate with
Peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate medium component, are added to the sea that step C is obtained by 5g/l dipotassium hydrogen phosphates
In algae digestive juice；Then
According to the 1.5% of marine alga digestive juice volume, the inoculum that step B is obtained is linked into fermentation tank, in temperature
Degree 28 DEG C, throughput 1:1.1st, constant temperature stirring 24h under conditions of pressure 0.05MPa and rotating speed 200rpm, marine alga digestive juice is complete
Fermentation, obtains described biological alga fertilizer.
Embodiment 3：The preparation of biology alga fertilizer of the invention
The implementation steps of the embodiment are as follows：
A. vibrios LX6-2 inclined-plane cultures
Prepare slant medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium, 2g/l dipotassium hydrogen phosphates and 20g/l agar powders, then sterilize 30min under conditions of 121 DEG C of temperature,
Obtain described slant medium；
Vibrios LX6-2 bacterial strain glycerol tube strains are inoculated on the inclined-plane of test tube slant solid medium, then in constant temperature
In incubator under conditions of 30 DEG C of temperature inclined-plane culture 2d, obtain slant culture；
B. seed culture
Prepare seed culture medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium and 2g/l dipotassium hydrogen phosphates, then sterilize 30min under conditions of 121 DEG C of temperature, obtains described kind
Sub- culture medium；
The slant culture that a ring step A is obtained is taken using oese, it is inoculated into described seed culture medium, so
1d is cultivated under conditions of 30 DEG C of temperature with rotating speed 180rpm in the constant-temperature table afterwards, obtains inoculum；
C. marine alga digestive juice is prepared
Mass ratio according to marine alga and weak base is 5:1, the sea-tangle raw material that dries of crushing is the carbonic acid of 0.001g/ml with concentration
Hydrogen aqueous solutions of potassium is well mixed, and then carries out digestion 1.0h under conditions of temperature 50 C with stirring, obtains a kind of marine alga digestion
Liquid；
D. the fermentation of biological marine alga liquid
In the fermentation tank equipped with marine alga digestive juice, according to 10g/l peptones, 7g/l sodium chloride, 0.1g/l magnesium sulfate with
Peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate medium component, are added to the sea that step C is obtained by 10g/l dipotassium hydrogen phosphates
In algae digestive juice；Then
According to the 1.8% of marine alga digestive juice volume, the inoculum that step B is obtained is linked into fermentation tank, in temperature
Degree 30 DEG C, throughput 1:1.2nd, constant temperature stirring 24h under conditions of pressure 0.06MPa and rotating speed 140rpm, marine alga digestive juice is complete
Fermentation, obtains described biological alga fertilizer.
Embodiment 4：The preparation of biology alga fertilizer of the invention
The implementation steps of the embodiment are as follows：
A. vibrios LX6-2 inclined-plane cultures
Prepare slant medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium, 2g/l dipotassium hydrogen phosphates and 20g/l agar powders, then sterilize 30min under conditions of 121 DEG C of temperature,
Obtain described slant medium；
Vibrios LX6-2 bacterial strain glycerol tube strains are inoculated on the inclined-plane of test tube slant solid medium, then in constant temperature
In incubator under conditions of 29 DEG C of temperature inclined-plane culture 3d, obtain slant culture；
B. seed culture
Prepare seed culture medium：Prepare and contain 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l sulfuric acid
The pH7.0 aqueous solution of magnesium and 2g/l dipotassium hydrogen phosphates, then sterilize 30min under conditions of 121 DEG C of temperature, obtains described kind
Sub- culture medium；
The slant culture that a ring step A is obtained is taken using oese, it is inoculated into described seed culture medium, so
2d is cultivated under conditions of 29 DEG C of temperature with rotating speed 180rpm in the constant-temperature table afterwards, obtains inoculum；
C. marine alga digestive juice is prepared
Mass ratio according to marine alga and weak base is 8:1, the yellow tang raw material that dries of crushing is the carbon of 0.004g/ml with concentration
Sour aqueous solutions of potassium is well mixed, and then carries out digestion 1.2h under conditions of temperature 60 C with stirring, obtains a kind of marine alga digestion
Liquid；
D. the fermentation of biological marine alga liquid
In the fermentation tank equipped with marine alga digestive juice, according to 8g/l peptones, 10g/l sodium chloride, 10g/l magnesium sulfate with
Peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate medium component, are added to the sea that step C is obtained by 5g/l dipotassium hydrogen phosphates
In algae digestive juice；Then
According to the 2.5% of marine alga digestive juice volume, the inoculum that step B is obtained is linked into fermentation tank, in temperature
Degree 29 DEG C, throughput 1:1.1st, constant temperature stirring 24h under conditions of pressure 0.05MPa and rotating speed 160rpm, marine alga digestive juice is complete
Fermentation, obtains described biological alga fertilizer.
The GA content of the biological alga fertilizer of embodiment 1-4 preparations, Yi Jicong are have detected using high performance liquid chromatography
That is bought in market is processed the liquid alga fertilizer product for obtaining and is sold from the market by chemical method by certain company's biological enzyme
The GA content of the liquid alga fertilizer product reference product of production, the results are shown in Table 1 for these.
Table 1：GA content analysis result in the alga fertilizer of distinct methods production
| Gibberellin (ppm) | |
| The liquid seaweed extract product of certain producer's biological enzyme production | 2284.535 |
| The liquid seaweed extract product of certain producer's chemical method production | 2564.006 |
| Liquid seaweed extract product prepared by embodiment 1 | 5503.142 |
| Liquid seaweed extract product prepared by embodiment 2 | 5211.688 |
| Liquid seaweed extract product prepared by embodiment 3 | 5475.269 |
| Liquid seaweed extract product prepared by embodiment 4 | 5383.867 |
The result that table 1 is listed shows, compared with conventional chemical processing method and other biological method, prepared by the inventive method
The GA content of product improves 1-2 times, more efficient therefore, it is possible to farthest protect the active material in marine alga Central Plains
Promote growing and improving crop yield for plant.
Test example 1：The application test of the compound bio alga fertilizer containing bacillus subtilis
The compound bio alga fertilizer contains 50% present invention biology alga fertilizer and 50% bacillus subtilis by volume
CGMCC No.2108 zymotic fluids.
Bacillus subtilis CGMCC No.2108 are the bacterial strain that a plant height produces macromolecular polyglutamic acid, macromolecular polyglutamic
Acid has obvious effect to improving soil granular structure.
This test example uses biological alga fertilizer prepared by embodiment 1；
The bacillus subtilis CGMCC No.2108 zymotic fluids that this test example is used are using leading biological husbantry stock
That part Co., Ltd possesses, the B of CN 101109010, denomination of invention《One plant of bacterial strain and its cultural method of product gamma-polyglutamic acid》
Described in fermentation process prepare zymotic fluid；
Using CN 201510127399.4, denomination of invention《One plant of bacillus licheniformis and its production gamma-polyglutamic acid
In purposes》Described in extraction and purification method, extracted from above-mentioned zymotic fluid and polyglutamic acid and purified.
The effect of I, polyglutamic acid to soil granular structure
By soil sample air-dry, bulk soil is broken into pieces with one's fingers with hand when soil sample is moist, remove root system of plant and finger stone, precise
Every part is the soil sample of 700g.
3 treatment are set, and its macromolecular polyglutamic acid mass concentration is respectively 0% (ck), 0.3%, 0.6%.To weigh
Air-dry soil sample and be well mixed with macromolecular polyglutamic acid and be fitted into basin alms bowl, pour 150ml water, be repeated 2 times, be classified according to Sha Weinuofu
The standard method detection of method (wet screening) soil granular structure after processing 10 months, its testing result is listed in Table 2 below.
Table 2：Improving effect of the polyglutamic acid sterling to soil granular structure
The result listed by table 2 is as can be seen that pure polyglutamic acid is significantly acted on promoting soil granular structure to have.
The effect of II, compound bio alga fertilizer to soil granular structure
Take for examination soil sample, it is the soil sample of 1000g that precise is a.By volume 50% the present invention biology alga fertilizer with
The compound bio alga fertilizer that 50% bacillus subtilis CGMCC No.2108 zymotic fluids are mixed to get contains 16g/l polyglutamic acids.
By this 100 times of compound bio alga fertilizer dilute with water, soil sample is poured onto by compound bio alga fertilizer is diluted according to each 300ml
In, two weeks, once continuous pouring 5 times was control with clear water pouring, according to the standard side of Sha Weinuofu stagings (wet screening)
Method detection soil granular structure after processing 10 months, its testing result is listed in Table 3 below.
Table 3：Improving effect of the compound bio alga fertilizer to soil granular structure
| Title | >5mm (%) | 2~5mm (%) | 1~2mm (%) | 0.5~1mm (%) | 0.25~0.5mm (%) | Collect (%) |
| ck | 1.25 | 5.998 | 11.164 | 16.768 | 20.818 | 55.998 |
| Compound bio alga fertilizer | 2.426 | 7.944 | 11.954 | 17.744 | 19.798 | 59.866 |
The result listed by table 2 can be seen that compound bio alga fertilizer of the present invention to be had significantly to soil granular structure
Improvement result.
Test example 2：The take-all controlling experiment of the compound bio alga fertilizer containing Pseudomonas fluorescens
The compound bio alga fertilizer contains 80% present invention biology alga fertilizer and 20% Pseudomonas fluorescens by volume
CGMCC No.8820 zymotic fluids, are detected using viable plate count method known to those skilled in the art, and fluorescence is false single
Born of the same parents bacterium CGMCC No.8820 are 7.2 hundred million/ml.
Wherein, the biological alga fertilizer that this test example is used is biological alga fertilizer prepared by embodiment 2；
The Pseudomonas fluorescens CGMCC No.8820 zymotic fluids that this test example is used are using leading biological husbantry stock
The CN 201410386364, denomination of invention of part Co., Ltd《One plant of ultraviolet mutagenesis type Pseudomonas fluorescens and application thereof》In retouch
Zymotic fluid prepared by the fermentation process stated.
Test site：Qinhuangdao Funing area, the serious plot of full rot morbidity.
Test method, the experiment sets three treatment：
CK：Base manure
Treatment 1：Base manure+present invention biology alga fertilizer
Treatment 2：Base manure+compound bio the alga fertilizer containing Pseudomonas fluorescens
Wherein：Base manure is every mu of N amounts 23kg, P2O5Amount 15kg, K2O measures 23kg.
Applied fertilizer since the wheat seeding stage and applied fertilizer altogether 2 times until tillering stage, each consumption is 20L/ mus, is poured, in wheat
Knot ear period is instituted an inquiry, and wheat growing way situation and the incidence of full rot are observed during this period.
Investigation result is listed in Table 4 below.
Table 4：The wheat growth gesture condition investigation of different disposal
200 take-all incidences are investigated respectively, and average prevention effect can be calculated according to following formula.
Preventive effect (%)=(control disease index-treatment group disease index)/control disease index × 100%
Investigation result is as follows：The take-all disease index of control treatment is 97%, and treatment 1 uses the biological sea of the present invention
Algae fertilizer is poured, and take-all disease index is 47%, is simply also improved in addition to growth is promoted using present invention biology alga fertilizer
The disease resistance of plant, protection effect is up to 51.5%；Treatment 2 has used the take-all of compound bio alga fertilizer of the present invention
Disease index is 10%, and protection effect reaches 89.7%.
The survey showed that for these, and the compound bio alga fertilizer containing Pseudomonas fluorescens of the invention is except to plant strain growth
Outside with obvious facilitation, fine prevention effect is also acted, improve fertilising efficiency, reduced with fertile cost.
Test example 3：The pakchoi application test of the compound bio alga fertilizer containing bacillus licheniformis
The compound bio alga fertilizer contains 70% present invention biology alga fertilizer and 30% bacillus licheniformis by volume
CGMCC No.8821 zymotic fluids, are detected, lichens gemma using viable plate count method known to those skilled in the art
Bacillus is 8.5 hundred million/ml；
Wherein, the biological alga fertilizer that this test example is used is bio-fermented liquid prepared by embodiment 3；
The compound bio alga fertilizer that this test example is used is using Leading Bio-agricultural Co., Ltd., CN
201510127399.4th, denomination of invention《One plant of bacillus licheniformis and its purposes in gamma-polyglutamic acid is produced》Described in
Fermentation culture method prepare zymotic fluid；
The experiment is carried out in bubble chamber, and influence of the fertilizer to pakchoi drought resisting is verified by controlling water.
Test method：
The experiment sets four treatment, and every group 2 parallel；
Treatment 1：Do not control water (clear water is poured, by normal water supply, water-holding capacity 65%~75%)
Treatment 2：Control water (clear water is poured, mild drought, and water-holding capacity 40%~50%)
Treatment 3：Control water (pouring present invention biology alga fertilizer, mild drought, water-holding capacity 40%~50%)
Treatment 4：Control water (pouring compound bio alga fertilizer of the present invention, mild drought, water-holding capacity 40%~50%)
9kg soil is filled in each bubble chamber；Design control water is mild drought (the 40%~50% of maximum water holding capacity),
Seedling stage starts to control water after applying fertilizer first, after after natural drought to the soil moisture content critical field for setting, every night 17:00~
18:00 determines soil moisture content with digital moisture meter, and moisturizing control water, 35 days whole growth periods, apply fertilizer altogether 2 times (20L/ mus
Consumption).
Result of the test is listed in Table 5 below.
Table 5：Each treatment wheat growth situation is investigated after growth period terminates：
| Treatment 1 | Treatment 2 | Treatment 3 | Treatment 4 | |
| Plant height cm | 27.78 | 18.56 | 29.16 | 30.8 |
| Fresh weight g/ plants | 16.95 | 5.36 | 20.96 | 28.17 |
As can be seen from the test results, compound bio alga fertilizer of the addition containing bacillus licheniformis zymotic fluid can be significantly improved
The drought resisting resistance of plant, remains to keep good growth tendency under conditions of drought and water shortage.
Test example 4：The flat board bacteriostatic test of the alga fertilizer of compound bio containing bacillus amyloliquefaciens
The compound bio alga fertilizer contains 60% present invention biology alga fertilizer and 40% bacillus amyloliquefaciens by volume
CGMCC No.8821 zymotic fluids, are detected using viable plate count method known to those skilled in the art, solve starch bud
The living bacteria count of spore bacillus is 8.3 hundred million/ml；
Wherein, the compound bio alga fertilizer that the test example is used is zymotic fluid prepared by embodiment 4；
The bacillus amyloliquefaciens zymotic fluid that the test example is used is to use Leading Bio-agricultural Co., Ltd.
, CN 201510894025, denomination of invention《One bacillus amyloliquefaciens LX-J1 and application thereof》Described in preparation method
The zymotic fluid for preparing.
Flat board bacteriostatic test is as follows：
Prepare PDA solid mediums：Peeled potatoes are placed in water according to 200g/L ratios boil 30min, cooled down, then use
2 layers of gauze are filtered, and then according to 20g/L sucrose and 20g/L agar, sucrose and agar are added toward the filtrate for obtaining, and are gone out
Bacterium, obtains described PDA solid mediums.
By Rhizoctonia cerealis, Rhizoctonia solani Kuhn, Pythium ultimum bacterium, Sclerotium rolfssi, the black hemorrhoid germ of potato bacteria cake
It is seeded in respectively in the PDA solid cultures of above-mentioned preparation, 4-5d is cultivated at 25 DEG C of temperature, treats that disease fungus covers with whole culture
Ware.
Antagonistic effect is determined：
The pathogen bacteria cake of its diameter about 6mm is taken from culture dish first with card punch, PDA plate center is placed in, respectively
Punched at the about 150mm of its left and right sides, and add 20 μ l compound bio alga fertilizers of the present invention containing bacillus amyloliquefaciens；Together
When to be not added with the compound bio alga fertilizer as control, cultivate 4-5d at 25 DEG C of temperature, treat that whole culture dish is covered with control,
To the bacteriostatic diameter of each pathogen, its result of the test is listed in Table 6 below record compound bio alga fertilizer, and is calculated according to following formula
Obtain corresponding bacteriostasis rate：
Colony growth inhibiting rate=(the net growth diameter of the control bacterium colony-net growth diameter for the treatment of bacterium colony)/control bacterium colony is given birth to only
Diameter × 100% long
Table 6：Compound bio alga fertilizer fungistatic effect of the present invention
| Pathogen | Cereal silk core | Miliary damping-off | Pythium ultimum | Peanut sclerotium rolfsii | Black scurf of potato |
| Bacteriostasis rate | 87.6% | 69.8% | 88.7% | 76.4% | 72.9% |
The result of table 6 clearly illustrates that bacillus amyloliquefaciens LX-J1 is compounded with present invention biology alga fertilizer, i.e. this hair
Bright compound bio alga fertilizer, to Rhizoctonia cerealis, Rhizoctonia solani Kuhn, to Pythium ultimum, peanut sclerotium rolfsii, the black hemorrhoid germ of potato
Still there is stronger inhibition, that is to say, that this functional marine alga fertilizer is except the basic effect with biological alga fertilizer after compounding
Outward, effectively preventing can also be played to plant disease, is achieved many things at one stroke.
Claims (10)
1. a kind of vibrios (Vibrio sp.) LX6-2, the bacterial strain is on March 23rd, 2015 in BeiChen West Road, Chaoyang District, BeiJing City
The preservation of No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms centers of Institute of Microorganism, Academia Sinica of No. 1 institute,
Its preserving number is CGMCCNo.10659.
2. purposes of the vibrio marinopraesens LX6-2 according to claim 1 in biological alga fertilizer is prepared.
3. purposes according to claim 2, it is characterised in that the preparation process of the biological alga fertilizer is as follows：
A. vibrios LX6-2 inclined-plane cultures
Prepare slant medium：Prepare containing 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l magnesium sulfate,
The pH7.0 aqueous solution of 2g/l dipotassium hydrogen phosphates and 20g/l agar powders, then sterilize 30min under conditions of 121 DEG C of temperature, obtains
To described slant medium；
Vibrios LX6-2 bacterial strain glycerol tube strains are inoculated on the inclined-plane of test tube slant solid medium, then incubated
In case under conditions of 28~30 DEG C of temperature 2~3d of inclined-plane culture, obtain slant culture；
B. seed culture
Prepare seed culture medium：Prepare containing 5g/l sodium alginates, 3g/l peptones, 3g/l sodium chloride, 0.1g/l magnesium sulfate with
The pH7.0 aqueous solution of 2g/l dipotassium hydrogen phosphates, then sterilize 30min under conditions of 121 DEG C of temperature, obtains described seed training
Support base；
The slant culture that a ring step A is obtained is taken using oese, it is inoculated into described seed culture medium, Ran Hou
1~2d is cultivated in constant-temperature table under conditions of 28~30 DEG C of temperature with rotating speed 180rpm, inoculum is obtained；
C. marine alga digestive juice is prepared
Dry marine alga raw material and be well mixed with weak base aqueous solution to crush, then 50~60 DEG C of temperature with stir under conditions of enter
Row 1~2h of digestion, obtains a kind of marine alga digestive juice；
D. the fermentation of biological marine alga liquid
In the fermentation tank equipped with marine alga digestive juice, according to 0.1~10g/l peptones, 0.1~10g/l sodium chloride, 0.1~
10g/l magnesium sulfate and 0.1~10g/l dipotassium hydrogen phosphates, peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate culture medium into
Divide and be added in the marine alga digestive juice that step C is obtained；
Then according to the 1.5~2.5% of marine alga digestive juice volume, the inoculum that step B is obtained is linked into fermentation tank,
In 28~30 DEG C of temperature, throughput 1:1.0~1.2, it is permanent under conditions of 100~200rpm of 0.04~0.06MPa of pressure and rotating speed
Temperature stirring 24h, marine alga digestive juice complete fermentation obtains described biological alga fertilizer.
4. purposes according to claim 2, it is characterised in that described biological alga fertilizer is also selected from macromolecular containing a kind of
Polyglutamic acid zymotic fluid, small molecule polyglutamic acid zymotic fluid, P. fluorescens fermentation liquid or bacillus amyloliquefaciens zymotic fluid.
5. purposes according to claim 4, it is characterised in that described macromolecular polyglutamic acid zymotic fluid is basis
The method of CN101109010B descriptions is the bacillus subtilis (Bacillus of CGMCC NO.2108 by deposit number
Subtillis) the zymotic fluid that fermented and cultured is obtained.
6. purposes according to claim 4, it is characterised in that described small molecule polyglutamic acid zymotic fluid is basis
The method of CN104694437A descriptions is the bacillus licheniformis (Bacillus of CGMCC NO.8821 by deposit number
Licheniformis, B.licheniformis) zymotic fluid that obtains of LW fermented and cultureds.
7. purposes according to claim 4, it is characterised in that described P. fluorescens fermentation liquid is basis
The method of CN104152380A descriptions is the ultraviolet mutagenesis type Pseudomonas fluorescens of CGMCC No.8820 by deposit number
The zymotic fluid that (Pseudomonasflorescens, P.florescens) F1 fermented and cultureds are obtained.
8. purposes according to claim 4, it is characterised in that described bacillus amyloliquefaciens zymotic fluid is basis
The method of CN105316266A descriptions is the bacillus amyloliquefaciens of CGMCC No.11263 by deposit number
(Bacillusamyloliquefaciens) zymotic fluid that LX-J1 fermented and cultureds are obtained.
9. purposes according to claim 4, it is characterised in that in the biological alga fertilizer containing the zymotic fluid, withered grass bud
Spore bacillus CGMCC NO.2108, bacillus licheniformis LW, ultraviolet mutagenesis type Pseudomonas fluorescens F1 and bacillus amyloliquefaciens
The living bacteria count of LX-J1 is respectively 500,000,000/more than ml.
10. purposes according to claim 4, it is characterised in that in the biological alga fertilizer containing the zymotic fluid, big point
The fermentation of sub- polyglutamic acid zymotic fluid, small molecule polyglutamic acid zymotic fluid, P. fluorescens fermentation liquid and bacillus amyloliquefaciens
The content of liquid is respectively with the biological alga fertilizer total volume meter 10%~50%.
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| CN113278661A (en) * | 2021-06-16 | 2021-08-20 | 通辽梅花生物科技有限公司 | Fermentation medium and fermentation method for increasing glutamic acid yield |
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| CN114304187A (en) * | 2022-02-23 | 2022-04-12 | 领先生物农业股份有限公司 | Compound microbial agent suitable for saline-alkali soil in arid and semi-arid regions and application thereof |
| CN114853546A (en) * | 2022-03-15 | 2022-08-05 | 领先生物农业股份有限公司 | Soil remediation microbial inoculum and preparation method and application thereof |
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| CN113278661A (en) * | 2021-06-16 | 2021-08-20 | 通辽梅花生物科技有限公司 | Fermentation medium and fermentation method for increasing glutamic acid yield |
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| CN114304187A (en) * | 2022-02-23 | 2022-04-12 | 领先生物农业股份有限公司 | Compound microbial agent suitable for saline-alkali soil in arid and semi-arid regions and application thereof |
| CN114304187B (en) * | 2022-02-23 | 2023-03-24 | 领先生物农业股份有限公司 | Compound microbial agent suitable for saline-alkali soil in arid and semi-arid regions and application thereof |
| CN114853546A (en) * | 2022-03-15 | 2022-08-05 | 领先生物农业股份有限公司 | Soil remediation microbial inoculum and preparation method and application thereof |
| CN114853546B (en) * | 2022-03-15 | 2024-03-22 | 领先生物农业股份有限公司 | Soil remediation microbial inoculum and preparation method and application thereof |
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