CN109456915A - One seed sand good fortune bacillus strain X3 and its application - Google Patents
One seed sand good fortune bacillus strain X3 and its application Download PDFInfo
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Abstract
The present invention provides a seed sand good fortune bacillus strain X3 and its applications, belong to the new application technical field of microorganism.The strain X 3 has dissolving P capacity, and the concentration for phosphorus of degrading reaches 476.6mg/L or more, it may be difficult to which the phosphorus utilized is converted into available phosphorus, increases the content of soil available phosphorus, improves the utilization rate of fertilizer, promotes the growth and development of plant and the absorption to fertilizer；The strain X 3 generates the concentration of heteroauxin up to 34.78 μ g/mL or more simultaneously.Application of the described husky good fortune bacillus strain X3 in planting is all significantly increased to the yield and quality of cereal crops wheat and corn, oil crops peanut, industrial crops tobacco.
Description
Technical field
The invention belongs to the new application technical fields of microorganism, and in particular to a seed sand good fortune bacillus strain X3 and its answer
With.
Background technique
Sandstone area permeability is strong, and shaken cultivation is good, and aerobic microorganism activity is dominant, can promote organic matter point
Solution, the mineralization quickening of organic matter.And loosing soil, Yi Gengzuo.Soil capillarity is strong, and moisture operation is fast, there is " Evening Tide " phenomenon.
The suitable cultivated phase is also grown, Yi Limiao；But nutrient content is low, and fertilizer conservation performance is poor, the easily de- fertile early ageing of crop later period.Currently, the soil big portion
It is allocated as cultivated land utilization, utilization rate yields two crops a year up to 92.3%, general year per mu yield grain 600kg or so.It targetedly improves from now on
Measure: because soil is planted, rational use of chemical fertilizer carries out rational application of fertilizers；Multipath applying organic manure；Farm field and forest network is carried out, is being protected
Under the premise of card grain-production grows steadily, develop the industrial crops such as Chinese yam, watermelon.
Plant growth-promoting rhizobacteria (Plant GrowthPromoting Bacteria, abbreviation PGPB) is defined as in certain condition
Under be conducive to bacterium of the free living in soil, rhizosphere, root table, phyllosphere of plant growth.These bacteriums can fixed nitrogen, Soluble phosphorus,
Molten iron, and plant hormone is generated, such as auxin, gibberellin, the basic element of cell division and ethylene.In addition, they can also improve plant
Resistance, including arid, with high salt, heavy metallic poison and pesticide.There are a large amount of phosphate solubilizing microorganisms in soil, they follow phosphorus
Ring and plant growth and development play an important role, and phosphate solubilizing microorganism can improve the titanium pigment content in soil, and then increase crop
Yield.Phosphate solubilizing bacteria also can increase absorption of the root system of plant to some micronutrient elements (such as zinc, copper etc.), enhance plant pair
The resilience of disease.Based on slightly solubility Phos in soil can be made to dissolve, phosphate solubilizing bacteria is widely used in Inoculant, to increase phosphorus
Uptake and crop yield.
Plant hormone be plant cell receive the induction of specific environment signal generate, low concentration when adjustable plant physiology it is anti-
The active material answered.In cell division and elongation, tissue and Organ Differentiation, bloom with solid, mature with aging, suspend mode and sprouting
And excised cotyledon etc., regulate and control the growth and development and differentiation of plant respectively or mutually coordinated.The kind of plant hormone
Class has very much, auxin (auxin), gibberellin (GA), the basic element of cell division (CTK), abscisic acid (abscisic acid, ABA),
Ethylene (ethylene, ETH) and rape element sterol (brassinosteroid, BR).In order to promote plant growth, some plants
Hormone then relies on plant itself secretion, some plant hormones can be by artificial synthesized, such as heteroauxin.Although artificial synthesized
Heteroauxin expands its source, but the mode sprayed, and content etc. needs strict control, and application is relatively complicated, limits Yin
The application range of indolylbutyric acid.Although with plant symbiosis effect rhizosphere bacterium be able to solve the problem, there is presently no about
Not only had the function of phosphorus decomposing but also there is the report for the rhizosphere bacterium for producing plant hormone.
Summary of the invention
In view of this, the purpose of the present invention is to provide a seed sand good fortune bacillus strain X3 and its application, the strain X 3
Not only has the function of stronger phosphorus decomposing, moreover it is possible to generate heteroauxin.The strain X 3 is applied in crop planting can not only be compared with
Big degree improves crop yield, moreover it is possible to improve the resistance such as the disease-resistant, drought resisting of plant.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides seed sand good fortune bacillus strain X3, a Bacillussafensis, the preservation of the strain X 3 is compiled
Number be CGMCCNo.12313.
The present invention provides application of the husky good fortune bacillus strain X3 in planting.
Preferably, the method for the planting, comprising the following steps:
(1) the husky good fortune bacillus strain X3 is expanded into culture, obtains the strain X 3 for expanding culture；
(2) entering field planting for 3 kinds of the strain X for expanding culture has in the soil of plant, keeps soil moisture content to field
The 50%~60% of maximum water-holding capacity.
Preferably, expand culture culture medium in the step (1), include following quality in every 1000ml PKO culture medium
The component of percentage composition: 1% carbon source and 0.05%~0.1% nitrogen source；The pH value for expanding culture culture medium is 5~8.
Preferably, carbon source described in the step (1) includes one in glucose, sucrose, mannitol, maltose and xylose
Kind is a variety of；
The nitrogen source includes one of ammonium nitrate, potassium nitrate, peptone, glutamic acid, yeast powder and ammonium sulfate or a variety of；
The inorganic salts include sodium chloride, potassium chloride, tricalcium phosphate, ammonium sulfate, bitter salt, manganese sulfate and seven water
Close one of ferrous sulfate or a variety of.
Preferably, the time for expanding culture in the step (1) is 20~60h.
Preferably, shaken cultivation is carried out during expanding culture in step (1) described in the step (1)；The oscillation training
Feeding revolving speed is 180~220rpm；
The splendid attire volume of culture medium is 25~75ml/250ml conical flask when the shaken cultivation.
Preferably, the microbial inoculum type for expanding the strain X 3 of culture in the step (2) includes bacterium aqua or microbial bacterial agent；
The inoculum concentration of the bacterium aqua is 1~9 × 108CFU/g soil.
Preferably, the concentration for expanding the strain X 3 of culture in the microbial bacterial agent is 1~9 × 1011CFU/g；
The amount of application of the microbial bacterial agent is 35~45kg/hm2。
Preferably, the plant includes crops；The crops include cereal crops, industrial crops or oil crops.
The present invention provides seed sand good fortune bacillus strain X3, a Bacillussafensis, the preservation of the strain X 3 is compiled
Number be CGMCCNo.12313.The strain X 3 has dissolving P capacity, and the concentration for phosphorus of degrading reaches 476.6mg/L or more, it may be difficult to benefit
Phosphorus is converted into available phosphorus, increases the content of soil available phosphorus, improves the utilization rate of fertilizer, and the growth of plant is promoted to send out
It educates and the absorption to fertilizer；The concentration that the strain X 3 generates heteroauxin simultaneously directly facilitates plant up to 34.78 μ g/mL or more
Object root elongation, to increase and the chance of the contact of nutriment in soil；The content of plant Endogenous IAA can be improved；
The expression of inducing plant defense gene, raising plant is disease-resistant, the resistance such as drought resisting.
Application of the husky good fortune bacillus strain X3 provided by the invention in planting.The strain X 3 is applied to
In the planting of crop field, have to the yield and quality of cereal crops wheat and corn, oil crops peanut, industrial crops tobacco
It is preferable to improve.Experiments have shown that: strain X 3 is applied in wheat potting, the root system of wheat can be made thicker longer with bigger
Surface area is conducive to absorption of the wheat root to moisture and nutrient, so that wheat overground part plant height and N-P-K content are equal
Higher than control treatment.In crop yield experiment, compared with the control, the processing of strain X 3 makes wheat increase yield 88.18%, increases corn
16.8% is produced, peanut yield increasing 11.18% is made.The better-than-average cigarette ratio of tobacco has reached 68.8%, compared with the control, improves
17.87%.The index potassium chlorine ratio of cigarette quality is characterized lower than control, and sugared alkali ratio is compareed with two sugar than being above.
Biomaterial preservation information
Husky good fortune bacillus (Bacillus safensis) is preserved in Chinese microorganism strain on March 28th, 2016 and protects
Administration committee's common micro-organisms center is hidden, unit abbreviation CGMCC, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica, deposit number CGMCCNo.12313, strain number X3.
Detailed description of the invention
Fig. 1 is the bacterium colony figure for being strain X 3 of the present invention；
Fig. 2 is the phylogenetic tree that the 16SrDNA sequence based on X3 and related strain uses adjacent method to establish；
Fig. 3 indicates influence of the different incubation times to 3 dissolving P capacity of strain X；
Fig. 4 indicates influence of the different pH to X3 bacterial strain dissolving P capacity；
Fig. 5 indicates influence of the different ventilatory capacities to X3 bacterial strain dissolving P capacity；
Fig. 6 indicates influence of the different carbon source to X3 bacterial strain dissolving P capacity；
Fig. 7 indicates influence of the different nitrogen sources to X3 bacterial strain dissolving P capacity.
Specific embodiment
The present invention provides seed sand good fortune bacillus strain X3, a Bacillussafensis, the preservation of the strain X 3 is compiled
Number be CGMCCNo.12313.
In the present invention, the colonial morphology of the strain X 3 is as follows: bacterium colony is white, and middle part is swelled and edge is complete, table
Face is smooth but transparent, and thallus is in elongated rod shape.The physio-biochemical characteristics of the strain X 3 are: gram-positive bacteria, Starch Hydrolysis
It is positive, amphimicrobian type bacterium, catalase test, V-P test, gelatin liquefaction and nitrate reduction are presented the positive, and first
The red reaction of base and citrate utilize test presentation negative reaction.
In the present invention, the strain X 3 passes through form and 16S rDNA Molecular Identification, and the strain X 3 is as the result is shown
Husky good fortune bacillus safensis.
In the present invention, the ability of phosphate solubilizing bacteria X3 decomposition Phos is strong, reaches 476.6mg/L or more.It is described simultaneously
Phosphate solubilizing bacteria X3 have the ability of secretion heteroauxin (IAA), up to 34.78 μ g/mL or more.
The present invention provides application of the husky good fortune bacillus strain X3 in planting.
In the present invention, the method for the planting, preferably includes following steps:
(1) the husky good fortune bacillus strain X3 is expanded into culture, obtains the strain X 3 for expanding culture；
(2) strain X 3 for expanding culture is inoculated with field planting has in the soil of plant, keeps soil moisture content to field
Between maximum water-holding capacity 50%~60%.
In the present invention, the expansion culture culture medium includes following quality in preferably every 1000ml PKO culture medium
The component of percentage composition: 1% carbon source and 0.05%~0.1% nitrogen source；The pH value for expanding culture culture medium is preferably 5~
8, more preferably 6.
In the present invention, carbon source described in the step (1) preferably includes glucose, sucrose, mannitol, maltose and wood
One of sugar is a variety of, more preferably glucose, sucrose or maltose；The nitrogen source preferably includes ammonium nitrate, potassium nitrate, egg
One of white peptone, glutamic acid, yeast powder and ammonium sulfate are a variety of, more preferably peptone or yeast powder.The inorganic salts are excellent
Choosing includes one in sodium chloride, potassium chloride, tricalcium phosphate, ammonium sulfate, bitter salt, manganese sulfate and green vitriol
Kind is a variety of.The present invention is not particularly limited the source of the carbon source, nitrogen source and inorganic salts, using those skilled in the art institute
The source of well known above-mentioned carbon source, nitrogen source and inorganic salts.
In the present invention, the time for expanding culture is preferably 20~60h, more preferably 50~56h.The expansion training
Feeding temperature is preferably 28~33 DEG C, and more preferably 30 DEG C.Shaken cultivation is preferably carried out during the expansion culture；The oscillation
The revolving speed of culture is 180~220rpm；More preferably 200rpm.The splendid attire volume of culture medium is preferably 25 when the shaken cultivation
~75ml/250ml conical flask, more preferably 50ml/250ml conical flask.
In the present invention, the microbial inoculum type of the strain X 3 for expanding culture includes bacterium aqua or microbial bacterial agent.It is described
The inoculum concentration of bacterium aqua is preferably 1~9 × 108CFU/g soil, more preferably 5 × 108CFU/g soil.The microbial bacterial agent
3 thallus of strain X and carrier including expanding culture.The concentration for expanding the strain X 3 of culture in the microbial bacterial agent is preferably 1
~9 × 1011CFU/g, more preferably 5 × 1011CFU/g.The present invention is not particularly limited the type of the carrier, using this
The carrier of microbial bacterial agent known to field.In embodiments of the present invention, the type of the carrier is bone meal.The present invention
The preparation method of the microbial bacterial agent is not particularly limited, using the preparation method of microbial bacterial agent known in the art
?.The amount of application of the microbial bacterial agent is preferably 35~45kg/hm2, more preferably 40kg/hm2。
In the present invention, the plant preferably includes crops.The crops preferably include cereal crops, industrial crops
Or oil crops.The cereal crops include wheat, millet, corn or sorghum etc..The industrial crops include tobacco, cotton,
Vegetables or fruit tree etc..Oil crops include peanut or rape etc..
Seed sand good fortune bacillus strain X3 provided by the invention and its application are carried out specifically below with reference to embodiment
It is bright, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
1, the configuration of culture medium
Prepare following several culture mediums:
(1) PKO fluid nutrient medium (inorganic phosphorus bacteria culture medium)
Sodium chloride 0.3g, glucose 10g, potassium chloride 0.3g, tricalcium phosphate 5g, ammonium sulfate 0.5g, bitter salt
0.3g, manganese sulfate 0.03g, green vitriol 0.03g, distilled water 1000mL adjust pH value and go out to 7.0~7.2,115 DEG C
Bacterium 30min.In culture solution plus 15g agar powder is PKO solid medium.
(2) LB liquid medium
Tryptone 10g, yeast powder 5g, sodium chloride 10g, water 1000mL adjust pH value to 7.0,121 DEG C of sterilizing 20min.
In culture solution plus 15g agar powder is LB solid medium.
The plant-growth promoting rhizobacteria that can secrete heteroauxin is filtered out by qualitative determination and quantitative determination again below.
Table 1 is for trying soil labile organic matter
2, qualitative determination
Microbionation after isolating and purifying from above-mentioned sandstone area (table 1) contains L-Trp (100mg/ in use
L LB liquid medium), 30 DEG C, 180rmin-1Shaking table culture 1d.Take 50 μ L bacterium aquas drop on whiteware plate, simultaneously
Add 50 μ L Salkowski color solution (50mL35%HClO4+1mL 0.5M FeCl3).50 μ L 50mg/L heteroauxins will be added
Color solution as positive control.Whiteware plate is observed after room temperature avoid light place 30min, and the color person of reddening indicates to divide
Secrete heteroauxin.
3, it quantitative determines
The bacterium for the producing IAA that primary dcreening operation obtains is quantitative determined, condition of culture is same as above.It is surveyed first with spectrophotometry
Determine the OD of bacterium aqua600Value, then by bacterium aqua with 10000rmin-1It is isometric that centrifugation 10min takes supernatant to be added
Salkowski color solution is protected from light and stands 30min, measures its OD530Value.Calculate bacteria concentration OD600When value is 1, unit volume fermentation
The content of heteroauxin in liquid.The drafting of standard curve is prepared using analytically pure heteroauxin gradient dilution.The result shows that institute
The bacterial strain for stating separation has producing IAA ability, reaches 34.78 μ gmL-1。
The obtained screening test for producing IAA bacterium and carrying out phosphorus decomposing situation, strains tested is inoculated in and fills 50mL slightly solubility
The 250mL triangular flask of Phos fluid nutrient medium, 30 DEG C, 200rmin-1After cultivating 72h, the culture solution 10mL of culture 72h is taken,
6000r/min is centrifuged 20min, and supernatant ultraviolet specrophotometer is taken to measure the content of wherein phosphorus.As a result the bacterial strain decomposes
The ability of Phos is strong, reaches 476.6mg/L.
The Strain Designation that above method screening is isolated is X3, according to Wan Bingbing (Wan Bingbing .2016, one plant of corn
The screening and identification and effect study of the multi-functional Promoting bacteria of rhizosphere) et al. report carry out 16SrDNA Molecular Identification, through Beijing U.S. hundred million
U.S. bioengineering Co., Ltd sequencing, according to the sequencing result of 16S rDNA, exists in http://www.ncbi.nlm.nih.gov
Line query analysis carries out tetraploid rice with other 16S rDNA sequences in GenBank using Blast software, selects phase
The 16S rDNA systematic evolution tree (Fig. 2) of the sequence of close sequence and X3 3 software building X3 of MEGAversion.
Embodiment 2
Aerobic test
Sterilized LB culture medium is poured into 3 sterilized test tubes, about at 2/3, on aseptic operating platform, is used
The strain X 3 of transfer needle picking inclined-plane culture, percutaneous puncture-inoculation (must be punctured to tube bottom) into above-mentioned culture medium.30 DEG C of cultures, point
Not in 3 days to 7 days observation results.The raw elder preferably oxygen bacterium on agar column surface, such as along the raw elder of puncture line for anaerobic bacteria or
Facultative anaerobic bacteria.
Test result performance, 3 bacterium colony of strain X are grown along agar column surface, also have bacterium colony growth in puncture line, are detested to be facultative
Oxygen (is shown in Table 2).
Embodiment 3
The measurement of catalase
The 1 drop 3%H of drop on clean slide2O2, 18 are taken~1 ring of strain X 3LB slant culture cultivated for 24 hours, in H2O2
Middle smearing is the positive if having bubble generation, is otherwise feminine gender.
Test result shows that strain X 3 is contact enzyme positive (being shown in Table 2).
Embodiment 4
Methyl red test (M.R test)
A. culture medium and reagent: peptone 5g, glucose 5g, sodium chloride 5g, distilled water 1000mL adjust pH value to 7.0
~7.2, test tube is dispensed, every pipe fills 4~5mL, 121 DEG C of sterilizing 20min.Reagent: methyl red 0.1g, 95% alcohol 300mL, distillation
Water 200mL.
B. in above-mentioned culture solution, 30 DEG C are cultivated l-2 days by Spawn incubation and result observation inoculating strain X3.In culture solution
A few drop methyl red reagents are added, are that methyl red is positive if red is presented in culture solution, yellow is negative (methyl red color change interval 4.4
Red -6.0 yellow).
Test result shows that strain X 3 is M.R negative (being shown in Table 2).
Embodiment 5
Second phthalein carbinol methine tests (VP test)
A. the same methyl red test of culture medium culture medium.
B. Spawn incubation and result observation inoculation and the same methyl red test of culture.When doing VP test, culture solution (about 2mL) is taken
It is mixed with the 40%NaOH solution of equivalent, adds a small amount of creatine, after sufficiently vibrating 2~5min, if red occurs in culture solution, as
VP is positive.
Test result shows that strain X 3 is VP positive (being shown in Table 2).
Embodiment 6
Starch Hydrolysis test
A. culture medium and reagent add 0.2% soluble starch in meat soup peptone agar, dispense triangular flask, and 121
DEG C sterilizing 20min it is spare.Road Ge Shi iodine solution: crystalline flake of iodine 1g, potassium iodide 2g first dissolve potassium iodide with a small amount of (3-5mL) distilled water, existing
The crystalline flake of iodine is added and is diluted with water to 300mL after iodine is completely dissolved.
B. Spawn incubation and result observation take X3 strain point to be connected on plate, and 30 DEG C are cultivated 2~4 days, after forming bacterium colony,
Ge Shi iodine solution in road is added dropwise on plate, to be paved with periphery of bacterial colonies as degree, plate is blue, and periphery of bacterial colonies is irised out if any colorless and transparent
It is existing, illustrate that starch has been hydrolyzed.The size of the size general remark hydrolysis starch ability of transparent circle.
Test result shows that strain X 3 is that Starch Hydrolysis is positive (being shown in Table 2).
Embodiment 7
Gelatin hydrolysis test
A. culture medium and reagent peptone 5g, gelatin 120g, distilled water 1000mL.PH value is adjusted to 7.2~7.4, is dispensed
Test tube, culture medium height are about 4~5cm, 121 DEG C of sterilizing 20min.
B. Spawn incubation and result observation are entreated in test tube with puncture method inoculating strain X3.One is cultivated in 30 DEG C of incubators
Month, whether observation gelatin liquefies.
Test result shows that strain X 3 is gelatin liquefaction positive (being shown in Table 2).
Embodiment 8
Nitrate reduction test
A. culture medium and reagent nitrate fluid nutrient medium: peptone 10g, KNO31g, distilled water 1000mL adjust pH value
To 7.0~7.4.Ge Lisishi (Gries) reagent: A liquid: p-aminobenzene sulfonic acid 0.5g, spirit of vinegar (10% or so) 150mL；B
Liquid: a- naphthols 0.1g, distilled water 20mL, spirit of vinegar (10% or so) 150mL.Diphenylamines reagent: diphenylamines 0.5g is dissolved in l00mL
In the concentrated sulfuric acid, diluted with 20mL distilled water.
B. strain X 3 is inoculated in nitrate fluid nutrient medium by Spawn incubation and result observation, and 30 DEG C are cultivated 1,3,5 day.
A little culture solution is poured into white porcelain dish aperture, then drop 1 drips reagent A and B liquid respectively wherein, when culture solution becomes pink
Whens color, rose, orange or brown etc., nitrite presence is indicated, be that nitrate reduction is positive, be otherwise feminine gender.
Test result bacterial strain shows that X3 is that nitrate reduction is positive (being shown in Table 2).
Embodiment 9
The utilization of citrate
A. culture medium and reagents citric acid sodium 2g, NaCl 5g, MgSO4·7H2O 0.2g, (NH4)2·HPO41g, 1% bromine
Thymol blue aqueous solution 10mL, agar 20g, distilled water 1000mL adjust pH value to 6.8~7.0,121 DEG C of sterilizing 20min.
B. Spawn incubation and result observation take young age (culture 18~for 24 hours) X3 strain to be inoculated on inclined-plane, and 30 DEG C of cultures 3~
7 days, culture medium was in that alkaline (blue) person is positive reaction, and constant person is then negative.
The test result that citrate utilizes shows that strain X 3 is negative (being shown in Table 2).
The physio-biochemical characteristics of 2 X3 bacterial strain of table
It can be seen from the above, isolated X3 bacterial strain is in Gram-positive, bacteria colony white, protuberance, edge is complete, and surface is smooth,
Bacterium colony is opaque.Aerobic or amphimicrobian.Optimum pH is 6, and nitrate reduction is positive.It is special according to the Physiology and biochemistry of the bacterial strain
Sign, is accredited as husky good fortune bacillus (Bacillussafensis).By the bacterial strain on March 28th, 2016 in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center preservation, deposit number CGMCCNo.12313.
Embodiment 10
Qualitative screening is carried out to strain X 3 and quantitative determination can get the phosphate solubilizing bacteria with phosphorus decomposing function
(1) qualitative determination: suitable gradient will be taken to be applied on PKO plate after strains tested X3 sterile water gradient dilution, set
In 30 DEG C of incubator cultures, transparent circle bacterium colony to appear is phosphate solubilizing bacteria, and phosphate solubilizing bacteria is saved after purification.Strain X 3 occurs transparent
Bacterium colony is enclosed, illustrates that strain X 3 provided by the invention is phosphate solubilizing bacteria.
(2) it quantitative determines: strain X 3 being inoculated in the triangular flask for the PKO fluid nutrient medium for filling 50mL, be placed in 30 DEG C
Culture solution is transferred in centrifuge tube by shaking table after 180r/min cultivates 96h, and 10000r/min is centrifuged 15min at 4 DEG C, by supernatant
Liquid is collected, with the content of available phosphorus in molybdenum blue colorimetric method measurement fermentation liquid.The Phos being difficult to be utilized can be converted to available
Phosphorus, there is solution Phos and organic phosphorus ability.As a result the ability of the bacterial strain decomposition Phos is strong, reaches 476.6mg/L.
Tobacco rhizosphere Promoting bacteria YC8, taxonomic identification are disclosed according to the Chinese patent application No. is 201410761484.1
For bacillus megaterium (Bacillus megaterium).3 taxonomic identification of strain X provided by the invention is husky good fortune bacillus
(Bacillus safensis) belongs to bacillus with bacterial strain YC8, but the dissolving P capacity of strain X 3 reaches 476.6mg/L
More than, the dissolving P capacity (YC8 reaches 163.16mg/L to the inversion quantity of tricalcium phosphate) compared to bacterial strain YC8 has obvious excellent
Gesture.
Embodiment 11
In order to further verify the phosphate solubilizing bacteria X3 dissolving P capacity and optimum condition that embodiment 10 obtains, below for different carbon
Source, different nitrogen sources, different shaken cultivation amounts, difference pH, different incubation times explore the influence to dissolving P capacity.
(1) different incubation times are to the growth of strain X 3 and the influence of dissolving P capacity
Strain X 3 is linked into PKO culture medium and carries out shaken cultivation under the conditions of 30 DEG C, the bacterial strain at each time point is taken
Sample measures the capacity of decomposition to phosphorus.
From the figure 3, it may be seen that the bacterial strain in 0~10h of strain X 3 is in growth phase therefore the dissolving P capacity of bacterial strain is not strong, phosphorus contains
It measures lower and increasess slowly.15~20h enters logarithmic phase, gradually increases to the capacity of decomposition of phosphorus, within the period of 20~36h
Phosphorus content enters plateau, and unit incrementss are reduced, and phosphorus content continues growing after 36h.
(2) difference pH is to the growth of strain X 3 and the influence of dissolving P capacity
PKO culture medium is respectively adjusted to different pH (4,5,6,7,8,9,10), 50mL is taken to be loaded on the triangular flask of 250mL
In, by the inoculation of 1% (v/v) inoculum concentration after the X3 of logarithmic growth phase, 30 DEG C are placed in, 180rmin-1Shaking table culture for 24 hours, is pressed
The content of the method measurement available phosphorus of quantitative determination, as a result as shown in figure 4, the optimal pH of the most strong dissolving P capacity of the strain is 6 left
Right slightly sour environment.
(3) different ventilatory capacities are to the growth of strain X 3 and the influence of dissolving P capacity
PKO fluid nutrient medium is pressed into 25ml, 50ml, 75ml, 100ml, 150ml are loaded in the triangular flask of 250mL, by 1%
(v/v) inoculum concentration inoculation is placed in 30 DEG C, 180rmin after the X3 of logarithmic growth phase-1Shaking table culture for 24 hours, presses quantitative determination
Method measurement available phosphorus content amount.As a result as shown in figure 5, ventilatory capacity influences bacterium since strain X 3 is amphimicrobian metabolism
The ability of strain phosphorus decomposing, when 50mL liquid amount, bacterial strain dissolving P capacity is most strong, and later as liquid amount is reduced, available phosphorus content is reduced
Illustrate that its dissolving P capacity is gradually reduced.
(4) different carbon source is to the growth of strain X 3 and the influence of dissolving P capacity
The carbon source of 1% (w/v) is separately added into PKO culture medium (being free of glucose), carbon source has glucose, xylose, sugarcane
Sugar, fructose, mannitol, lactose, maltose take 50ml loaded in the triangular flask of 250ml, are in by the inoculation of 1% (v/v) inoculum concentration
After the X3 of logarithmic growth phase, 30 DEG C are placed in, 180rmin-1Shaking table culture measures available phosphorus for 24 hours, by the method for quantitative determination
Content.As a result as shown in fig. 6, the bacterial strain is when supplying maltose, dissolving P capacity is most strong, followed by sucrose.
(5) different nitrogen sources are to the growth of strain X 3 and the influence of dissolving P capacity
The nitrogen source of 0.1% (w/v) is separately added into PKO culture medium (not including ammonium sulfate), nitrogen source includes ammonium nitrate, sulphur
Sour ammonium, potassium nitrate, peptone, yeast powder, alanine, urea etc. take 50ml to be loaded in the triangular flask of 250ml and connect by 1% (v/v)
Kind amount inoculation is placed in 30 DEG C, 180rmin after the X3 of logarithmic growth phase-1Shaking table culture for 24 hours, by the method for quantitative determination
Measure available phosphorus content.As a result as shown in fig. 7, available phosphorus content is most, followed by albumen when illustrating that taking yeast powder is nitrogen source
Peptone.
Embodiment 12
Strain X 3 of the present invention has obvious growth promoting function to wheat, is illustrated below by pot experiment.
It takes for examination soil about 200g loaded in basin alms bowl, testing wheat breed used is Zheng wheat 9023, and wheat surface is disappeared
Poison, it is then multiple with sterile water repeated flushing, vernalization 2 days, the consistent seed that germinates was chosen by 4 Wheat Species of every basin in basin,
Final singling is to 3 after one week.Bacterium aqua will be made after strains tested X3 culture, according to 108The inoculum concentration of CFU/g is inoculated into soil
In, it is control with not inoculating strain, 4 repetitions of each processing keep water content to the 60% of maxmun field capacity, by potting
It is placed in illumination cultivation room.30 days post-samplings, wheat root obtain image with root scanner (LA1600+scanner, Canada)
Afterwards, related root index is analyzed with root system analysis software (Winrhizo2003b, Canada), and it is basic to measure soil
Physicochemical property and Nitrogen and phosphorus content.The result shows that strain X 3 is applied in wheat potting, the root system of wheat can be made thicker
It is longer that there is bigger surface area (table 3), be conducive to absorption of the wheat root to moisture and nutrient, so that wheat overground part strain
High and N-P-K content is above control treatment (table 4).
Influence of 3 strain X 3 of table to wheat potting root system
Influence of 4 strain X 3 of table to wheat potted plant
Embodiment 13
Microbial inoculum is made by carrier of bone meal in strain X 3, is 10 containing living bacteria count11CFU/g carries out field experiment
The effect of verifying X3, respectively at the plantation time plantation cereal crops wheat and corn that various crops are suitable for, oil crops peanut,
Industrial crops tobacco.Test is respectively provided with 3 repetitions, random district's groups arrangement, 6 × 3m of plot area2.15- is applied in all processing
The compound fertilizer of 15-15 makees base manure, amount of application 600kghm-2, X3 microbial bacterial agent 40kghm is administered simultaneously-2, at control
Reason application equivalent bone meal, in harvest time measurement yield and the corresponding index of quality.
The result shows that compared with the control, the processing of strain X 3 makes wheat increase yield 87.85% (table 5), make corn yield increasing
16.55% (table 6) makes peanut yield increasing 10.89% (table 7).The better-than-average cigarette ratio of tobacco has reached 65.1%, compared with the control,
Improve 11.86% (table 8).The index potassium chlorine ratio of cigarette quality is characterized lower than control, and sugared alkali ratio and two sugar ratios are above pair
According to (table 9).
Influence of 5 X3 of table to Yield Traits of Wheat
Influence of 6 X3 of table to corn yield and its Components
Influence of 7 X3 of table to peanut yield and its Components
Influence of 8 X3 of the table processing to yield of flue-cured tobacco character
Influence of 9 X3 of table to tobacco middle leaf routine chemical components
As can be seen from the above embodiments, application of the strain X 3 provided by the invention in planting, can significantly improve plant
The speed of growth of object improves the quality of crop yield and product.
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 (10)
1. the bacillus strain of seed sand good fortune an X3, Bacillus safensis, which is characterized in that the deposit number of the strain X 3
For CGMCC No.12313.
2. application of the sand good fortune bacillus strain X3 described in claim 1 in planting.
3. application according to claim 2, which is characterized in that the method for the planting, comprising the following steps:
(1) the husky good fortune bacillus strain X3 is expanded into culture, obtains the strain X 3 for expanding culture；
(2) strain X 3 for expanding culture is inoculated with field planting and had in the soil of plant, keep soil moisture content to field most
The 50%~60% of big water-holding capacity.
4. application according to claim 3, which is characterized in that expand culture culture medium in the step (1), often
It include the component of following mass percentage: 1% carbon source and 0.05%~0.1% nitrogen source in 1000ml PKO culture medium；It is described
The pH value for expanding culture culture medium is 5~8.
5. application according to claim 3, which is characterized in that carbon source described in the step (1) includes glucose, sugarcane
One of sugar, mannitol, maltose and xylose are a variety of；
The nitrogen source includes one of ammonium nitrate, potassium nitrate, peptone, glutamic acid, yeast powder and ammonium sulfate or a variety of；
The inorganic salts include sodium chloride, potassium chloride, tricalcium phosphate, ammonium sulfate, bitter salt, manganese sulfate and seven hydration sulphur
One of sour ferrous iron is a variety of.
6. application according to claim 3, which is characterized in that the time for expanding culture in the step (1) is 20~
60h。
7. according to application described in claim 3~6 any one, which is characterized in that during expanding culture in the step (1)
Carry out shaken cultivation；The revolving speed of the shaken cultivation is 180~220rpm；
The splendid attire volume of culture medium is 25~75ml/250ml conical flask when the shaken cultivation.
8. application according to claim 3, which is characterized in that expand the microbial inoculum of the strain X 3 of culture in the step (2)
Type includes bacterium aqua or microbial bacterial agent；
The inoculum concentration of the bacterium aqua is 1~9 × 108CFU/g soil.
9. application according to claim 8, which is characterized in that expand the dense of the strain X 3 of culture in the microbial bacterial agent
Degree is 1~9 × 1011CFU/g；
The amount of application of the microbial bacterial agent is 35~45kg/hm2。
10. the application according to any one of claim 2~6,8 and 9, which is characterized in that the plant includes farming
Object；The crops include cereal crops, industrial crops or oil crops.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154669A (en) * | 2019-11-25 | 2020-05-15 | 根力多生物科技股份有限公司 | Bacillus safensis and application thereof |
| WO2022120503A1 (en) | 2020-12-08 | 2022-06-16 | Blanes Spa | Agricultural formulation comprising at least one bacterial strain b. safensis rgm 2450, and/or a bacterial strain b. siamensis rgm 2529 and agricultural excipients; use of the formulation and method for stimulating growth and/or increasing crop yields and/or protecting crops against diseases and pests |
| CN114836335A (en) * | 2021-11-11 | 2022-08-02 | 施可丰化工股份有限公司 | Bacillus safensis T1-5 and application thereof |
| CN117887634A (en) * | 2024-02-29 | 2024-04-16 | 昆明诺沃医学检验实验室有限公司 | Bacterium with growth promoting effect and application thereof |
-
2018
- 2018-11-20 CN CN201811381569.1A patent/CN109456915B/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154669A (en) * | 2019-11-25 | 2020-05-15 | 根力多生物科技股份有限公司 | Bacillus safensis and application thereof |
| CN111154669B (en) * | 2019-11-25 | 2022-04-05 | 根力多生物科技股份有限公司 | Bacillus safensis and application thereof |
| WO2022120503A1 (en) | 2020-12-08 | 2022-06-16 | Blanes Spa | Agricultural formulation comprising at least one bacterial strain b. safensis rgm 2450, and/or a bacterial strain b. siamensis rgm 2529 and agricultural excipients; use of the formulation and method for stimulating growth and/or increasing crop yields and/or protecting crops against diseases and pests |
| CN114836335A (en) * | 2021-11-11 | 2022-08-02 | 施可丰化工股份有限公司 | Bacillus safensis T1-5 and application thereof |
| CN114836335B (en) * | 2021-11-11 | 2023-09-05 | 施可丰化工股份有限公司 | Bacillus saxifragilis T1-5 and application thereof |
| CN117887634A (en) * | 2024-02-29 | 2024-04-16 | 昆明诺沃医学检验实验室有限公司 | Bacterium with growth promoting effect and application thereof |
| CN117887634B (en) * | 2024-02-29 | 2024-06-04 | 昆明诺沃医学检验实验室有限公司 | Bacterium with growth promoting effect and application thereof |
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| CN109456915B (en) | 2020-08-21 |
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