CN110079484A - A kind of bacillus subtilis and its application in agricultural production - Google Patents
A kind of bacillus subtilis and its application in agricultural production Download PDFInfo
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- CN110079484A CN110079484A CN201910451861.4A CN201910451861A CN110079484A CN 110079484 A CN110079484 A CN 110079484A CN 201910451861 A CN201910451861 A CN 201910451861A CN 110079484 A CN110079484 A CN 110079484A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/20—Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses
Abstract
The present invention relates to functional microorganism screening and applied technical fields, specifically provide one plant of new bacillus subtilis and its application in agricultural production.For the bacillus subtilis screened from calacareous soil, deposit number is CCTCC NO:M2019402.The bacterial strain has stronger enzymatic productivity, not only can effectively improve the content of available phosphorus in soil, increases crop yield, can also effectively prevent the frequently seen plants disease such as root rot, shot hole, significant effect.
Description
Technical field
The present invention relates to the screening of functional microorganism and applied technical fields, and in particular to one plant of novel bacillus subtilis
And its application in agricultural production.
Background technique
Phosphorus is nutrient necessary to plant growth, but P elements are easy to be fixed available phosphorus is caused to lack in soil,
So how to be soluble available phosphorus by insoluble phosphate-solubilizing in soil being the key that solves the problems, such as phosphorus shortage.Phosphate solubilizing microorganism can
With activating soil insoluble phosphorus, have become one of hot spot of current research.
Phosphate solubilizing microorganism (Phosphate-Solubilizing-Microorganisms, PSM) can be by utilizing itself
The phosphate transfection of slightly solubility in soil can be turned to plant available benefit by metabolite or synergistic effect with other biological
Phosphate.The type of phosphate solubilizing microorganism is relatively more in soil, including bacterium, fungi and actinomyces etc..Wherein bacterial species
It at most, mainly include bacillus (Bacillus), enterobacteria category (Enterbacter), Salmonella
(Salmonella), Erwinia (Erwinia), alcaligenes (Alcaligenes), azotobacter
(Azotobacter), pseudomonas (Pseudomonas), Agrobacterium (Agrobacterium), Serratia
(Serratia), Flavobacterium (Flavobacterium), Micrococcus (Micrococcus), rhizobium
(Bradyrhizobium), Chromobacterium (Clromobacterium), Thiobacillus (Thiobacilus), Escherichia
(Escherichia), the bacterium of Arthrobacter (Arthrobacter) etc. 16 categories.Phosphorus decomposing fungi is also the micro- life of phosphorus decomposing in soil
The important component of object, but its quantity can not show a candle to phosphate-solubilizing bacteria, and type is also relatively fewer.At present it has been reported that phosphorus decomposing fungi
It mainly include aspergillus (Aspergillus), rhizopus (Rhizopus), Penicillium (Penicillium), Fusarium
(Fusarium), Sclerotium (Sclerotium) etc., wherein studying more is aspergillus and Penicillium.In addition table is studied
Bright, mycorrhizal fungi (Arbuscular mycorrhiza) can also promote absorption and utilization of the host to soil indissoluble state phosphorus, with
Phosphate solubilizing bacteria is inoculated with the content that can significantly improve available phosphorus in soil simultaneously.
The most important purposes of phosphate solubilizing microorganism is exactly to be used as microorganism fertilizer, is applied in agricultural production.By phosphate solubilizing microorganism
Using to after soil, it can be obviously improved crop yield and quality, further increase the utilization efficiency of phosphorus in soil, moreover it is possible to help
Plant adsorbs the microelements such as calcium, magnesium, the iron of rhizosphere.Some phosphate solubilizing bacterias are quickly bred around crop rhizosphere, can inhibit it
The growth of his pathogenic microorganism plays the effect of mitigating crop disease, effectively improves crop yield.Zhao buys fine jade and will filter out
Efficient phosphate-solubilizing bacterium pseudomonas Y2 be equipped with again in conjunction with bacillus amyloliquefaciens T-5, NJN-6 it is a certain proportion of inorganization
Fertilizer develops composite microbiological fertilizer by disc granulation method, equal to tomato, eggplant, potato, corn, tobacco etc. as the result is shown
With preferable growth-promoting effect.Microbial inoculum is made in phosphate solubilizing microorganism by Feng Ruizhang etc., and field experiment is the result shows that application microbial inoculum and half
Amount phosphate fertilizer combination processing can significantly improve the biomass of oat.In addition during developing phosphate solubilizing microorganism fertilizer, Ke Yishi
The recycling of the agricultural wastes such as existing agricultural crop straw, feces of livestock and poultry.
Both at home and abroad experienced to the research of phosphate solubilizing microorganism the long period, but many kinds of due to phosphate solubilizing microorganism, very
More researchs are still not mature enough.The development of China's phosphate solubilizing microorganism product at present is still in initial stage, commercial product type phase
To less, using effect is unstable, and it is larger to promote difficulty.Therefore, the microbial resources for screening efficient phosphate-solubilizing are still mesh
The top priority of preceding phosphate solubilizing microorganism product development.
Summary of the invention
The present invention is to solve prior art problem, provides one plant of new bacillus subtilis and its in agricultural production
Using.The bacillus subtilis has stronger enzymatic productivity, not only can effectively improve soil screened from calacareous soil
The content of middle available phosphorus increases crop yield, can also effectively prevent the frequently seen plants disease such as root rot, shot hole, significant effect.
One aspect of the present invention provides a kind of bacillus subtilis LLH-3 (Bacillus subtilis LLH-3), in
On May 27th, 2019 is preserved in the China typical culture collection center of Wuhan, China Wuhan University, deposit number CCTCC
NO:M2019402.
Application of the bacillus subtilis in bio-fertilizer.
Application of the bacillus subtilis in control of plant disease.
Another aspect of the present invention provides a kind of microorganism formulation, includes above-mentioned bacillus subtilis LLH-3.
The microorganism formulation also include bacillus, pseudomonad, agrobacterium, nitrogen-fixing bacteria, rhizobium, Penicillium notatum,
Aspergillus, rhizopus, any one or two or more of combinations in streptomycete.
The viable bacteria amount of bacillus subtilis LLH-3 is at least 10 in the microorganism formulation9CFU/g。
The present invention also provides application of the mentioned microorganism preparation in bio-fertilizer.
The present invention also provides application of the mentioned microorganism preparation in control of plant disease.
The plant disease includes root rot, southern blight, powdery mildew, shot hole, gray mold, wilt disease, brown spot and fruit
Any one in maize ear rot.
Beneficial effect
The bacillus subtilis LLH-3 that the present invention screens can be by indissoluble phosphorus (Ca3(PO4)2) resolve into the effective of solubility
Phosphorus, phosphorus decomposing efficiency are up to 85%, and the bacterial strain has a stronger enzymatic productivity, fermented and cultured 28-32h, phytic acid in supernatant
Enzyme enzyme activity is up to 13.5U/mL, and cellulose enzyme activity is up to 4.04U/mL.Bacillus subtilis LLH-3 can also significantly improve rice
Quality of Seedlings, the plant height of rice seedling, stem thickness, radical, leaf age, the second leaf is long, and chlorophyll content has been respectively increased 24.1%,
27.3%, 29.3%, 30.9%, 10.3%, 10.2%.In addition, bacillus subtilis LLH-3 is to root rot, southern blight, scab
Disease and brown spot have apparent control efficiency, wherein being more than 80% to the prevention and treatment efficiency of root rot and shot hole, to southern blight
Prevention and treatment efficiency with brown spot is more than 60%, and control efficiency is significantly better than traditional chemical bactericide.
Bacillus subtilis LLH-3 provided by the invention can be used alone or have effects that phosphorus decomposing or biological and ecological methods to prevent plant disease, pests, and erosion effect with other
Antimicrobial composition improve soil environment, increase soil fertility as bio-fertilizer and biocontrol agent, prevent and treat plant Common Diseases,
And it is environmentally friendly, be conducive to the quality for promoting crops, push transformation of the traditional agriculture to the ecological agriculture, green agriculture,
Realize agricultural health, sustainable development.
Specific embodiment
The present invention is further explained combined with specific embodiments below.For specific method or material used in embodiment
Material, those skilled in the art can carry out conventional replacement according to existing technology and select on the basis of the technology of the present invention thinking
It selects, is not limited solely to the specific record of the embodiment of the present invention.Equipment selected by the present invention and reagent can be selected from commercially available any
It is a kind of.
Embodiment 1 has the screening of phosphorus decomposing functional microorganism
1, pedotheque: the calacareous soil in Shandong Province, mountain area, the Qingzhou City west and south.
2, the preparation of soil dilution liquid: weigh 0.5g pedotheque be dissolved in be made in 4.5ml sterile water 1:10 soil it is molten
Then liquid is therefrom drawn the 0.5ml soil liquid and is placed in 4.5ml sterile water, the soil liquid of 1:100 is made, in this approach class
It pushes away, prepares 1:106-107Soil dilution solution.
0.1ml soil dilution solution is taken to be spread evenly across slightly solubility Phos solid medium (glucose 10g, (NH4)2SO40.5g,NaCl 0.3g,KC1 0.3g,MgSO4·7H20 0.3g,FeSO4.7H20 0.03g,MnSO4.4H20 0.03g,
Ca3(PO4)25.0g, distilled water 1000ml, pH 7.0-7.5, agar 20g, 115 DEG C of sterilizing 30min) on, it is fallen in 30 DEG C of incubators
Culture 3 days is set, the bacterium colony grown on culture medium is observed, wherein there are 12 periphery of bacterial colonies to produce apparent color change, and has
Transparent circle generates, and is respectively designated as LLH-1, LLH-2, LLH-3 ... ..., LLH-12.
The secondary screening of 2 phosphate solubilizing microorganism of embodiment
12 plants of bacterium that 1 primary dcreening operation of embodiment is obtained are inoculated into respectively on slightly solubility Phos solid medium, 30 DEG C of cultures 3
After it, the size of periphery of bacterial colonies transparent circle is observed, as a result, it has been found that the maximum three plants of bacterium of transparent circle are respectively LLH--2, LLH--3,
LLH--10。
The maximum three plants of bacterium of above-mentioned transparent circle are inoculated into 50mL slightly solubility Phos fluid nutrient medium (glucose respectively
10g,(NH4)2SO4 0.5g,NaCl 0.3g,KC1 0.3g,MgSO4.7H20 0.3g,FeSO4.7H20 0.03g,
MnSO4.4H20 0.03g, Ca3(PO4)25.0g, distilled water 1000ml, pH 7.0-7.5,115 DEG C of sterilizing 30min) in, 30 DEG C,
200rpm is cultivated 6 days, while the liquid phosphorus decomposing culture medium of any bacterium is not added as a control group；It detects in culture solution respectively
Number of viable.
The drafting of 2.1 phosphorus standard curves
Successively draw 5mg/l phosphorus standard solution 0.0,0.2,0.4,0.8,1.6,2.0,3.2,4.0ml in test tube, so
The anti-color developing agent 2ml of molybdenum antimony is respectively added afterwards, distilled water is settled to 20ml, shakes up and stands 20min, measures absorbance under 700nm wavelength.
The concentration of phosphorus is divided into each pipe at this time: 0.00,0.05,0.10,0.20,0.40,0.50,0.80,1.00mg/l.It is with phosphorus concentration
Abscissa, absorbance are ordinate, draw phosphorus standard curve.
The measurement of 2.2 culture solution available phosphorus contents
Culture solution each 5ml, the 8000rpm of above-mentioned three plants of bacterium are aseptically taken respectively, are centrifuged 5min, are taken supernatant,
It is diluted to suitable concentration, draws 0.5ml dilution into test tube, add 5ml distilled water, add 2 drops 2,4- dinitrophenol dinitrophenolate indicator,
Add the anti-color developing agent of 2ml molybdenum antimony, be then settled to 20ml with distilled water, shakes up and stand 20min, the colorimetric under 700nm wavelength, extinction
Angle value substitutes into the available phosphorus content in standard curve calculating supernatant, and concrete outcome is shown in Table 1.
The phosphorus decomposing effect of 1 different strains of table
| Sample | Viable count (CFU/mL) | Available phosphorus content (mg/L) |
| Blank control group | 0 | 0 |
| LLH--2 | 106-107 | 332 |
| LLH--3 | 107-108 | 856 |
| LLH--10 | 107-108 | 562 |
It can be seen that three plants of bacterium that applicant screens from pedotheque from the data in table 1 and all have stronger solution
Phosphorus ability, can be by the indissoluble phosphorus (Ca in culture medium3(PO4)2) resolve into soluble available phosphorus, and significant effect.Wherein,
The phosphorus decomposing efficiency of LLH-3 bacterial strain is up to 85%, and available phosphorus content is up to 856mg/L, achieves unexpected effect.
The identification of 3 LLH-3 bacterial strain of embodiment
1) colonial morphology of LLH-3 bacterial strain:
The bacterium colony of the LLH-3 bacterial strain is flat, and rough surface is opaque, roughening after edge initial stage is smooth, and bacterium colony is presented
White is to dirty white, and gemma size is about 0.5-0.9 × 0.8-1.3 μm, ellipse or column, middle life or close middle raw；Gram
Reacting positive, catalase reaction is positive, V.P reacting positive, and Starch Hydrolysis test is positive, and indole test is positive, available
Glucose, arabinose, xylose and mannitol, 20-45 DEG C of growth temperature range, pH range 5-10.
2) molecular biology identification of LLH-3 bacterial strain:
The LLH-3 bacterial strain that above-mentioned screening obtains is identified using the method for molecular biology, measures its 16s rDNA
Sequence SEQ ID NO:1, and blast comparison is carried out in GenBank nucleic acid database.
SEQ ID NO:1 is as follows:
In conjunction with the colonial morphology and 16srDNA comparison result of LLH-3 bacterial strain, applicant determined that LLH-3 bacterial strain is withered grass bud
Spore bacillus (Bacillus subtilis) is named as bacillus subtilis LLH-3 (Bacillus subtilis LLH-3).
Applicant is on May 27th, 2019 by above-mentioned bacillus subtilis LLH-3 (Bacillus subtilis LLH-
3) it is preserved in the China typical culture collection center of Wuhan, China Wuhan University, deposit number is CCTCC NO:M2019402.
The producing enzyme vitality test of 4 bacillus subtilis LLH-3 of embodiment
1, the preparation of bacterium solution
Choose the fresh bacillus subtilis LLH-3 bacterium mud of a ring, be inoculated in 100mL LB culture medium (1000ml water, 10.0
Peptone, 5.0 yeast extracts, 5.0gNaCl, pH7.0-7.2) in, 30 DEG C, 200rpm culture 12-16h seed liquor；It will kind
Sub- liquid is seeded in bactericidal nurishing broth bouillon with 5% inoculum concentration, 30 DEG C, 200rpm culture 28-32h, microscopy gemma yield
Stop fermented and cultured at 95% or more, obtaining viable bacteria content is 108-109The fermentation liquid of cfu/ml.
2, Enzyme activity assay
By fermentation liquid under the conditions of 4 DEG C, 8000rpm is centrifuged 5min, takes supernatant, by the following method measurement fermentation respectively
Phytase and cellulose enzyme activity in supernatant.The results show that the present invention, which screens the bacillus subtilis LLH-3 obtained, to be had
Stronger enzymatic productivity, phytase activity is up to 13.5U/mL in the fermented supernatant fluid, and cellulose enzyme activity is up to 4.04U/
ML, unexpected technical results have been achieved.
(1) phytase activity measuring method
The definition of enzyme-activity unit: under conditions of 30 DEG C, pH value are 5.0, the sodium phytate for being per minute 5mg/ml from concentration
It is an enzyme activity unit U that enzyme amount required for 1 μm of ol Phos is discharged in solution.
Measuring method: taking the sodium phytate solution that 4ml concentration is 7.5mmol/L, (pH5.0 0.25mol/L acetate buffer is matched
System), it is added in colorimetric cylinder, 30 DEG C of balance 5min add 2ml and suitably dilute through pH5.0 0.25mol/L acetate buffer
And the phytase enzyme solution balanced through 30 DEG C, it mixes in 30 DEG C of accurate insulation reaction 30min.After reaction, 4ml is added to terminate
Liquid (2 parts of nitric acid solutions (nitric acid: water=1:2), 1 part of 100g/L ammonium molybdate solution, 1 part of 2.35g/L Ammonium Vanadate Solution), mix with
Terminate reaction.Then it is placed at room temperature for 10min colour developing, measures light absorption value at spectrophotometer 415nm.
Enzyme activity calculation formula:
U=(A-A0-0.0016)×F/(0.0415×30)
In formula: A is the light absorption value of sample；A0For the light absorption value of blank sample；F is total dilution times before the reaction of practical sample liquid
Number；30 be enzyme digestion reaction time, min.
(2) Cellulase Activity Measurement Methods
The definition of enzyme-activity unit: under conditions of 50 DEG C, pH value are 6.0, the methylol for being per minute 5mg/ml from concentration
Enzyme amount required for 1 μm of ol reduced sugar of degradation release is an enzyme activity unit U in sodium cellulosate solution, and reduced sugar is with glucose
Equivalent.
Measuring method: taking three test tubes that 0.5mL CMC substrate is respectively added, 50 DEG C of water-bath preheatings together with enzyme solution to be measured
5min.0.5mL prepare liquid, and timing are respectively added in the first and second test tube, reacts 15min in 50 DEG C of water-baths.Three after having reacted
1.5mL DNS reagent is respectively added in branch test tube, and always adds the enzyme solution to be measured of 0.5mL in third branch test tube.It takes out and shakes up three
After branch test tube, 5min is reacted in boiling water bath.It is rapidly cooled to room temperature, it is fixed to 5.0mL with water.It is pair with third branch test tube test solution
The absorbance that the first and second test tube test solution is surveyed under 540nm wavelength condition is impinged upon, absorbance is advisable between 0.25-0.35.Enzyme to be measured
The absolute value of the difference of the absorbance of liquid reaction solution and horizontal control enzyme solution reaction solution absorbance is no more than 0.015.
Enzyme activity X=(magnitudes/180/15/0.5 such as glucose) × n
Wherein: X --- enzyme activity unit, IU/g (mL)；
180 --- glucose is converted into micromole from microgram；
15 --- the reaction time of prepare liquid and substrate；
0.5 --- the enzyme liquid amount to be measured of reaction is added；
N --- extension rate.
Application of the 5 bacillus subtilis LLH-3 of embodiment in the experiment of spinach field production
1, place: Qingdao City Laixi spinach planting greenhouse, soil integral status are more uniform.
2, experimentation
30 test blocks are set, and each test block is the square region of 2m × 2m, and 1 meter is kept between each test block
Interval.
Experiment sets 3 groups altogether: 1. blank control group: not adding any substance；2. medium treatment group: in each test block
In press 40mL/m2Ratio uniform spray the nutrient broth medium of above-mentioned sterilizing, then by the soil of surface layer 5-10cm thickness into
Row effectively mixes；3. bacillus subtilis LLH-3 processing group: pressing 60mL/m in each test block2Ratio uniform spray it is real
Apply bacillus subtilis LLH-3 zymocyte liquid (10 described in example 48-109Cfu/ml), then the soil of surface layer 5-10cm thickness is carried out
Effectively mix.10 test blocks of each group of random selection.
1) seed treatment: with 5% sodium hypochlorite surface sterilizing spinach seed 10min, wash with distilled water 3-4 removing
30min natural drying is placed after sodium hypochlorite at normal temperature.
2) sowing and harvest: 25g spinach seed, regular watering and management are uniformly sowed in each test block, does not apply fertilizer
Material.After sowing 50 days, whole spinach is harvested, and detect the fresh weight and dry weight of each test block spinach respectively, calculate each processing
The mean fresh and average dry weight of group spinach, are compared.
3) while harvesting spinach, the soil sample of each test block is acquired respectively, using available phosphorus in Olsen method detection soil sample
Content, and be compared.
Test result shows: compared with blank control group, the mean fresh and dry weight of medium treatment group spinach mention respectively
High by 4.8% and 4.1%, available phosphorus content improves 3.2%；The mean fresh of bacillus subtilis LLH-3 processing group spinach
295.7% and 228.2% has been respectively increased than blank control group with dry weight, 290.9% He is respectively increased than medium treatment group
224.1%, available phosphorus content improves 78.5% than space management group, improves 75.3% than medium treatment group.
The above results show that the bacillus subtilis LLH-3 that the present invention screens extremely significant can increase soil fertility, thus
The yield for greatly improving long-term cropping can be used as bio-fertilizer and be widely used in agricultural production.
Application of the 6 bacillus subtilis LLH-3 of embodiment in seedling cultivation of rice
1, prepared by bacterium powder
The fresh bacillus subtilis LLH-3 bacterium mud of a ring is chosen, is inoculated in 100mL LB culture medium, 30 DEG C, 200rpm
Cultivate 12-16h seed liquor；Seed liquor is seeded in bactericidal nurishing broth bouillon with 5% inoculum concentration, 30 DEG C,
200rpm cultivates 28-32h, and microscopy gemma yield stops fermented and cultured at 95% or more；By fermentation liquid centrifugation (8000rpm,
Thallus 5min) is collected, dextrin is added and stirs and evenly mixs, vacuum freeze drying, it is 10 that bacterium amount, which is made,9-1010The withered grass gemma of CFU/g
Bacillus LLH-3 bacterium powder.
2, experimentation
(1) place: the seedling raising greenhouse of Dandong City, Liaoning Province is tested.
(2) experimental design:
1. blank control group: only with seedling medium (strengthening development in agricultural science and technology Co., Ltd purchased from Harbin Miao Sheng)；
2. bacillus subtilis CGMCC1.8886 control group: 1.5kg bacillus subtilis CGMCC1.8886 bacterium powder (109-
1010CFU/g it) is mixed with 1 ton of seedling medium even；
3. bacillus subtilis LLH-3 processing group: 1.5kg bacillus subtilis LLH-3 bacterium powder (109-1010CFU/g) with 1
Ton seedling medium is mixed even；
The management such as sowing, irrigation and fertilising of two above processing group is identical, and entire nursery stage is about 40 days.
3, experimental result and analysis
(1) rice seedling form and chlorophyll content evaluation
100 plants of rice shoots are extracted from the central randomization of each processing group seedlings nursing plate, carry out examining seedling after being rinsed well with clear water, and examine
Survey the chlorophyll content of the second leaf.It the results are shown in Table 3.
The influence that 2 bacillus subtilis LLH-3 of table grows rice seedlings strain
From the data of table 2 it is found that compared with blank control group, bacillus subtilis LLH-3 processing group provided by the invention
Rice seedling plant height, stem thickness, radical, leaf age, the second leaf is long, and chlorophyll content has been respectively increased 24.1%, 27.3%,
29.3%, 30.9%, 10.3%, 10.2%, effect highly significant.And use commercially available bacillus subtilis CGMCC1.8886
Treated rice seedling, only plant height and stem thickness have been respectively increased 10.2% and 8.9%, and radical, leaf age, the second leaf be long and leaf
Chlorophyll contents increase unobvious.To illustrate, bacillus subtilis LLH-3 provided by the invention can significantly improve educating for rice
Seedling quality, effect are substantially better than commercially available bacillus subtilis.
(2) Seedling Quality in Rice is evaluated
Hundred plant dry weights and seedling vigorous index are the important indicators of seedling quality height.
Seedling vigorous index=(stem thickness × 100 plant rice shoot complete stool dry weight)/plant height.
Hundred plant dry weight of rice seedling and nursery index of bacillus subtilis LLH-3 processing group provided by the invention compare blank
Control group has increased separately 38.56% and 58.07%, increases separately than bacillus subtilis CGMCC1.8886 control group
30.48% and 48.76%, unexpected technical results have been achieved.
7 bacillus subtilis LLH-3 of embodiment measures bacteriostasis
1, pathomycete is cultivated:
In aseptic operating platform, peanut root rot bacterium, the southern blight of the fritter of about 0.5cm × 0.5cm are taken respectively with tweezers
Bacterium, Streptomyces scabies and brown patch germ are seeded in PDA culture medium, and 28 DEG C of inversions are cultivated 3 days.
2, inoculation face-off bacterium:
It is long to when accounting for about culture dish 1/3 when pathomycete, it is being inoculated with bacillus subtilis LLH- respectively at fungi 2cm
3, the fungi not to be inoculated with bacillus subtilis LLH-3 measures after continuation is cultivated 3 days under the conditions of 28 DEG C respectively as control
The colony radius of each pathomycete calculates bacteriostasis rate.
Bacteriostasis rate=[(control fungi growth radius-processing fungi grows radius)/control fungi grows radius] ×
100%.
The results show that bacillus subtilis LLH-3 has apparent antagonism to above-mentioned four pathomycetes of cultivating peanut.Its
In, LLH-3 is most strong to the inhibiting effect of peanut root rot bacterium and Streptomyces scabies, and inhibiting rate is up to 87.4% and 81.6% respectively；
Relatively weak to the inhibiting effect of Sclerotium rolfssi and brown spot, inhibiting rate is respectively 63.3% and 59.8%.
Application of the 8 bacillus subtilis LLH-3 of embodiment in peanut disease prevention and treatment
1, place is tested:
The peanut continuous cropping field in dune ridge village after the Pingdu of Qingdao, peanut root rot, southern blight, shot hole and brown spot occur tight
Weight.
2, experimental design:
It is randomly provided test block, each test block is the rectangular region of 6m × 10m, and keeps 3 between each test block
Meter or more interval.The line-spacing of peanut is 40cm, spacing in the rows 20cm.Three parallel laboratory test areas are arranged in each experimental group.
(1) blank control group: clear water；
(2) fungicide processing group: 50% 800 times of carbendazim liquid；
(3) bacillus subtilis CGMCC1.8886 processing group: respectively in sowing time, after planting 15d, 30d, 45d be using withered
Careless bacillus LLH-3 fermentation liquid (108-109Cfu/ml) pouring root, every plant is poured 100 times of fermentation liquid about 30mL of dilution every time；
(4) bacillus subtilis LLH-3 processing group: respectively sowing time, after planting 15d, 30d, 45d utilize withered grass gemma
Bacillus LLH-3 fermentation liquid (108-109Cfu/ml) pouring root, every plant is poured 100 times of fermentation liquid about 30mL of dilution every time.
Other field management investigate incidence after 75 days, as a result as shown in Table 3-6 with normal production.
Root rot grade scale:
0 grade: equal disease-free spot on stem foot and main fibrous root；
1 grade: having a small amount of scab on stem foot and main root；
3 grades: scab is more on stem foot and main root, and lesion area accounts for the 1/4~1/2 of stem foot and the root gross area；
5 grades: scab is more and big on stem foot and main root, and lesion area accounts for the 1/2~3/4 of stem foot and the root gross area；
7 grades: scab in flakes, is formed around stem phenomenon, but root system is not dead on stem foot and main root；
9 grades: root system necrosis, plant above ground portion wilts or death.
Southern blight grade scale:
0 grade: plant is asymptomatic；
1 grade: only generating scab in basal part of stem；
2 grades: basal part of stem generates contracting symptom of hanging, and systemic symptom (withered, dead, wilting is showed below the one third of whole strain
Deng)；
3 grades: 2/3rds or less whole strains show systemic symptom；
4 grades: 2/3rds or more representation system symptoms of whole strain.
Shot hole grade scale:
0 grade: healthy plant
1 grade: occurring small scab on top tender leaf and carpopodium
2 grades: occurring small scab on tender leaf, carpopodium, stem
3 grades: tender leaf edge upsweeps, and occurs scab shape on peanut stem and carpopodium
4 grades: carpopodium, stem severe bends, plant show calcination shape
Brown spot grade scale:
0 grade: disease-free symptom；
1 grade: aggrieved blade area accounts for 1/10 or less investigation blade area；
2 grades: aggrieved blade area accounts for 1/4 or less investigation blade area；
3 grades: aggrieved blade area accounts for 1/2 or less investigation blade area；
4 grades: aggrieved blade area accounts for 1/2 or more of investigation blade area, fallen leaves.
Diseased plant rate=morbidity strain number/total strain number × 100%
Disease index=∑ (morbidity grade typical value × diseased plant number at different levels) × 100/ (fall ill generation by investigation total strain number × superlative degree
Tabular value)
Prevent and treat efficiency=[(control disease index-processing disease index)/control disease index] × 100%
3 peanut root rot control efficiency of table compares
4 peanut sclerotium rolfsii control efficiency of table compares
5 Peanut Scab control efficiency of table compares
6 cercospora brown spot of peanut control efficiency of table compares
It can be seen that bacillus subtilis LLH-3 provided by the invention to peanut root-rot from the field experiment data of table 3-6
Disease, southern blight, shot hole and brown spot have apparent control efficiency, wherein being more than to the prevention and treatment efficiency of root rot and shot hole
80%, the prevention and treatment efficiency to southern blight and brown spot is more than 60%, is significantly higher than the control efficiency of chemicals treatment group carbendazim.And
Commercially available bacillus subtilis CGMCC1.8886 has certain prevention and treatment to peanut root rot, southern blight, shot hole and brown spot
Effect, but preventing and treating efficiency is only 11.7%-13.6%, is far below bacillus subtilis LLH-3 of the present invention.
The above results show that bacillus subtilis LLH-3 provided by the invention wants the control efficiency of peanut Common Diseases
It is significantly better than traditional chemical bactericide, and environmentally friendly, is conducive to the quality for promoting crops, can be widely applied to green
In agricultural production.
Sequence table
<110>Qingdao force favour biotech inc
<120>a kind of bacillus subtilis and its application in agricultural production
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 960
<212> DNA
<213>bacillus subtilis (Bacillus subtilis)
<400> 1
ctctagggtt ttcagaggat gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa 60
accacatgct ccaccgcttt tgcgggcctc cgtcaattcc tttgagtttt agccttgcgg 120
ccgtactccc caggcggagt ggttaatgcg ttaacttcag cactaaaggg cggaaaccct 180
ctaacactta gaactcatcc tttacggcgt ggactaccag ggtatctaat cctgtttgct 240
ccccacgctt tcgcgcctca gtgtcagtta cagaccagaa agtcgccttc gccactggtg 300
ttcctccata tctctacgca tttcaccgct acacatggaa ttccactttc ctcttctgca 360
ctcaagtctc ccagtttcca atgaccctcc acggttgagc cgtgggcttt cacatcagac 420
ttaagaaacc acctgcgcgc gctttacgcc caataattcc ggataacgct tgccacctac 480
gtattaccgc ggctgctggc acgtagttag ccgtggcttt ctggttaggt accgtcaagg 540
tgccagctta ttcaactagc acttgttctt ccctaacaac agagttttac gacccgaaag 600
ccttcatcac tcacgcggcg ttgctccgtc agactttcgt ccattgcggg ggattccgtc 660
ctgctgcctc ccctaggagg ctgggccgtg tctcagtccc agtgtggccg atcaccctct 720
caggtcggct acgcatcgtt gccttggtga gccgttacct caccaactag ctaatgcgac 780
gcgggtccat ccataagtga cagccgaagc cgcctttcaa tttcgaacca tgcagttcaa 840
aatgttatcc ggtattagcc ccggtttccc ggagttaccc cagtcttatg ggcaggttac 900
ccacgtgtta ctcacccgtc cgccgctaac tcactcgagc atgctactag cttttgcccc 960
Claims (9)
1. a kind of bacillus subtilis, which is characterized in that the deposit number of the bacillus subtilis is CCTCC NO:
M2019402。
2. application of the bacillus subtilis described in claim 1 in bio-fertilizer.
3. application of the bacillus subtilis described in claim 1 in control of plant disease.
4. a kind of microorganism formulation, which is characterized in that the microorganism formulation includes bacillus subtilis described in claim 1
Bacterium.
5. microorganism formulation as claimed in claim 4, which is characterized in that the microorganism formulation also include bacillus,
Pseudomonad, agrobacterium, nitrogen-fixing bacteria, rhizobium, Penicillium notatum, Aspergillus, rhizopus, any one or two kinds in streptomycete
Or a variety of combination.
6. microorganism formulation as described in claim 4 or 5, which is characterized in that bacillus subtilis in the microorganism formulation
Viable bacteria Liang≤10 of bacterium9 CFU/g。
7. application of any microorganism formulation of claim 4-6 in bio-fertilizer.
8. application of any microorganism formulation of claim 4-6 in control of plant disease.
9. application as claimed in claim 8, which is characterized in that the plant disease be root rot, southern blight, powdery mildew,
Any one in shot hole, gray mold, wilt disease, brown spot or fruit rot.
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