CN108034620A - One plant of bacillus megaterium bacterial strain, peanut drop cadmium agent and its application - Google Patents
One plant of bacillus megaterium bacterial strain, peanut drop cadmium agent and its application Download PDFInfo
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Abstract
The present invention provides one plant of bacillus megaterium bacterial strain, peanut drop cadmium agent and its application, belong to Crop securify production technical field.The content provided by the invention that cadmium in Ecological Property of Peanut Seeds can be reduced including the drop cadmium agent of the peanut of bacillus megaterium zymotic fluid or bacillus megaterium solid pharmaceutical preparation.P content, Ecological Property of Peanut Seeds yield and the Peanut Root System biomass in blade can also be improved at the same time.Further, drop cadmium agent provided by the invention further includes the LaCL that mass concentration is 1~2g/L3Solution, can further improve the removal rate to cadmium in Ecological Property of Peanut Seeds.Compared with not applying drop cadmium agent, using drop cadmium agent cultivation peanut provided by the invention, cadmium content in Ecological Property of Peanut Seeds is set to reduce by 39.06%~42.65%, phosphorus content increase at the same time 16.4%~22.22%, Peanut Root System dry weight improves 29.50%~33.2%, Ecological Property of Peanut Seeds output increased 20.32%~23.5%.
Description
Technical field
The invention belongs to Crop securify production technical field, and in particular to one plant of bacillus megaterium bacterial strain, peanut drop
Cadmium agent and its application.
Background technology
With industrial expansion and the modernization of agricultural production, heavy metals in farmland pollution getting worse, contaminated area is year by year
Expand.Heavy metal with features such as not degradable, easy enrichments due to receiving significant attention.Because having high toxicity and high migration,
Cadmium (Cd) is considered as most important environmental pollutants, and excessive cadmium can cause Itai-itai diseases, kidney trouble, liver damage in human body
A series of diseases such as evil.After cadmium enters soil, preferentially combined with carbonate, form the cadmium carbonate of indissoluble, there is larger movement
Property and plant availability, so as to be easier to be absorbed by plants into food chain, harm is produced to health.
Peanut is to inhale one of most strong field crop of cadmium ability, its absorption to cadmium in soil and the efficiency to seed transfer
Higher than many crops.According to investigations, northern China producing region Ecological Property of Peanut Seeds cadmium average content is 0.2-0.3mg/kg, investigates the soil in place
Earth is most beyond the nuisanceless peanut Cd contents 0.05mg/kg (NY5303-2005) of country, portion without obvious pollution condition
Divide and exceeded green food standard 0.4mg/kg limitations (NY/T420-2009).Ecological Property of Peanut Seeds cadmium content is exceeded to have become restriction
An important factor for China peanut outlet and related industry development.
At present, special crop varieties are selected, screening or selection and breeding low cadmium-accumulation kind are the cadmium accumulations of abatement plant edible part
A kind of mode, but such a mode, due to the particularity of plant variety, there may be certain defect, such as yield for its biological character
It is low, resistance is poor etc., limit being widely applied for kind.
The content of the invention
In view of this, it is an object of the invention to provide one plant of bacillus megaterium bacterial strain, reduction Ecological Property of Peanut Seeds cadmium content
Preparation, can reduce Ecological Property of Peanut Seeds Cd accumulation, improve Ecological Property of Peanut Seeds drop cadmium efficiency.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The present invention provides a kind of bacillus megaterium bacterial strain BaCillus megaterium PP84, deposit number is
CGMCC No.12798。
Cultivate peanut present invention also offers one and drop cadmium agent, including bacillus megaterium zymotic fluid or bacillus megaterium solid
Preparation；
The preparation method of the bacillus megaterium zymotic fluid, comprises the following steps：
Bacillus megaterium bacterial strain described in such scheme is activated and expands culture, obtains bacillus megaterium fermentation seed
Liquid, the bacillus megaterium fermentation seed liquid is inoculated into beef extract-peptone fluid nutrient medium by 1~3% inoculum concentration
Liquid fermentation 36h~60h under conditions of 25~30 DEG C, 150~200r/min of rotating speed, obtains bacillus megaterium zymotic fluid；
The preparation method of the bacillus megaterium solid pharmaceutical preparation, comprises the following steps：
A. bacillus megaterium bacterial strain described in such scheme is activated and expands culture, obtains bacillus megaterium fermentation kind
Sub- liquid, the bacillus megaterium fermentation seed liquid is inoculated into wheat bran medium 25 by 2.5%~7.5% inoculum concentration
3~5d of solid fermentation culture under the conditions of~30 DEG C, stirring, obtains mixture；
B. the mixture obtained in the step a is continued into 1.5~2.5d of fermented and cultured at 25~30 DEG C, obtained huge
Bacillus solid pharmaceutical preparation.
Preferably, the drop cadmium agent further includes the LaCl that the mass concentration independently dispensed is 1~2g/L3Solution.
The present invention provides application of the peanut drop cadmium agent in peanut is planted described in a kind of above method.
Preferably, the bacillus megaterium zymotic fluid is applied in the rhizosphere of plantation peanut；The bacillus megaterium hair
The applied amount of zymotic fluid is 16~20L/ mus
Preferably, the application number of the bacillus megaterium zymotic fluid is 3 times；The time applied for the first time goes out for peanut
14~16d of after seedling, second of the time applied, the time that third time applies went out for peanut for 29~31d after peanut emergence
44~46d of after seedling.
Preferably, the bacillus megaterium solid pharmaceutical preparation is applied in the soil surface of plantation peanut；The huge gemma
The applied amount of bacillus solid pharmaceutical preparation is 7~9kg/ mus.
Preferably, the application number of the bacillus megaterium solid pharmaceutical preparation is 1 time, and the time of application is after peanut is emerged
7~10d.
Preferably, in peanut florescence by LaCl3Solution is sprayed to be sprayed once in the blade face of plantation peanut, every 9~11d,
Continuously spray three times.
Preferably, the LaCl3The each amount of spraying of solution is 8~12L/ mus.
The present invention provides one plant of bacillus megaterium bacterial strain Bacillus megaterium PP84, deposit number is
CGMCC No.12798.Peanut provided by the invention including bacillus megaterium zymotic fluid or bacillus megaterium solid pharmaceutical preparation
Drop cadmium agent improves peanut phosphorus nourishing situation, promotes peanut growth, reduces absorption of the peanut to Cd, while reduce Cd from root system
Shifted to seed, so as to reduce the content of cadmium in Ecological Property of Peanut Seeds.Not apply the agent of drop cadmium as a control group, apply drop
Cadmium agent can significantly reduce Ecological Property of Peanut Seeds Cd contents.Apply Cd contents in the Ecological Property of Peanut Seeds planted after drop cadmium agent and be less than control group
Cd contents 26.56%~27.9%.
Meanwhile drop cadmium agent provided by the invention can also dramatically increase the content of blade P.In the peanut leaf for applying drop cadmium agent
P content is higher than 14.55%~16.7% of P content in control group.In addition, drop cadmium agent provided by the invention can also improve peanut
Grain yield and Peanut Root System biomass.Compared with control group, apply drop cadmium agent weight of root system it is higher than control group by 23.9%~
24.6%, grain yield is higher than control group by 16.4%~17.1%.
Further, drop cadmium agent provided by the invention contains the LaCl for including that mass concentration is 1~2g/L3Solution.Will be above-mentioned
The LaCl that bacillus megaterium bacterial strain preparation and mass concentration described in scheme are 1~2g/L3Solution is used cooperatively.Huge gemma
Bacillus strain preparation and LaCl3Solution is used cooperatively, and effectively improves peanut phosphorus nourishing situation, improves peanut and Cd is resisted
Property, promote peanut growth, reduce absorption of the peanut to Cd, while reduce Cd and shifted from root system to seed, so as to more
The further content for reducing cadmium in Ecological Property of Peanut Seeds, applies and plants Cd contents in Ecological Property of Peanut Seeds after the preparation and be less than control group
39.06%~42.65%.
Meanwhile peanut drop cadmium agent provided by the invention can also further improve the content of blade P, compared with control group,
Blade P content is higher than control group 16.4%~22.22%.After applying peanut drop cadmium agent, weight of root system is higher than control group by 29.50%
~33.2%, grain yield is higher than control group by 20.32%~23.5%.
Biological deposits explanation
Bacillus megaterium (Bacillus megaterium), is preserved in China Microbiological bacterial strain preservation administration committee
Common micro-organisms center, preservation address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation mechanism is referred to as：CGMCC, preservation day
Phase is on July 20th, 2016, and biological deposits numbering is CGMCCNo.12798, strain number：PP84.
Embodiment
The present invention provides a kind of bacillus megaterium bacterial strain Bacillus megaterium PP84, deposit number is
CGMCC No.12798。
Cultivate peanut present invention also offers one and drop cadmium agent, including bacillus megaterium zymotic fluid or bacillus megaterium solid
Preparation；
The preparation method of the bacillus megaterium zymotic fluid, comprises the following steps：
Bacillus megaterium bacterial strain described in such scheme is activated and expands culture, obtains bacillus megaterium fermentation seed
Liquid, beef extract-peptone fluid nutrient medium is inoculated into by obtained bacillus megaterium fermentation seed liquid by 1~3% inoculum concentration
In under conditions of 25~30 DEG C, 150~200r/min of rotating speed liquid fermentation 36h~60h, obtain bacillus megaterium fermentation
Liquid；
The preparation method of the bacillus megaterium solid pharmaceutical preparation, comprises the following steps：
A. bacillus megaterium bacterial strain described in such scheme is activated and expands culture, obtains bacillus megaterium fermentation kind
Sub- liquid, obtained bacillus megaterium fermentation seed liquid is inoculated into wheat bran medium by 2.5%~7.5% inoculum concentration
3~5d of solid fermentation culture under the conditions of 25~30 DEG C, stirring, obtains mixture；
B. the mixture obtained in the step a is continued into 1.5~2.5d of fermented and cultured at 25~30 DEG C, obtained huge
Bacillus solid pharmaceutical preparation.
The present invention is preferably activated bacillus megaterium bacterial strain, the bacillus megaterium bacterial strain after being activated.Institute
It is beef extract-peptone slant medium that activation, which is stated, with culture medium.The preparation method of the beef extract-peptone slant medium
For：Beef extract 3g, peptone 5g, agar 18g are weighed, is settled to 1000ml with deionized water, 121 DEG C of sterilizing 20min, obtain ox
Meat extract peptone slant medium.The temperature of the activation is preferably 25~32 DEG C, more preferably 28 DEG C.The time of the activation
Preferably 16~32h, more preferably 24h.
In the present invention, after the bacillus megaterium bacterial strain after being activated, the present invention preferably will be huge after the activation
Bacillus strain is enlarged culture, obtains bacillus megaterium fermentation seed liquid.It is described expand culture mode be：Picking
Bacillus megaterium after the activation of one ring is linked into beef extract-peptone fluid nutrient medium at 29~31 DEG C, and rotating speed 170~
14h~18h is cultivated under conditions of 190r/min.The temperature for expanding culture is preferably 30 DEG C.The rotating speed is preferably 180r/
min.The time of the culture is preferably 18h.
After obtaining bacillus megaterium fermentation seed liquid, the present invention by the bacillus megaterium fermentation seed liquid by 1~
3% inoculum concentration is inoculated into beef extract-peptone fluid nutrient medium the liquid under conditions of 25~30 DEG C, 150~200r/min of rotating speed
Body fermentation 36h~60h, obtains bacillus megaterium zymotic fluid.In obtained bacillus megaterium zymotic fluid viable count for 8 ×
107~2 × 109CFU/mL。
In the present invention, the inoculum concentration of the bacillus megaterium fermentation seed liquid is preferably 2.5%.The liquid fermentation
Temperature is preferably 28 DEG C.The time of the liquid fermentation is preferably 48h.The rotating speed during liquid fermentation is preferably 180r/
min。
In the present invention, the preparation method of the beef extract-peptone fluid nutrient medium is：Weigh beef extract 3g, peptone
5g, is settled to 1000ml, 121 DEG C of sterilizing 20min, obtain beef extract-peptone fluid nutrient medium with deionized water.
The present invention is not particularly limited the source of the beef extract, peptone and agar, using this area conventional commercial
Product.The beef extract-peptone slant medium and fluid nutrient medium each component used in the embodiment of the present invention is purchased from state
Medicine group chemical reagent Beijing Co., Ltd.
In the present invention, the bacillus megaterium zymotic fluid improves peanut phosphorus nourishing situation, promotes peanut growth, drop
Absorption of the low peanut to Cd, while reduce Cd and shifted from root system to seed, so as to reduce the Cd contents in Ecological Property of Peanut Seeds.
It is of the invention by institute after obtaining bacillus megaterium fermentation seed liquid when preparing bacillus megaterium solid pharmaceutical preparation
Bacillus megaterium fermentation seed liquid is stated to be inoculated into the wheat bran medium at 25~30 DEG C by 2.5%~7.5% inoculum concentration
Under the conditions of solid fermentation 3~5d of culture, stirring, obtain mixture.In the present invention, the inoculum concentration is preferably 5.0%.It is described solid
Body fermented and cultured temperature is preferably 28 DEG C.The solid fermentation incubation time is preferably 4d.
After obtaining mixture, the mixture is continued 36~60h of solid fermentation by the present invention at 25~30 DEG C, is obtained huge
Bacterium anthracoides solid pharmaceutical preparation.Viable count is 5 × 10 in obtained bacillus megaterium zymotic fluid7~9 × 108CFU/mL。
After obtaining bacillus megaterium solid pharmaceutical preparation, the present invention preferably stirs the bacillus megaterium solid pharmaceutical preparation
Mix, the operation repetition of 36~60h of solid fermentation 2~3 times, make bacillus megaterium equal in wheat bran medium at 25~30 DEG C
Even distribution.
In the present invention, the mode of the mixing is preferably to stir.The rotating speed of the stirring is preferably 150~200r/min,
More preferably 180r/min.The time of the stirring is preferably 3~5min, more preferably 4min.The time of the fermentation is preferred
For 2d.
In the present invention, percentage, the component of the wheat bran medium is：87.9 parts of wheat bran, dusty yeast 1.6
Part, 8.0 parts of rice bran, KH2PO40.5 part, FeSO40.3 part, (NH4)2SO41.1 parts and MgSO40.6 part.
The water content of the wheat bran medium is preferably 40%~60wt%, more preferably 50wt%.The wheat bran culture
Base preferably sterilizes before inoculation.The sterilising conditions temperature is 121 DEG C.The sterilization time is preferably 30min.
The present invention is not particularly limited the source of the wheat bran medium, using this area conventional commercial product.
Described in the embodiment of the present invention in wheat bran medium, wheat bran is purchased from Qingdao wheat drifting fragrance flour Co., Ltd, remaining medicine is purchased from state
Medicine group chemical reagent Beijing Co., Ltd.
In the present invention, the bacillus megaterium solid pharmaceutical preparation improves peanut phosphorus nourishing situation, promotes peanut growth,
Absorption of the peanut to Cd is reduced, while reduces Cd and is shifted from root system to seed, so that the Cd reduced in Ecological Property of Peanut Seeds contains
Amount.
Drop cadmium agent provided by the invention preferably further includes the LaCl independently dispensed3Solution.The LaCl3The concentration of solution is excellent
Elect 1~2g/L as, more preferably 1.4~1.6g/L, be most preferably 1.5g/L.In the present invention, the LaCl3Solution carries after
The resistance that high peanut poisons Cd, while La3+With Cd2+Antagonism is produced, absorption of the peanut to Cd is reduced, promotes
Peanut growth.In the present invention, to the LaCl3The source of solution is not particularly limited, using this area conventional commercial product.
Using the LaCl of Chinese medicines group chemical reagent Beijing Co., Ltd production in the embodiment of the present invention3。
The present invention provides application of the drop cadmium agent in peanut is planted described in a kind of such scheme.
In the present invention, the bacillus megaterium zymotic fluid is applied in plantation peanut rhizosphere.The bacillus megaterium hair
The applied amount of zymotic fluid is preferably 16~20L/ mus, more preferably 17~19L/ mus, is most preferably 18L/ mus.The huge gemma bar
Fermented liquid is preferably diluted before administration.The diluted multiple is preferably 100 times.The bacillus megaterium zymotic fluid
Application number be 3 times.The time that the bacillus megaterium zymotic fluid applies for the first time be preferably after peanut emergence 14~
16d, more preferably 15d.Second of the time applied is preferably 29~31d, more preferably 30d.The time that third time applies is excellent
Elect 44~46d as, more preferably 45d.In the present invention, the bacillus megaterium zymotic fluid preferably includes to inactivate huge gemma bar
Fermented liquid and bacillus megaterium zymotic fluid is not inactivated, do not inactivate bacillus megaterium zymotic fluid more preferably.
In the present invention, the bacillus megaterium solid pharmaceutical preparation is applied in the soil surface of plantation peanut.The huge bud
The each applied amount of spore bacillus solid pharmaceutical preparation is 7~9kg/ mus, more preferably 7.5~8.5kg/ mus, is most preferably 8kg/ mus.Institute
The application time for stating bacillus megaterium solid pharmaceutical preparation is preferably 7~10d after peanut emergence, more preferably 8d.In the present invention
The mode of the application is preferably to spread fertilizer over the fields.In the present invention, solid that the bacillus megaterium solid pharmaceutical preparation does not inactivate preferably
Preparation.
In the present invention, the LaCl3Solution is preferably sprayed in peanut florescence in the blade face of plantation peanut, every 9~11d sprays
Apply once, more preferably sprayed once every 10d.The number sprayed is preferably three times.The LaCl3The each amount of spraying of solution
Preferably 8~12L/ mus, more preferably 9~11L/ mus, are most preferably 10L/ mus.In the present invention, the LaCl3Solution is spraying
It is preceding to be preferably diluted.The diluted concentration is preferably 50 times.
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.Based on the reality in the present invention
Apply example, those of ordinary skill in the art's all other embodiments obtained without making creative work, all belong to
In the scope of protection of the invention.
Embodiment 1
For trying soil：
Qingdao City's Pingdu City contaminated soil (sandy loam) is picked up from, its basic physical and chemical is：(Tu Shui is than 1 by pH 6.4：
2.5), organic matter 14.8g/kg, cation exchange capacity (CEC) 15.9cmol/kg, total cadmium content 3.05mg/kg.Pedotheque natural wind
It is dry, it is levigate to cross 2mm (10 mesh) sieves, for potted plant simulation test.(depth of cultivated soil is in 20cm or so in crop field, according to soil
The density (2.65g/m3) of earth and the depth of cultivated soil are calculated, and the weight of soil is 353t or so in field per acre.Root
The additive amount of cadmium agent drops in standard conversion pot experiment accordingly.All embodiments according to said method convert in the present invention)
For trying plant
Peanut (rich to spend No. 3), purchased from Shandong Academy of Agricultural Sciences.
Strains tested
Bacillus megaterium (BaCillus megaterium) bacterial strain PP84 (deposit numbers：CGMCC No.12798), bud
1.0~1.2 × 1.5~2.0 microns of spore, ellipse, middle life or the life of secondary end.
Culture medium
Beef extract-peptone slant medium：Beef extract 3g is weighed, peptone 5g, agar 18g, is settled to deionized water
1000ml, 121 DEG C of sterilizing 20min.
Dried beef peptone fluid nutrient medium：Beef extract 3g is weighed, peptone 5g, 1000ml is settled to deionized water,
121 DEG C of sterilizing 20min.
The preparation of bacillus megaterium bacterial strain fermentation liquor
Bacillus megaterium PP84 is inoculated on beef extract-peptone slant medium activation culture is carried out at 30 DEG C
After 24h, accessed with one ring of oese picking in beef extract-peptone fluid nutrient medium, 180r/min, 30 DEG C of culture 16h, are
Bacillus megaterium fermentation seed liquid.Seed liquor is inoculated into beef extract-peptone fluid nutrient medium according to 2.5% inoculum concentration
In under conditions of 28 DEG C, rotating speed 180r/min liquid fermentation 48h, obtain bacillus megaterium zymotic fluid (the huge bud
Spore bacillus fermentation liquid viable count is 2 × 109CFU/mL it is) and stand-by after 100 times of dilutions of progress.
Pot experiment is implemented
Load in per basin after air-drying and cross the soil sample 21.0kg of 2mm sieves, add distilled water to water-retaining quantity among field of soil 16%, protect
Hold after soil moisture content balances 1 week and start to sow.
Choose full seed, soaked with the liquor natrii hypochloritis that volumetric concentration is 1% take out after seed 10min spend from
After sub- water cleaning seed 5~7 times, the consistent cave of depth is dug in the soil in basin in advance, has uniformly sowed 6 seeds.Then
Soil is covered on seed.Spray deionized water 20mL.Thinning when plant grows 2 true leaves, ensures there are 3 flowers in each basin
It is raw.The 15d after peanut emergence, the bacillus megaterium zymotic fluid 100mL after the plant rhizosphere of every basin applies 100 times of dilution.
In planting process, soil moisture content is kept as 65% or so of maximum field capacity.When cultivating 30d and 45d, respectively to
Plant rhizosphere adds bacillus megaterium zymotic fluid, and dosage is 100mL.After plant maturation, whole strain is harvested, with stainless steel scissors
Plant is divided into overground part, underground part and pod, measures plant fresh weight.After deionized water rinsing, 105 DEG C of 20min that finish, 70
DEG C dry to constant weight, crushed 100 mesh sieves.
Embodiment 2
For examination soil, the preparation for examination plant, strains tested, the method for medium treatment and bacillus megaterium zymotic fluid
Method is the same as embodiment 1
Pot experiment is implemented
Load in per basin after air-drying and cross the soil sample 21.0kg of 2mm sieves, add distilled water to water-retaining quantity among field of soil 16%, protect
Hold after soil moisture content balances 1 week and start to sow.
Choose full seed, soaked with the liquor natrii hypochloritis that volumetric concentration is 1% take out after seed 10min spend from
After sub- water cleaning seed 5~7 times, the consistent cave of depth is dug in the soil in basin in advance, has uniformly sowed 6 seeds.Then
Soil is covered on seed.Spray deionized water 20mL.Thinning when plant grows 2 true leaves, ensures there are 3 flowers in each basin
It is raw.The 15d after peanut emergence, the bacillus megaterium zymotic fluid 100mL after the plant rhizosphere of every basin applies 100 times of dilution.
In planting process, soil moisture content is kept as 65% or so of maximum field capacity.When cultivating 30d and 45d, respectively to
Plant rhizosphere adds bacillus megaterium zymotic fluid, and dosage is 100mL.It is to blade spraying concentration in peanut florescence at the same time
30mg·L-1LaCl3Solution, sprays 30mL/ basins every time, sprays once within every 10 days, continuously sprays 3 times.After plant maturation, receive
Whole strain is obtained, plant is divided into overground part, underground part and pod with stainless steel scissors, measures plant fresh weight.Use deionized water rinsing
Afterwards, 105 DEG C of water-removing 20min, 70 DEG C dry to constant weight, and crushed 100 mesh sieves.
Embodiment 3
For examination soil, for examination plant, strains tested, medium treatment method with embodiment 1.
The preparation of bacillus megaterium zymotic fluid
Bacillus megaterium PP84 is inoculated on beef extract-peptone slant medium activation culture is carried out at 32 DEG C
16h, is accessed in beef extract-peptone fluid nutrient medium, 190r/min with one ring of oese picking, and 29 DEG C are cultivated 18h, as huge
Bacterium anthracoides fermentation seed liquid.Seed liquor is inoculated into beef extract-peptone fluid nutrient medium according to 3% inoculum concentration
25 DEG C, liquid fermentation 36h under conditions of rotating speed 150r/min, obtains bacillus megaterium zymotic fluid (the huge gemma bar
Fermented liquid viable count is 8 × 108CFU/mL), it is and spare after 100 times of dilution.
Pot experiment is implemented
Load in per basin after air-drying and cross the soil sample 21.0kg of 2mm sieves, add distilled water to water-retaining quantity among field of soil 16%, protect
Hold after soil moisture content balances 1 week and start to sow.
Choose full seed, soaked with the liquor natrii hypochloritis that volumetric concentration is 1% take out after seed 10min spend from
After sub- water cleaning seed 5~7 times, the consistent cave of depth is dug in the soil in basin in advance, has uniformly sowed 6 seeds.Then
Soil is covered on seed.Spray deionized water 20mL.Thinning when plant grows 2 true leaves, ensures there are 3 flowers in each basin
It is raw.The 14d after peanut emergence, the bacillus megaterium zymotic fluid 95mL after the plant rhizosphere of every basin applies dilution.Planting
During, soil moisture content is kept as 65% or so of maximum field capacity.When cultivating 29d and 44d, respectively to plant roots
Border adds bacillus megaterium zymotic fluid, and dosage is 95mL.At the same time peanut florescence to blade spraying concentration be 20mg
L-1LaCl3Solution, sprays 36mL/ basins every time, sprays once within every 11 days, continuously sprays 3 times.After plant maturation, whole strain is harvested,
Plant is divided into overground part, underground part and pod with stainless steel scissors, measures plant fresh weight.After deionized water rinsing, 105 DEG C
Finish 20min, and 70 DEG C dry to constant weight, and crushed 100 mesh sieves.
Embodiment 4
For examination soil, for examination plant, strains tested, medium treatment method with embodiment 1.
The preparation of bacillus megaterium zymotic fluid
Bacillus megaterium PP84 is inoculated on beef extract-peptone slant medium activation culture is carried out at 25 DEG C
32h, is accessed in beef extract-peptone fluid nutrient medium, 170r/min with one ring of oese picking, and 31 DEG C are cultivated 14h, as huge
Bacterium anthracoides fermentation seed liquid.Seed liquor is inoculated into beef extract-peptone fluid nutrient medium according to 1% inoculum concentration
30 DEG C, liquid fermentation 60h under conditions of rotating speed 200r/min, obtains bacillus megaterium zymotic fluid (the huge gemma bar
Fermented liquid viable count is 5 × 108CFU/mL), it is and spare after 100 times of dilution.
Pot experiment is implemented
Load in per basin after air-drying and cross the soil sample 21.0kg of 2mm sieves, add distilled water to water-retaining quantity among field of soil 16%, protect
Hold after soil moisture content balances 1 week and start to sow.
Choose full seed, soaked with the liquor natrii hypochloritis that volumetric concentration is 1% take out after seed 10min spend from
After sub- water cleaning seed 5~7 times, the consistent cave of depth is dug in the soil in basin in advance, has uniformly sowed 6 seeds.Then
Soil is covered on seed.Spray deionized water 20mL.Thinning when plant grows 2 true leaves, ensures there are 3 flowers in each basin
It is raw.The 16d after peanut emergence, the bacillus megaterium zymotic fluid 119mL after the plant rhizosphere of every basin applies dilution.Planting
During, soil moisture content is kept as 65% or so of maximum field capacity.When cultivating 31d and 46d, respectively to plant roots
Border adds bacillus megaterium zymotic fluid, and dosage is 100mL.It is to blade spraying concentration in peanut florescence at the same time
40mg.L-1LaCl3Solution, sprays 24mL/ basins every time, sprays once within every 9 days, continuously sprays 3 times.After plant maturation, harvest
Whole strain, is divided into overground part, underground part and pod by plant with stainless steel scissors, measures plant fresh weight.After deionized water rinsing,
105 DEG C of water-removing 20min, 70 DEG C dry to constant weight, and crushed 100 mesh sieves.
Comparative example 1
For examination soil, for examination plant with embodiment 1, pot experiment embodiment, which is removed, does not apply bacillus megaterium zymotic fluid
Other outer operating procedures are identical with embodiment 1.
Comparative example 2
The same implementation of method prepared for examination soil, for examination plant, strains tested, culture medium and bacillus megaterium zymotic fluid
Example 2, pot experiment embodiment other operating procedures and 2 complete phase of embodiment in addition to bacillus megaterium zymotic fluid is not applied
Together.
Comparative example 3
In addition to bacillus megaterium zymotic fluid is the zymotic fluid of inactivation, other operating procedures are identical with embodiment 2.It is huge
Bacterium anthracoides zymotic fluid inactivation treatment mode is：Bacillus megaterium zymotic fluid is taken to add 6mol/L salt acid for adjusting pH to 2.5
And 85 DEG C are heated to, constant temperature 1h, then it is adjusted to original pH with 6mol/L sodium hydroxide solutions.
Embodiment 5
Weight of root system, grain yield and P and Cd in Plant samples are measured in embodiment 1~4 and comparative example 1~3 respectively
Content, the assay method of wherein P and Cd is：Using wet digestion (dense HNO3With HClO4Volume ratio is 4:1), ICP-MS is measured
P, Cd contents, sample analysis matter is controlled in digestion and continuous mode with national standard material (GBW07603GSV-2) for internal standard
Amount.Concrete outcome is as shown in Tables 1 and 2.
Peanut Root System dry weight and grain yield under 1 different disposal of table
As can be seen from Table 1, bacillus megaterium zymotic fluid provided by the invention can significantly improve weight of root system and seed
Grain yield, the bacillus megaterium zymotic fluid not inactivated is more excellent compared to the effect after inactivation, and compared with comparative example 1, root system is done
Again than comparative example 1 high 24.6%, grain yield is than comparative example 1 high 16.4%.Wherein using provided by the invention by huge gemma
Bacillus fermentation liquid and LaCl3Preparation best results made of solution.Compared with comparative example 1, weight of root system raising 29.50%~
32.0%, grain yield is than comparative example 1 high 20.32%~21.8%.
Ecological Property of Peanut Seeds cadmium content and blade phosphorus content under 2 different disposal of table
As can be seen from Table 2, bacillus megaterium zymotic fluid provided by the invention can significantly reduce Ecological Property of Peanut Seeds cadmium and contain
Amount, while blade P content is dramatically increased, the bacillus megaterium zymotic fluid not inactivated is more excellent compared to the effect after inactivation, with
Comparative example 1 is compared, and Cd contents low 27.9%, blade P content is high by 16.7%.Wherein using provided by the invention by huge gemma bar
Fermented liquid and LaCl3Preparation best results made of solution.Compared with comparative example 1, Cd contents low 39.71%~42.65%,
Blade P content is high by 20.4%~22.22%.
Embodiment 6
For trying soil
Qingdao City Chengyang District contaminated soil (Shajiang black soil) is picked up from, its basic physical and chemical is：(Tu Shui is than 1 by pH 7.11：
2.5), organic matter 17.9g/kg, cation exchange capacity (CEC) 15.1cmol/kg, total cadmium content 3.31mg/kg.Pedotheque natural wind
It is dry, it is levigate to cross 2mm (10 mesh) sieves, for potted plant simulation test.
For trying plant
Peanut (rich to spend No. 3), purchased from Shandong Academy of Agricultural Sciences.
Strains tested
Bacillus megaterium (BaCillus megaterium) bacterial strain PP84 (deposit numbers：CGMCC No.12798), bud
1.0~1.2 × 1.5~2.0 microns of spore, ellipse, middle life or the life of secondary end.
Culture medium
Beef extract-peptone slant medium：Beef extract 3g is weighed, peptone 5g, agar 18g, is settled to deionized water
1000ml, 121 DEG C of sterilizing 20min.
Dried beef peptone fluid nutrient medium：Beef extract 3g is weighed, peptone 5g, 1000ml is settled to deionized water,
121 DEG C of sterilizing 20min.
The preparation of bacillus megaterium solid pharmaceutical preparation
Bacillus megaterium PP84 is inoculated on beef extract-peptone slant medium activation culture is carried out at 30 DEG C
24h, is accessed in beef extract-peptone fluid nutrient medium, 180r/min with one ring of oese picking, and 30 DEG C are cultivated 16h, as huge
Bacterium anthracoides fermentation seed liquid.Seed liquor is inoculated into wheat bran medium in 28 DEG C of conditions according to the inoculum concentration by 5.0%
Lower solid fermentation 4d, stirring, obtains mixture；
The mixture that the step obtains is continued to the 48h that ferments at 28 DEG C, obtains bacillus megaterium solid pharmaceutical preparation, institute
The bacillus megaterium solid pharmaceutical preparation viable count stated is 9 × 108CFU/g。
Pot experiment is implemented
Load in per basin after air-drying and cross the soil sample 21.0kg of 2mm sieves, add distilled water to water-retaining quantity among field of soil, keep soil
Earth water content starts to sow after balancing 1 week.Full seed is chosen, is taken out after soaking seed 10min with 1% liquor natrii hypochloritis
After cleaning seed 5~7 times with deionized water, the consistent cave of depth has been dug in the soil in basin in advance, has uniformly sowed 6 kinds
Soil, is then covered on seed by son.Spray deionized water 20mL.Thinning when plant grows 2 true leaves.Ensure per basin alms bowl 3
Peanut.After emergence 8 days, spread fertilizer over the fields bacillus megaterium solid pharmaceutical preparation 0.5g in soil surface, after plant maturation, harvest whole strain,
Plant is divided into overground part, underground part and pod with stainless steel scissors, measures plant fresh weight.After deionized water rinsing, 105 DEG C
Finish 20min, and 70 DEG C dry to constant weight, and crushed 100 mesh sieves.
Embodiment 7
For trying soil
Qingdao City Chengyang District contaminated soil (Shajiang black soil) is picked up from, its basic physical and chemical is：(Tu Shui is than 1 by pH 7.11：
2.5), organic matter 17.9g/kg, cation exchange capacity (CEC) 15.1cmol/kg, total cadmium content 3.31mg/kg.Pedotheque natural wind
It is dry, it is levigate to cross 2mm (10 mesh) sieves, for potted plant simulation test.
For trying plant
Peanut (rich to spend No. 3), purchased from Shandong Academy of Agricultural Sciences.
Strains tested
Bacillus megaterium (BaCillus megaterium) bacterial strain PP84 (deposit numbers：CGMCC No.12798), bud
1.0~1.2 × 1.5~2.0 microns of spore, ellipse, middle life or the life of secondary end.
Culture medium
Beef extract-peptone slant medium：Beef extract 3g is weighed, peptone 5g, agar 18g, is settled to deionized water
1000ml, 121 DEG C of sterilizing 20min.
Dried beef peptone fluid nutrient medium：Beef extract 3g is weighed, peptone 5g, 1000ml is settled to deionized water,
121 DEG C of sterilizing 20min.
The preparation of bacillus megaterium solid pharmaceutical preparation
Bacillus megaterium PP84 is inoculated on beef extract-peptone slant medium activation culture is carried out at 30 DEG C
24h, is accessed in beef extract-peptone fluid nutrient medium, 180r/min with one ring of oese picking, and 30 DEG C are cultivated 16h, as huge
Bacterium anthracoides fermentation seed liquid.Seed liquor is inoculated into wheat bran medium in 28 DEG C of conditions according to the inoculum concentration by 5.0%
Lower solid fermentation 4d, stirring, obtains mixture；
The mixture that the step obtains is continued to the 48h that ferments at 28 DEG C, obtains bacillus megaterium solid pharmaceutical preparation, institute
The bacillus megaterium solid pharmaceutical preparation viable count stated is 9 × 108CFU/g。
Pot experiment is implemented
Load in per basin after air-drying and cross the soil sample 21.0kg of 2mm sieves, add distilled water to water-retaining quantity among field of soil, keep soil
Earth water content starts to sow after balancing 1 week.Full seed is chosen, is taken out after soaking seed 10min with 1% liquor natrii hypochloritis
After cleaning seed 5~7 times with deionized water, the consistent cave of depth has been dug in the soil in basin in advance, has uniformly sowed 6 kinds
Soil, is then covered on seed by son.Spray deionized water 20mL.Thinning when plant grows 2 true leaves.Ensure per basin alms bowl 3
Peanut.After emergence 8 days, bacillus megaterium solid pharmaceutical preparation 0.5g is spread fertilizer over the fields in soil surface, in planting process, keeps soil
Water content is 65% or so of maximum field capacity.At the same time peanut florescence to blade spraying concentration be 30mgL-1's
LaCl3Solution, sprays 30mL/ basins every time, sprays once within every 10 days, continuously sprays 3 times.After plant maturation, whole strain is harvested, with not
Plant is divided into overground part, underground part and pod by rust steel scissors, measures plant fresh weight.After deionized water rinsing, 105 DEG C of water-removings
20min, 70 DEG C dry to constant weight, and crushed 100 mesh sieves.
Embodiment 8
For examination soil, for examination plant, strains tested, medium treatment method with embodiment 6.
The preparation of bacillus megaterium solid pharmaceutical preparation
Bacillus megaterium PP84 is inoculated on beef extract-peptone slant medium activation culture is carried out at 32 DEG C
16h, is accessed in beef extract-peptone fluid nutrient medium, 190r/min with one ring of oese picking, and 29 DEG C are cultivated 18h, as huge
Bacterium anthracoides fermentation seed liquid.Seed liquor is inoculated into wheat bran medium in 30 DEG C of conditions according to the inoculum concentration by 7.5%
Lower solid fermentation 5d, stirring, obtains mixture；
The mixture that the step obtains is continued to the 60h that ferments at 25 DEG C, obtains bacillus megaterium solid pharmaceutical preparation, institute
The bacillus megaterium solid pharmaceutical preparation viable count stated is 3 × 108CFU/g。
Pot experiment is implemented
Load in per basin after air-drying and cross the soil sample 21.0kg of 2mm sieves, add distilled water to water-retaining quantity among field of soil, keep soil
Earth water content starts to sow after balancing 1 week.Full seed is chosen, is taken out after soaking seed 10min with 1% liquor natrii hypochloritis
After cleaning seed 5~7 times with deionized water, the consistent cave of depth has been dug in the soil in basin in advance, has uniformly sowed 6 kinds
Soil, is then covered on seed by son.Spray deionized water 20mL.Thinning when plant grows 2 true leaves.Ensure per basin alms bowl 3
Peanut.After emergence 7 days, bacillus megaterium solid pharmaceutical preparation 0.41g is spread fertilizer over the fields in soil surface, in planting process, keeps soil
Earth water content is 65% or so of maximum field capacity.At the same time peanut florescence to blade spraying concentration be 20mgL-1's
LaCl3Solution, sprays 36mL/ basins every time, sprays once within every 9 days, continuously sprays 3 times.After plant maturation, whole strain is harvested, with not
Plant is divided into overground part, underground part and pod by rust steel scissors, measures plant fresh weight.After deionized water rinsing, 105 DEG C of water-removings
20min, 70 DEG C dry to constant weight, and crushed 100 mesh sieves.
Embodiment 9
For examination soil, for examination plant, strains tested, medium treatment method with embodiment 6.
The preparation of bacillus megaterium solid pharmaceutical preparation
Bacillus megaterium PP84 is inoculated on beef extract-peptone slant medium activation culture is carried out at 30 DEG C
24h, is accessed in beef extract-peptone fluid nutrient medium, 170r/min with one ring of oese picking, and 30 DEG C are cultivated 16h, as huge
Bacterium anthracoides fermentation seed liquid.Seed liquor is inoculated into wheat bran medium in 28 DEG C of conditions according to the inoculum concentration by 2.5%
Lower solid fermentation 3d, stirring, obtains mixture；
The mixture that the step obtains is continued to the 36h that ferments at 28 DEG C, obtains bacillus megaterium solid pharmaceutical preparation, institute
The bacillus megaterium solid pharmaceutical preparation viable count stated is 9 × 107CFU/g。
Pot experiment is implemented
Load in per basin after air-drying and cross the soil sample 21.0kg of 2mm sieves, add distilled water to water-retaining quantity among field of soil, keep soil
Earth water content starts to sow after balancing 1 week.Full seed is chosen, is taken out after soaking seed 10min with 1% liquor natrii hypochloritis
After cleaning seed 5~7 times with deionized water, the consistent cave of depth has been dug in the soil in basin in advance, has uniformly sowed 6 kinds
Soil, is then covered on seed by son.Spray deionized water 20mL.Thinning when plant grows 2 true leaves.Ensure per basin alms bowl 3
Peanut.After emergence 10 days, bacillus megaterium solid pharmaceutical preparation 0.54g is spread fertilizer over the fields in soil surface, in planting process, keeps soil
Earth water content is 65% or so of maximum field capacity.At the same time peanut florescence to blade spraying concentration be 40mgL-1's
LaCl3Solution, sprays 24mL/ basins every time, sprays once within every 11 days, continuously sprays 3 times.After plant maturation, whole strain is harvested, with not
Plant is divided into overground part, underground part and pod by rust steel scissors, measures plant fresh weight.After deionized water rinsing, 105 DEG C of water-removings
20min, 70 DEG C dry to constant weight, and crushed 100 mesh sieves.
Comparative example 4
For examination soil, for examination plant, with embodiment 5, pot experiment embodiment is not except applying bacillus megaterium solid
Other outer operating procedures of preparation are identical with embodiment 5.
Comparative example 5
For examination soil, for trying the method for plant, strains tested, culture medium and the preparation of bacillus megaterium solid pharmaceutical preparation with real
Example 6 is applied, pot experiment embodiment other operating procedures and embodiment 6 in addition to bacillus megaterium solid pharmaceutical preparation is not applied is complete
It is identical.
Comparative example 6
In addition to bacillus megaterium solid pharmaceutical preparation is the solid pharmaceutical preparation of inactivation, other operating procedures and comparative example 6 are complete
It is identical.
Embodiment 10
Weight of root system, grain yield and P and Cd in Plant samples are measured in embodiment 6~9 and comparative example 4~6 respectively
Content, the assay method of wherein P and Cd is：Using wet digestion (dense HNO3With HClO4Volume ratio is 4:1), ICP-MS is measured
P, Cd contents, sample analysis matter is controlled in digestion and continuous mode with national standard material (GBW07603 GSV-2) for internal standard
Amount.Concrete outcome is as shown in Tables 3 and 4.
Peanut Root System dry weight and grain yield under 3 different disposal of table
As can be seen from Table 3, bacillus megaterium zymotic fluid provided by the invention can significantly improve weight of root system and seed
Grain yield, the bacillus megaterium zymotic fluid not inactivated is more excellent compared to the effect after inactivation, and compared with comparative example 4, root system is done
Again than comparative example 4 high 23.9%, grain yield is than comparative example 4 high 17.5%.Wherein using provided by the invention by huge gemma
Bacillus fermentation liquid and LaCl3Preparation best results made of solution.Compared with comparative example 4, weight of root system raising 32.60%~
33.2%, grain yield is than comparative example 4 high 23.27%~23.5%.
Ecological Property of Peanut Seeds cadmium content and blade phosphorus content under 4 different disposal of table
As can be seen from Table 4, bacillus megaterium zymotic fluid provided by the invention can significantly reduce Ecological Property of Peanut Seeds cadmium and contain
Amount, while blade P content is dramatically increased, the bacillus megaterium zymotic fluid not inactivated is more excellent compared to the effect after inactivation, with
Comparative example 4 is compared, and Cd contents low 26.56%, blade P content is high by 14.55%.Wherein using provided by the invention by huge gemma
Bacillus fermentation liquid and LaCl3Preparation best results made of solution.Compared with comparative example 4, Cd contents are low 39.06%~
40.63%, blade P content is high by 16.4%~18.18%.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. one plant of bacillus megaterium bacterial strain Bacillus megaterium PP84, deposit number are CGMCC No.12798.
2. one, which cultivates peanut, drops cadmium agent, it is characterised in that including bacillus megaterium zymotic fluid or bacillus megaterium solid pharmaceutical preparation；
The preparation method of the bacillus megaterium zymotic fluid, comprises the following steps：
Bacillus megaterium bacterial strain described in claim 1 is activated and expands culture, obtains bacillus megaterium fermentation seed liquid,
The bacillus megaterium fermentation seed liquid is inoculated into beef extract-peptone fluid nutrient medium by 1~3% inoculum concentration
25~30 DEG C, liquid fermentation 36h~60h under conditions of 150~200r/min of rotating speed, obtains bacillus megaterium zymotic fluid；
The preparation method of the bacillus megaterium solid pharmaceutical preparation, comprises the following steps：
A. bacillus megaterium bacterial strain described in claim 1 is activated and expands culture, obtains bacillus megaterium fermentation seed
Liquid, the bacillus megaterium fermentation seed liquid is inoculated into wheat bran medium 25 by 2.5%~7.5% inoculum concentration~
3~5d of solid fermentation culture under the conditions of 30 DEG C, stirring, obtains mixture；
B. the mixture obtained in the step a is continued into 1.5~2.5d of fermented and cultured at 25~30 DEG C, obtains huge gemma
Bacillus solid pharmaceutical preparation.
3. drop cadmium agent according to claim 2, it is characterised in that it is 1~2g/L to further include the mass concentration independently dispensed
LaCl3Solution.
4. application of the drop cadmium agent in peanut is planted described in Claims 2 or 3.
5. application according to claim 4, it is characterised in that the bacillus megaterium zymotic fluid is applied in plantation peanut
Rhizosphere；The applied amount of the bacillus megaterium zymotic fluid is 16~20L/ mus.
6. application according to claim 5, it is characterised in that the application number of the bacillus megaterium zymotic fluid is 3
It is secondary；The time applied for the first time is 14~16d after peanut emergence, second time applied for 29 after peanut emergence~
31d, the time that third time applies are 44~46d after peanut emergence.
7. application according to claim 4, it is characterised in that the bacillus megaterium solid pharmaceutical preparation is applied in plantation flower
Raw soil surface；The applied amount of the bacillus megaterium solid pharmaceutical preparation is 7~9kg/ mus.
8. application according to claim 7, it is characterised in that the application number of the bacillus megaterium solid pharmaceutical preparation is
1 time, the time of application is 7~10d after peanut emergence.
9. according to the application described in claim 5~8 any one, it is characterised in that in peanut florescence by LaCl3Solution sprays
Apply and spray once, continuously spray three times in the blade face of plantation peanut, every 9~11d.
10. application according to claim 9, it is characterised in that the LaCl3The each amount of spraying of solution is 8~12L/
Mu.
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