CN108034620B - 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 applications, belong to Crop securify production technical field.Peanut drop cadmium agent provided by the invention including bacillus megaterium fermentation liquid or bacillus megaterium solid pharmaceutical preparation can reduce the content of cadmium in Ecological Property of Peanut Seeds.P content, Ecological Property of Peanut Seeds yield and the Peanut Root System biomass in blade can also be improved simultaneously.Further, drop cadmium agent provided by the invention further includes the LaCL that mass concentration is 1~2g/L3Solution can further increase the removal rate to cadmium in Ecological Property of Peanut Seeds.Compared with not applying drop cadmium agent, peanut is cultivated using drop cadmium agent provided by the invention, cadmium content in Ecological Property of Peanut Seeds is set to reduce by 39.06%~42.65%, phosphorus content increases by 16.4%~22.22% simultaneously, 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 fields, and in particular to one plant of bacillus megaterium bacterial strain, peanut drop
Cadmium agent and its application.
Background technique
With the development of industry with the modernization of agricultural production, heavy metals in farmland, which pollutes, to be got worse, and contaminated area is year by year
Expand.Heavy metal is due to having many characteristics, such as that not degradable, easy enrichment receives significant attention.Because having high toxicity and high migration,
Cadmium (Cd) is considered as most important environmental pollutants, and excessive cadmium can lead to Itai-itai diseases, kidney trouble, liver damage in human body
A series of diseases such as evil.After cadmium enters soil, preferentially in conjunction with carbonate, the cadmium carbonate of indissoluble is formed, there is biggish movement
Property and plant availability to be easier to be absorbed by plants into food chain harm is generated to human health.
Peanut is to inhale one of strongest field crop of cadmium ability, the absorption to cadmium in soil and the efficiency to seed transfer
Higher than many crops.According to investigations, Ecological Property of Peanut Seeds cadmium average content in northern China producing region is 0.2-0.3mg/kg, investigates the soil in place
For earth without obvious pollution condition, majority has exceeded the nuisanceless peanut Cd content 0.05mg/kg (NY5303-2005) of country, portion
Dividing has been more than green food standard 0.4mg/kg limitation (NY/T420-2009).Ecological Property of Peanut Seeds cadmium content is exceeded to have become restriction
An important factor for outlet of China's peanut and related industry development.
Currently, selecting special crop varieties, screening or breeding low cadmium-accumulation kind are abatement plant edible part cadmium accumulations
A kind of mode, but such mode, due to the particularity of plant variety, there may be certain defects, such as yield for biological character
It is low, resistance is poor etc., limit being widely applied for kind.
Summary of the invention
In view of this, the purpose of the present invention is to provide one plant of bacillus megaterium bacterial strain, reducing Ecological Property of Peanut Seeds cadmium content
Preparation, can reduce Ecological Property of Peanut Seeds Cd accumulation, improve Ecological Property of Peanut Seeds and drop cadmium efficiency.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of bacillus megaterium bacterial strain BaCillus megaterium PP84, deposit number is
CGMCC No.12798。
It cultivates peanut the present invention also provides one and drops cadmium agent, including bacillus megaterium fermentation liquid or bacillus megaterium solid
Preparation；
The preparation method of the bacillus megaterium fermentation liquid, comprising the following steps:
Bacillus megaterium bacterial strain described in above scheme is activated and expands culture, obtains bacillus megaterium fermentation seed
The bacillus megaterium fermentation seed liquid is inoculated into beef extract-peptone fluid nutrient medium by liquid by 1~3% inoculum concentration
Liquid fermentation 36h~60h under conditions of 25~30 DEG C, 150~200r/min of revolving speed, obtains bacillus megaterium fermentation liquid；
The preparation method of the bacillus megaterium solid pharmaceutical preparation, comprising the following steps:
A. bacillus megaterium bacterial strain described in above scheme is activated and expands culture, obtains bacillus megaterium fermentation kind
The bacillus megaterium fermentation seed liquid is inoculated into wheat bran medium by 2.5%~7.5% inoculum concentration 25 by sub- liquid
3~5d of solid fermentation culture under the conditions of~30 DEG C, stirring, obtains mixture；
B. mixture obtained in the step a is continued at 25~30 DEG C 1.5~2.5d of fermented and cultured, obtained huge
Bacillus solid pharmaceutical preparation.
Preferably, it is the LaCl of 1~2g/L that the drop cadmium agent, which further includes the mass concentration independently dispensed,3Solution.
The present invention provides a kind of application of the drop cadmium agent of peanut described in above method in plantation peanut.
Preferably, the bacillus megaterium fermentation liquid 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 fermentation liquid is 3 times；The time applied for the first time goes out for peanut
14~16d after seedling, second of the time applied, the time that third time applies went out for 29~31d after peanut emergence for peanut
44~46d 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 sprays on the blade face of plantation peanut, and every 9~11d is sprayed once,
Continuous spraying is 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 fermentation liquid 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 reducing Cd from root system
It is shifted to seed, so as to reduce the content of cadmium in Ecological Property of Peanut Seeds.Not apply drop cadmium agent as a control group, apply drop
Ecological Property of Peanut Seeds Cd content can be significantly reduced in cadmium agent.Apply Cd content in the Ecological Property of Peanut Seeds planted after drop cadmium agent and is lower than control group
Cd content 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 the 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 mass concentration for 1~2g/L3Solution.It will be above-mentioned
Bacillus megaterium bacterial strain preparation and mass concentration described in scheme are the LaCl of 1~2g/L3Solution is used cooperatively.Huge gemma
Bacillus strain preparation and LaCl3Solution is used cooperatively, and effectively improves peanut phosphorus nourishing situation, is improved peanut and is resisted to Cd
Property, promote peanut growth, reduce absorption of the peanut to Cd, while reducing Cd and being 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 in Ecological Property of Peanut Seeds Cd content after the preparation lower than control group
39.06%~42.65%.
Meanwhile peanut drop cadmium agent provided by the invention can also further increase the content of blade P, compared with the 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 abbreviation: CGMCC, preservation day
Phase is on July 20th, 2016, and biological deposits number is CGMCCNo.12798, strain number: PP84.
Specific embodiment
The present invention provides a kind of bacillus megaterium bacterial strain Bacillus megaterium PP84, deposit number is
CGMCC No.12798。
It cultivates peanut the present invention also provides one and drops cadmium agent, including bacillus megaterium fermentation liquid or bacillus megaterium solid
Preparation；
The preparation method of the bacillus megaterium fermentation liquid, comprising the following steps:
Bacillus megaterium bacterial strain described in above scheme is activated and expands culture, obtains bacillus megaterium fermentation seed
Obtained bacillus megaterium fermentation seed liquid is inoculated into beef extract-peptone fluid nutrient medium by 1~3% inoculum concentration by liquid
In under conditions of 25~30 DEG C, 150~200r/min of revolving speed liquid fermentation 36h~60h, obtain bacillus megaterium fermentation
Liquid；
The preparation method of the bacillus megaterium solid pharmaceutical preparation, comprising the following steps:
A. bacillus megaterium bacterial strain described in above scheme is activated and expands culture, obtains bacillus megaterium fermentation kind
Obtained bacillus megaterium fermentation seed liquid is inoculated into wheat bran medium by sub- liquid 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. mixture obtained in the step a is continued at 25~30 DEG C 1.5~2.5d of fermented and cultured, obtained huge
Bacillus solid pharmaceutical preparation.
The present invention preferably activates 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
Are as follows: 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, and more preferably 28 DEG C.The time of the activation
Preferably 16~32h, more preferably for 24 hours.
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 expands culture, and obtains bacillus megaterium fermentation seed liquid.The mode for expanding culture are as follows: picking
Bacillus megaterium after the activation of one ring is linked into beef extract-peptone fluid nutrient medium at 29~31 DEG C, and revolving speed 170~
14h~18h is cultivated under conditions of 190r/min.The temperature for expanding culture is preferably 30 DEG C.The revolving 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 revolving speed
Body fermentation 36h~60h, obtains bacillus megaterium fermentation liquid.In obtained bacillus megaterium fermentation liquid viable count be 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 revolving speed when liquid fermentation is preferably 180r/
min。
In the present invention, the preparation method of the beef extract-peptone fluid nutrient medium are as follows: weigh beef extract 3g, peptone
5g is settled to 1000ml with deionized water, and 121 DEG C of sterilizing 20min obtain beef extract-peptone fluid nutrient medium.
The present invention is not particularly limited the source of the beef extract, peptone and agar, using this field 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 fermentation liquid improves peanut phosphorus nourishing situation, promotes peanut growth, drop
Absorption of the low peanut to Cd, while reducing Cd and being shifted from root system to seed, to reduce the Cd content in Ecological Property of Peanut Seeds.
When preparing bacillus megaterium solid pharmaceutical preparation, after obtaining bacillus megaterium fermentation seed liquid, the present invention is by institute
Bacillus megaterium fermentation seed liquid is stated to be inoculated into the wheat bran medium by 2.5%~7.5% inoculum concentration at 25~30 DEG C
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 fermentation liquid7~9 × 108CFU/mL。
After obtaining bacillus megaterium solid pharmaceutical preparation, the present invention preferably stirs the bacillus megaterium solid pharmaceutical preparation
It mixes, the operation of 36~60h of solid fermentation repeats 2~3 times at 25~30 DEG C, keeps bacillus megaterium equal in wheat bran medium
Even distribution.
In the present invention, the mixed mode is preferably stirred.The revolving 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 are as follows: 87.9 parts of wheat bran, yeast powder 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 field conventional commercial product.
In wheat bran medium described in the embodiment of the present invention, wheat bran is purchased from Qingdao wheat drifting fragrance flour Co., Ltd, remaining drug 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 reducing Cd and being shifted from root system to seed, so that the Cd reduced in Ecological Property of Peanut Seeds contains
Amount.
It is also preferable to include the LaCl independently dispensed for drop cadmium agent provided by the invention3Solution.The LaCl3The concentration of solution is excellent
It is selected as 1~2g/L, more preferably 1.4~1.6g/L, most preferably 1.5g/L.In the present invention, the LaCl3Solution mentions after
The resistance that high peanut poisons Cd, while La3+With Cd2+Antagonism is generated, 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 field 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 a kind of application of the drop cadmium agent described in above scheme in plantation peanut.
In the present invention, the bacillus megaterium fermentation liquid 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, 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 fermentation liquid
Application number be 3 times.The time that the bacillus megaterium fermentation liquid 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
It is selected as 44~46d, more preferably 45d.In the present invention, the bacillus megaterium fermentation liquid preferably includes to inactivate huge gemma bar
Fermented liquid and bacillus megaterium fermentation liquid is not inactivated, do not inactivate bacillus megaterium fermentation liquid 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, most preferably 8kg/ mus.Institute
State bacillus megaterium solid pharmaceutical preparation application time be preferably peanut emergence after 7~10d, more preferably 8d.In the present invention
The mode of the application is preferably 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 preferably sprays in peanut florescence in the blade face of plantation peanut, every 9~11d spray
It applies once, is 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, 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 embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1
For trying soil:
Pick up from Qingdao City's Pingdu City contaminated soil (sandy loam), basic physical and chemical are as follows: pH 6.4 (Tu Shui is than 1:
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) sieve, for potting 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) is purchased from Shandong Academy of Agricultural Sciences.
Strains tested
Bacillus megaterium (BaCillus megaterium) bacterial strain PP84 (deposit number: CGMCC No.12798), bud
1.0~1.2 × 1.5~2.0 microns of spore, ellipse, middle life or secondary end are given birth to.
Culture medium
Beef extract-peptone slant medium: weighing beef extract 3g, peptone 5g, and agar 18g is settled to deionized water
1000ml, 121 DEG C of sterilizing 20min.
Dried beef peptone fluid nutrient medium: weighing beef extract 3g, and peptone 5g is settled to 1000ml with 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 and carries out activation culture at 30 DEG C
It after for 24 hours, is accessed in beef extract-peptone fluid nutrient medium with one ring of oese picking, 180r/min, 30 DEG C of culture 16h, as
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, revolving speed 180r/min liquid fermentation 48h, obtain bacillus megaterium fermentation liquid (the huge bud
Spore bacillus fermentation liquid viable count is 2 × 109CFU/mL) and stand-by after 100 times of dilutions of progress.
Pot experiment is implemented
It is packed into the soil sample 21.0kg for crossing 2mm sieve after air-drying in every basin, distilled water is added to water-retaining quantity among field of soil 16%, protects
It holds after soil moisture content balances 1 week and starts to sow.
Choose full seed, with volumetric concentration be 1% liquor natrii hypochloritis impregnate seed 10min after take out spend from
After sub- water cleans seed 5~7 times, the consistent cave of depth has been 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 guarantees there are 3 flowers in each basin
It is raw.The 15d after peanut emergence, the bacillus megaterium fermentation liquid 100mL after the plant rhizosphere of every basin applies 100 times of dilution.
In planting process, keeping soil moisture content is 65% or so of maximum field capacity.When cultivating 30d and 45d, respectively to
Plant rhizosphere adds bacillus megaterium fermentation liquid, 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 being rinsed with deionized water, 105 DEG C of water-removing 20min, 70
It DEG C dries to constant weight, crushing sieves with 100 mesh sieve.
Embodiment 2
For examination soil, for the preparation of examination plant, strains tested, the method for medium treatment and bacillus megaterium fermentation liquid
Method is the same as embodiment 1
Pot experiment is implemented
It is packed into the soil sample 21.0kg for crossing 2mm sieve after air-drying in every basin, distilled water is added to water-retaining quantity among field of soil 16%, protects
It holds after soil moisture content balances 1 week and starts to sow.
Choose full seed, with volumetric concentration be 1% liquor natrii hypochloritis impregnate seed 10min after take out spend from
After sub- water cleans seed 5~7 times, the consistent cave of depth has been 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 guarantees there are 3 flowers in each basin
It is raw.The 15d after peanut emergence, the bacillus megaterium fermentation liquid 100mL after the plant rhizosphere of every basin applies 100 times of dilution.
In planting process, keeping soil moisture content is 65% or so of maximum field capacity.When cultivating 30d and 45d, respectively to
Plant rhizosphere adds bacillus megaterium fermentation liquid, and dosage is 100mL.It is to blade spraying concentration in peanut florescence simultaneously
30mg·L-1LaCl3Solution sprays 30mL/ basin every time, sprays within every 10 days once, continuous spraying 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.It is rinsed with deionized water
Afterwards, 105 DEG C of water-removing 20min, 70 DEG C dry to constant weight, and crushing sieves with 100 mesh sieve.
Embodiment 3
For examination soil, for examination plant, strains tested, medium treatment method with embodiment 1.
The preparation of bacillus megaterium fermentation liquid
Bacillus megaterium PP84 is inoculated on beef extract-peptone slant medium and carries out activation culture at 32 DEG C
16h is accessed in beef extract-peptone fluid nutrient medium, 190r/min with one ring of oese picking, 29 DEG C of culture 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 revolving speed 150r/min obtains bacillus megaterium fermentation liquid (the huge gemma bar
Fermented liquid viable count is 8 × 108CFU/mL), and it is spare after 100 times of dilution.
Pot experiment is implemented
It is packed into the soil sample 21.0kg for crossing 2mm sieve after air-drying in every basin, distilled water is added to water-retaining quantity among field of soil 16%, protects
It holds after soil moisture content balances 1 week and starts to sow.
Choose full seed, with volumetric concentration be 1% liquor natrii hypochloritis impregnate seed 10min after take out spend from
After sub- water cleans seed 5~7 times, the consistent cave of depth has been 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 guarantees there are 3 flowers in each basin
It is raw.The 14d after peanut emergence, the bacillus megaterium fermentation liquid 95mL after the plant rhizosphere of every basin applies dilution.It is planting
In the process, keeping soil moisture content is 65% or so of maximum field capacity.When cultivating 29d and 44d, respectively to plant roots
Border adds bacillus megaterium fermentation liquid, and dosage is 95mL.Simultaneously peanut florescence to blade spraying concentration be 20mg
L-1LaCl3Solution sprays 36mL/ basin every time, sprays within every 11 days once, continuous spraying 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 being rinsed with deionized water, 105 DEG C
Finish 20min, and 70 DEG C dry to constant weight, and crushing sieves with 100 mesh sieve.
Embodiment 4
For examination soil, for examination plant, strains tested, medium treatment method with embodiment 1.
The preparation of bacillus megaterium fermentation liquid
Bacillus megaterium PP84 is inoculated on beef extract-peptone slant medium and carries out activation culture at 25 DEG C
32h is accessed in beef extract-peptone fluid nutrient medium, 170r/min with one ring of oese picking, 31 DEG C of culture 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 revolving speed 200r/min obtains bacillus megaterium fermentation liquid (the huge gemma bar
Fermented liquid viable count is 5 × 108CFU/mL), and it is spare after 100 times of dilution.
Pot experiment is implemented
It is packed into the soil sample 21.0kg for crossing 2mm sieve after air-drying in every basin, distilled water is added to water-retaining quantity among field of soil 16%, protects
It holds after soil moisture content balances 1 week and starts to sow.
Choose full seed, with volumetric concentration be 1% liquor natrii hypochloritis impregnate seed 10min after take out spend from
After sub- water cleans seed 5~7 times, the consistent cave of depth has been 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 guarantees there are 3 flowers in each basin
It is raw.The 16d after peanut emergence, the bacillus megaterium fermentation liquid 119mL after the plant rhizosphere of every basin applies dilution.It is planting
In the process, keeping soil moisture content is 65% or so of maximum field capacity.When cultivating 31d and 46d, respectively to plant roots
Border adds bacillus megaterium fermentation liquid, and dosage is 100mL.It is to blade spraying concentration in peanut florescence simultaneously
40mg.L-1LaCl3Solution sprays 24mL/ basin every time, sprays within every 9 days once, continuous spraying 3 times.After plant maturation, harvest
Plant is divided into overground part, underground part and pod with stainless steel scissors, measures plant fresh weight by whole strain.After being rinsed with deionized water,
105 DEG C of water-removing 20min, 70 DEG C dry to constant weight, and crushing sieves with 100 mesh sieve.
Comparative example 1
For examination soil, for examination plant with embodiment 1, pot experiment embodiment, which is removed, does not apply bacillus megaterium fermentation liquid
Other outer operating procedures are identical with embodiment 1.
Comparative example 2
Implement for examination soil, for the method for examination plant, strains tested, culture medium and the preparation of bacillus megaterium fermentation liquid is same
Example 2, pot experiment embodiment other operating procedures and the complete phase of embodiment 2 in addition to not applying bacillus megaterium fermentation liquid
Together.
Comparative example 3
In addition to bacillus megaterium fermentation liquid is the fermentation liquid of inactivation, other operating procedures are identical with embodiment 2.It is huge
Bacterium anthracoides fermentation liquid inactivation treatment mode are as follows: take bacillus megaterium fermentation liquid that 6mol/L salt acid for adjusting pH is added to 2.5
And 85 DEG C are heated to, constant temperature 1h, then original pH is adjusted to 6mol/L sodium hydroxide solution.
Embodiment 5
Weight of root system, grain yield and P and Cd in Plant samples are measured in Examples 1 to 4 and comparative example 1~3 respectively
Content, the wherein measuring method of P and Cd are as follows: use wet digestion (dense HNO3With HClO4Volume ratio is 4:1), ICP-MS measurement
P, Cd content, digestion and continuous mode in national standard substance (GBW07603GSV-2) be internal standard control sample analysis matter
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 fermentation liquid provided by the invention can significantly improve weight of root system and seed
Grain yield, the bacillus megaterium fermentation liquid not inactivated is more excellent compared to the effect after inactivation, and compared with comparative example 1, root system is dry
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 effect made of solution is best.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 fermentation liquid 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 fermentation liquid not inactivated is more excellent compared to the effect after inactivation, with
Comparative example 1 is compared, and Cd content 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 effect made of solution is best.Compared with comparative example 1, Cd content low 39.71%~42.65%,
Blade P content is high by 20.4%~22.22%.
Embodiment 6
For trying soil
Pick up from Qingdao City Chengyang District contaminated soil (Shajiang black soil), basic physical and chemical are as follows: pH 7.11 (Tu Shui is than 1:
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) sieve, for potting simulation test.
For trying plant
Peanut (rich to spend No. 3) is purchased from Shandong Academy of Agricultural Sciences.
Strains tested
Bacillus megaterium (BaCillus megaterium) bacterial strain PP84 (deposit number: CGMCC No.12798), bud
1.0~1.2 × 1.5~2.0 microns of spore, ellipse, middle life or secondary end are given birth to.
Culture medium
Beef extract-peptone slant medium: weighing beef extract 3g, peptone 5g, and agar 18g is settled to deionized water
1000ml, 121 DEG C of sterilizing 20min.
Dried beef peptone fluid nutrient medium: weighing beef extract 3g, and peptone 5g is settled to 1000ml with 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 and carries out activation culture at 30 DEG C
For 24 hours, it is accessed in beef extract-peptone fluid nutrient medium with one ring of oese picking, 180r/min, 30 DEG C of culture 16h are as huge
Bacterium anthracoides fermentation seed liquid.Seed liquor is inoculated into wheat bran medium according to the inoculum concentration by 5.0% in 28 DEG C of conditions
Lower solid fermentation 4d, stirring, obtains mixture；
The mixture that the step obtains is continued into 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
It is packed into the soil sample 21.0kg for crossing 2mm sieve after air-drying in every basin, distilled water is added to water-retaining quantity among field of soil, keeps soil
Earth water content starts to sow after balancing 1 week.Full seed is chosen, is taken out after impregnating seed 10min with 1% liquor natrii hypochloritis
The consistent cave of depth has been dug after cleaning seed 5~7 times with deionized water, in the soil in basin in advance, has uniformly sowed 6 kinds
Then soil is covered on seed by son.Spray deionized water 20mL.Thinning when plant grows 2 true leaves.Guarantee every 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, after plant maturation, harvests whole strain,
Plant is divided into overground part, underground part and pod with stainless steel scissors, measures plant fresh weight.After being rinsed with deionized water, 105 DEG C
Finish 20min, and 70 DEG C dry to constant weight, and crushing sieves with 100 mesh sieve.
Embodiment 7
For trying soil
Pick up from Qingdao City Chengyang District contaminated soil (Shajiang black soil), basic physical and chemical are as follows: pH 7.11 (Tu Shui is than 1:
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) sieve, for potting simulation test.
For trying plant
Peanut (rich to spend No. 3) is purchased from Shandong Academy of Agricultural Sciences.
Strains tested
Bacillus megaterium (BaCillus megaterium) bacterial strain PP84 (deposit number: CGMCC No.12798), bud
1.0~1.2 × 1.5~2.0 microns of spore, ellipse, middle life or secondary end are given birth to.
Culture medium
Beef extract-peptone slant medium: weighing beef extract 3g, peptone 5g, and agar 18g is settled to deionized water
1000ml, 121 DEG C of sterilizing 20min.
Dried beef peptone fluid nutrient medium: weighing beef extract 3g, and peptone 5g is settled to 1000ml with 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 and carries out activation culture at 30 DEG C
For 24 hours, it is accessed in beef extract-peptone fluid nutrient medium with one ring of oese picking, 180r/min, 30 DEG C of culture 16h are as huge
Bacterium anthracoides fermentation seed liquid.Seed liquor is inoculated into wheat bran medium according to the inoculum concentration by 5.0% in 28 DEG C of conditions
Lower solid fermentation 4d, stirring, obtains mixture；
The mixture that the step obtains is continued into 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
It is packed into the soil sample 21.0kg for crossing 2mm sieve after air-drying in every basin, distilled water is added to water-retaining quantity among field of soil, keeps soil
Earth water content starts to sow after balancing 1 week.Full seed is chosen, is taken out after impregnating seed 10min with 1% liquor natrii hypochloritis
The consistent cave of depth has been dug after cleaning seed 5~7 times with deionized water, in the soil in basin in advance, has uniformly sowed 6 kinds
Then soil is covered on seed by son.Spray deionized water 20mL.Thinning when plant grows 2 true leaves.Guarantee every 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.Simultaneously peanut florescence to blade spraying concentration be 30mgL-1's
LaCl3Solution sprays 30mL/ basin every time, sprays within every 10 days once, continuous spraying 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 being rinsed with deionized water, 105 DEG C of water-removings
20min, 70 DEG C dry to constant weight, and crushing sieves with 100 mesh sieve.
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 and carries out activation culture at 32 DEG C
16h is accessed in beef extract-peptone fluid nutrient medium, 190r/min with one ring of oese picking, 29 DEG C of culture 18h, as huge
Bacterium anthracoides fermentation seed liquid.Seed liquor is inoculated into wheat bran medium according to the inoculum concentration by 7.5% in 30 DEG C of conditions
Lower solid fermentation 5d, stirring, obtains mixture；
The mixture that the step obtains is continued into 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
It is packed into the soil sample 21.0kg for crossing 2mm sieve after air-drying in every basin, distilled water is added to water-retaining quantity among field of soil, keeps soil
Earth water content starts to sow after balancing 1 week.Full seed is chosen, is taken out after impregnating seed 10min with 1% liquor natrii hypochloritis
The consistent cave of depth has been dug after cleaning seed 5~7 times with deionized water, in the soil in basin in advance, has uniformly sowed 6 kinds
Then soil is covered on seed by son.Spray deionized water 20mL.Thinning when plant grows 2 true leaves.Guarantee every 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.Simultaneously peanut florescence to blade spraying concentration be 20mgL-1's
LaCl3Solution sprays 36mL/ basin every time, sprays within every 9 days once, continuous spraying 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 being rinsed with deionized water, 105 DEG C of water-removings
20min, 70 DEG C dry to constant weight, and crushing sieves with 100 mesh sieve.
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 and carries out activation culture at 30 DEG C
For 24 hours, it is accessed in beef extract-peptone fluid nutrient medium with one ring of oese picking, 170r/min, 30 DEG C of culture 16h are as huge
Bacterium anthracoides fermentation seed liquid.Seed liquor is inoculated into wheat bran medium according to the inoculum concentration by 2.5% in 28 DEG C of conditions
Lower solid fermentation 3d, stirring, obtains mixture；
The mixture that the step obtains is continued into 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
It is packed into the soil sample 21.0kg for crossing 2mm sieve after air-drying in every basin, distilled water is added to water-retaining quantity among field of soil, keeps soil
Earth water content starts to sow after balancing 1 week.Full seed is chosen, is taken out after impregnating seed 10min with 1% liquor natrii hypochloritis
The consistent cave of depth has been dug after cleaning seed 5~7 times with deionized water, in the soil in basin in advance, has uniformly sowed 6 kinds
Then soil is covered on seed by son.Spray deionized water 20mL.Thinning when plant grows 2 true leaves.Guarantee every 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.Simultaneously peanut florescence to blade spraying concentration be 40mgL-1's
LaCl3Solution sprays 24mL/ basin every time, sprays within every 11 days once, continuous spraying 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 being rinsed with deionized water, 105 DEG C of water-removings
20min, 70 DEG C dry to constant weight, and crushing sieves with 100 mesh sieve.
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 examination plant, strains tested, culture medium and the preparation of bacillus megaterium solid pharmaceutical preparation method with real
Example 6 is applied, pot experiment embodiment other operating procedures and embodiment 6 in addition to not applying bacillus megaterium solid pharmaceutical preparation 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 wherein measuring method of P and Cd are as follows: use wet digestion (dense HNO3With HClO4Volume ratio is 4:1), ICP-MS measurement
P, Cd content, digestion and continuous mode in national standard substance (GBW07603 GSV-2) be internal standard control sample analysis matter
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 fermentation liquid provided by the invention can significantly improve weight of root system and seed
Grain yield, the bacillus megaterium fermentation liquid not inactivated is more excellent compared to the effect after inactivation, and compared with comparative example 4, root system is dry
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 effect made of solution is best.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 fermentation liquid 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 fermentation liquid not inactivated is more excellent compared to the effect after inactivation, with
Comparative example 4 is compared, and Cd content 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 effect made of solution is best.Compared with comparative example 4, Cd content is low 39.06%~
40.63%, blade P content is high by 16.4%~18.18%.
The above is only a 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 are also answered
It is considered as protection scope of the present invention.
Claims (8)
- Cadmium agent drops 1. cultivating peanut, which is characterized in that including bacillus megaterium fermentation liquid or bacillus megaterium solid pharmaceutical preparation；The preparation method of the bacillus megaterium fermentation liquid, comprising the following steps:Bacillus megaterium bacterial strain is activated and expands culture, bacillus megaterium fermentation seed liquid is obtained, by the huge bud Spore bacillus fermentation seed liquor is inoculated into beef extract-peptone fluid nutrient medium by 1~3% inoculum concentration at 25~30 DEG C, revolving speed Liquid fermentation 36h~60h under conditions of 150~200r/min obtains bacillus megaterium fermentation liquid；The preparation method of the bacillus megaterium solid pharmaceutical preparation, comprising the following steps:A. bacillus megaterium bacterial strain is activated and expands culture, obtains bacillus megaterium fermentation seed liquid, will be described huge Fermentation of bacillus seed liquor is inoculated into wheat bran medium solid under the conditions of 25~30 DEG C by 2.5%~7.5% inoculum concentration Body 3~5d of fermented and cultured, stirring, obtains mixture；B., mixture obtained in step a is continued to 1.5~2.5d of fermented and cultured at 25~30 DEG C, obtains bacillus megaterium Solid pharmaceutical preparation；The bacillus megaterium is bacillus megaterium bacterial strain Bacillus megaterium PP84, and deposit number is CGMCC No.12798；It is the LaCl of 1~2g/L that the peanut drop cadmium agent, which further includes the mass concentration independently dispensed,3Solution.
- 2. application of the drop cadmium agent described in claim 1 in plantation peanut.
- 3. application according to claim 2, which is characterized in that the bacillus megaterium fermentation liquid is applied in plantation peanut Rhizosphere；The applied amount of the bacillus megaterium fermentation liquid is 16~20L/ mus.
- 4. application according to claim 3, which is characterized in that the application number of the bacillus megaterium fermentation liquid is 3 It is secondary；The time applied for the first time is 14~16d after peanut emergence, second time for applying be after peanut emergence 29~ 31d, the time that third time applies are 44~46d after peanut emergence.
- 5. application according to claim 2, which is characterized 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.
- 6. application according to claim 5, which is characterized 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.
- 7. according to application described in claim 3~6 any one, which is characterized in that in peanut florescence by LaCl3Solution spray It applies on the blade face of plantation peanut, every 9~11d is sprayed once, and continuous spraying is three times.
- 8. application according to claim 7, which is characterized in that the LaCl3The each amount of spraying of solution is 8~12L/ mus.
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| CN110305674B (en) * | 2019-07-10 | 2021-03-02 | 青岛农业大学 | Modified biochar, soil remediation agent and preparation method and application thereof |
| CN110408562B (en) * | 2019-07-12 | 2021-05-04 | 南京农业大学 | Preparation method and application of compound microbial agent for repairing cadmium-polluted soil and promoting plant growth |
| CN111592997A (en) * | 2020-01-08 | 2020-08-28 | 佛山市植宝生态科技有限公司 | Bacillus megaterium Z-101, composite microbial cadmium inhibitor thereof and preparation method |
| CN111548230A (en) * | 2020-05-12 | 2020-08-18 | 四川嘉道博文生态科技有限公司 | Ligusticum wallichii cadmium-reducing special fertilizer and application method thereof |
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