CN104928212B - Bacillus megaterium X3 and preparation method thereof, application - Google Patents

Bacillus megaterium X3 and preparation method thereof, application Download PDF

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CN104928212B
CN104928212B CN201510296589.9A CN201510296589A CN104928212B CN 104928212 B CN104928212 B CN 104928212B CN 201510296589 A CN201510296589 A CN 201510296589A CN 104928212 B CN104928212 B CN 104928212B
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bacillus megaterium
bacterium
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plant
strain
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CN104928212A (en
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蔡燕飞
张娅娅
李永涛
熊汉琴
曹禺
郭真真
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South China Agricultural University
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Abstract

The invention discloses one plant of bacillus megaterium Bacillus megaterium strain X3 and the application in terms of plant growth-promoting.The bacterial strain deposit number is CGMCC No.10803, and China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) is preserved on May 11st, 2015;The bacillus megaterium X3 of the present invention is isolated to produce heteroauxin (IAA), possesses production siderophore, Soluble phosphorus, production ammonia, salt tolerant and the sick characteristic of suppression, can remarkably promote the growth of crop, improve corn yield.Strain X 3 can also increase Soil Microorganism diversity and bacterial abundance at the same time.The culture propagation speed is fast, and simple production process, high-output stress-resistance is easy to maintain, and beneficial to industrialized production, bio-fertilizer is prepared using the bacterial strain, can not only prevent soil-borne disease but also can promote plant growth, have a extensive future.

Description

Bacillus megaterium X3 and preparation method thereof, application
Technical field
The present invention relates to a kind of microorganism and its application, and in particular to one plant of bacillus megaterium X3 (Bacillus Megateriumstrain) and preparation method thereof, application.
Background technology
Heteroauxin (IAA) is the semiochemicals for producing coordinate plant growth element, is the life of the generally existing in plant Long element, plays plant growth the effect of key.Bacterium carries not only by producing IAA also by fixed nitrogen Soluble phosphorus, production iron Body, suppress soil surface characters and increase growth-promoting functions of the number of ways such as the soil beneficial microbe arrival to plant.Related patents Document shows that bacillus megaterium SJ-7 bacterial strains have dissolving phosphor and dissolving potassium and prevent the function of fungal disease.Bacillus megaterium JX15 Possess the characteristics such as production heteroauxin, fixed nitrogen and Soluble phosphorus, it is possible to increase utilization rate of fertilizer, promotes peanut growth.Cheng Yuanyuan etc. is screened Bacillus megaterium WXD-3-1 and bacillus subtilis WXD-3-2 there is phosphorus decomposing, production IAA and thermophilic iron element ability, can promote Growth of Lettuce.Jiang Yun etc. filters out plant height production IAA (10.2mgL-1) Su Jin cloud bacillus, there is phosphorus decomposing, potassium decomposing, solid Nitrogen and production siderophore characteristic.The bacillus amyloliquefaciens GJ3 and bacillus subtilis NS178 of the screenings such as Wang Xixiang have wider 14 kinds of pathogens are had antagonistic effect by antimicrobial spectrum.It is and multi-functional to efficiently production IAA, Soluble phosphorus, antagonism, salt tolerant, production ammonia etc. at present Bacillus megaterium research not yet report.
In recent years, researches show that for bacterium using urea production ammonia, the circulation for not only directly affecting N sources in soil is sharp in soil With, and can be used for the improvement for being acidified soil property.Kingdom is emerging to wait research to show that some bacteriums have urease-producing ability, passes through urea Ammonia approach is produced, promotes the validation of Soil Nitrogen, meets that plant growth to N element nutritional needs, improves plant products and quality.Also Research shows that there is a variety of enzyme systems of bacterial secretory application value, Ma Guizhen etc. to filter out one plant of production protease ability strongerly Clothing Bacillus GD-2-2, its hydrolyzable protein are utilized for plant.Mo Qin etc., which has screened 9 plants, has starch the thin of degradation capability Bacterium is belonging respectively to native bacillus, anaerobic spore-bearing bacilli and bacillus.Chen Xin etc. filters out one plant and produces the withered of catalase Careless bacillus, and optimize its fermentation condition.The strain X 3 of this research has production ammonia characteristic, while also amylase, protease With catalase characteristic, there is good trans-utilization ability to organic matter, soil nitrogen availability can be improved, promote crop nitrogen Plain nutrition.
Many document report bacillus production IAA existing at present, Soluble phosphorus, produces siderophore, antibacterial to wait beneficial aspects, these are special Property with plant growth maintain close ties with.Xu Wensi etc. filters out one plant of production IAA content and reaches 22.6mgL-1Bacillus megaterium JX15, is applied to peanut plant, its fresh weight, plant height, full nitrogen, phosphorus, potassium and Peanut Root System total length, root surface area and tip of a root number are equal Dramatically increase.With after bacillus megaterium WXD-3-1 and bacillus subtilis WXD-3-2 processing romaine lettuce, it is planted Cheng Yuanyuan etc. Plant height, blade are wide, plant fresh weight and plant weights compared with the control respectively increase by 21.5% and 8.9%, 31.9% and 14.5%, 41.3% and 13.6%, 42.8% and 26.4%.Luo Huan etc. apply bacillus megaterium CJLC2, tomato plant root long, plant height and Fresh weight adds 17.1%, 18.0% and 15.8% respectively.Wang Mengliang etc. applies appropriate bacillus megaterium agent, promotes rape Root growth, reduces the nitrate content in rape, adds Oilseed rape biomass and yield.Khaled etc. applies gemma microbial inoculum The research of S4 shows the root long of corn and stem length adds the result table that 62% and 54%, Hamdy etc. apply growth promoting bacteria agent respectively 34% is added on the fresh weight of bright corn.Bio-bacterial manure is acted on corn by Zhu Yunna etc. so that conventional corn and corn Yield adds 18.2% and 11.6% respectively.The strain X 3 of this research is remarkably improved pakchoi, romaine lettuce and corn growth, tool There is significant plant growth-promoting superiority.
In addition, plant growth-promoting rhizobacteria can also produce a large amount of active materials, after being manured into soil, Soil Microorganism can be adjusted Fauna composition, improve soil micro-ecosystem system.The bacillus megaterium NCT-2 of the screenings such as week training acts on secondary salinization soil In earth, the content of nitrate in soil can be made significantly reduce, the AWCD values in soil significantly improve, so as to improve edaphon Diversity, improve the richness of microorganism.Yu Xianmei etc. analyzes bacillus subtilis Bs- by BIOLOG ECO Microdilution plate methods 15 have good colonization ability, improve the overall activity of Chinese chestnut and jujube tree edaphon, enrich edaphon kind Group.Bacillus subtilis HL-1 is also remarkably improved microbial diversity.These results of study are expected to speculate, bacillus megaterium Agent, which also has, improves diversity of soil microorganism, optimizes the effect of soil micro-ecosystem structure, waits to study.
For this reason, this technology is for above progress and there are problem, the multi-functional and efficient huge gemma of separation screening Bacillus, enriches microorganism manure strain resource, and widening microbial manure is improving soil nutrient and soil ecology, promoting plant life Long, many functionalization development such as salt tolerant is degeneration-resistant.This technology will be to improving crop yield and quality, promoting agriculture sustainable development Open up, reduce environmental pollution and ensure that human health has important theory and practice meaning.
The content of the invention
For above-mentioned deficiency, it is an object of the invention to provide one kind to have growth-promoting, volume increase, diseases prevention concurrently with administering salination work( The bacillus megaterium of energy.
Its technical solution is:One plant of bacillus megaterium (Bacillus megaterium strain) X3, abbreviation X3 bacterium. The bacterial strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on May 11st, 2015, Deposit number CGMCC No.10803, preservation address:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism Research institute.
The X3 bacterium are to be separated in Guangzhou, Guangdong Agricultural University Of South China farm soil, purify gained, and it is blue to belong to leather Family name's positive bacteria, there is gemma;Thalline is rod-shaped, there is motility, aerobic;Formed after cultivating 24h on beef-protein medium Bacterium colony to be circular or irregular, all circles of bacterium colony after 48h, milky, diameter is about 2~3mm, and edge is irregular, Flat moistening.
By sequencing, the 16S rDNA nucleotide sequences such as SEQ ID NO of the X3 bacterium:Shown in 1.
The method for preserving of X3 bacterium, the component of its storage medium are beef extract 3.0g, peptone 5.0g, sodium chloride 5.0g, Agar 18.0g, distilled water 1000mL, or the culture medium being configured to according to this ratio, pH value are 7.0~7.2.Routinely strain is protected Hide temperature conservation.
It is a further object of the present invention to provide the preparation method of above-mentioned bacterial strains, this method comprises the following steps successively:
(1) separate
Using the soil on Tianhe District, Guangzhou City, Guangdong Province Agricultural University Of South China farm as screening soil sample, screening soil sample is weighed, is put Enter in the triangular flask equipped with sterile water, shaking table vibration makes cell fully dispersed, stands 20~30s, takes supernatant to carry out 10 times and passs Enleanment is released, using 103~105Dilution factor, dilution is drawn with liquid-transfering gun respectively, is coated on beef extract-peptone tablet, 28 DEG C Culture 48h is inverted, carries out inclined-plane preservation, it is spare;
(2) screening of heteroauxin is produced
Produce the qualitative determination of IAA bacteriums:By the microbionation of step 1) screening in the LB Liquid Cultures containing L-Trp In base, 30 DEG C, 180rmin-1Under the conditions of shaking table culture 1d, take 50 μ L bacteria suspensions drop on whiteware plate, while add 50 μ L Salkowski color solutions, using the color solution of 50 μ L IAA of addition as positive control, whiteware plate is in room temperature, lucifuge condition Observed after lower placement 30min, the color person of reddening represents that IAA can be produced;
The quantitative determination of IAA in nutrient solution:Spectrophotometry bacteria suspension D is used first600Value, then bacteria suspension with from Heart 10min, takes supernatant to add isometric Salkowski color solutions, and lucifuge is stood, and measures its D530Value, use are analytically pure IAA gradient dilutions assay method draws standard curve, IAA contents in unit of account volume zymotic fluid.
(3) purify
IAA will be produced up to 5mgL-1Bacterial strain purification storage, altogether preserve 13 plants of bacterial strains, and strain X 3 produce IAA highests, will The X3 bacterium filtered out after purification, are stored on nutrient agar slant medium using plate streak.
Final object of the present invention is to provide the application of the bacillus megaterium X3, specifically with as follows:
Applications of the above-mentioned bacillus megaterium X3 in terms of production auxin or/and siderophore or/and production ammonia.
Applications of the above-mentioned bacillus megaterium X3 in terms of insoluble phosphorus or salt tolerant is decomposed.
Applications of the above-mentioned bacillus megaterium X3 in banana blight is prevented.
Above-mentioned bacillus megaterium X3 applications in terms of production catalase or/and amylase or protease.
Applications of the above-mentioned bacillus megaterium X3 in terms of plant seed germination is promoted.
Applications of the above-mentioned bacillus megaterium X3 in terms of promoting plant growth or/and improving yield
Above-mentioned bacillus megaterium X3 answering in increase soil microbial community diversity or/and increase bacterial abundance With.
Compared with prior art, the invention has the advantages that:
The present invention filters out one plant of bacillus megaterium (Bacillus megaterium strain) X3, this bacterial strain can High-content auxin (IAA) and siderophore are produced, promotes plant growth and improves yield, particularly corn;And there is Soluble phosphorus, Ammonia is produced, high-salt tolerance, can administer salinization soil;And there is good antagonistic effect to banana blight, biology can be used as Pesticide is used.
Brief description of the drawings
Fig. 1 .X3 bacterium Gram's staining microphotos;
Fig. 2 .X3 bacterium spore staining microphotos;
Fig. 3 bacterial strains produce the qualitative comparison diagram of IAA;
Fig. 4 bacterial strains produce the quantitative contrast figure of IAA;
Fig. 5 .X3 bacterial strains produce the qualitative test lab diagram of siderophore;
Fig. 6 .X3 bacterial strains produce the quantitative test lab diagram of siderophore;
Fig. 7 .X3 bacterial strain salt tolerance lab diagrams;
Fig. 8 .X3 bacterial strains produce ammonia ability comparison diagram;
Preventive and therapeutic effect comparison diagram of Fig. 9 .X3 bacterium to banana blight;
Figure 10 bacterial strains produce the comparison diagram of water-soluble phosphorus concentration under indissoluble Phos culture medium;
Figure 11 .X3 bacterium produce catalase test photo, and wherein HL-1 is positive control;
Figure 12 .X3 fungi degradation starch test photos, wherein HL-1 are positive control;
Figure 13 .X3 bacterium are production Protease assays;
Figure 14 .X3 bacterium promote Growth of Cabbage pot experiment photo;
Figure 15 .X3 bacterium promote corn growth pot experiment photo;
The curve map of Figure 16 different disposal soil microbial community metabolic activity AWCD values change
Figure 17 .X3 bacterium promote Growth of Lettuce field experiment photo;
Figure 18 .X3 bacterium promote corn growth field experiment photo;
Figure 19 .X3 bacterium promote corncob growth test photo.
Embodiment
With reference to embodiment, the claim of the present invention is described in further detail, but is not formed pair Any restrictions of the present invention, the modification of anyone limited number of time made within the claims in the present invention protection domain, still at this Within the claims of invention.
In following embodiments unless otherwise indicated, it is normal experiment method and operating procedure in the art.
Separation, the purifying of 1 bacterial strain of embodiment
(1) separate
Prepare beef-protein medium:Peptone 10.0g, beef extract powder 3g, sodium chloride 5g, distilled water 1000mL, PH7.0~7.5.
It is accurate using spread plate using the soil on Tianhe District, Guangzhou City, Guangdong Province Agricultural University Of South China farm as screening soil sample Screening soil sample 10.0g is really weighed, is put into the 250mL triangular flasks equipped with 90mL sterile waters and (adds bead), shaking table vibration 30min, makes cell fully dispersed, stands 20~30s, takes supernatant to carry out 10 times of dilutions of successively decreasing, using 103~105Dilution factor, Draw dilution 0.1mL respectively with liquid-transfering gun, be coated on beef extract-peptone tablet, 28 DEG C are inverted culture 48h.Selection is suitable Concentration tablet, according to form, size, color, the typical single bacterium colony of picking different strains, right on culture medium after purification The bacterial strain screened carries out inclined-plane preservation, spare.
(2) screening of heteroauxin (IAA) is produced
Produce the qualitative determination of IAA bacteriums:By the microbionation of preliminary screening in containing L-Trp (100mgL-1) LB In fluid nutrient medium, 30 DEG C, 180rmin-1Under the conditions of shaking table culture 1d.50 μ L bacteria suspensions drop is taken on whiteware plate, together When add 50 μ L Salkowski color solutions (HClO4+1mL 0.5mol·L-1FeCl3), 50 μ L will be added IAA(50mg·L- 1) color solution as positive control.Whiteware plate is seen after placing 30min under the conditions of room temperature, lucifuge Examine, the color person of reddening represents that IAA can be produced.
The quantitative determination of IAA in nutrient solution:Condition of culture is same as above.Spectrophotometry bacteria suspension OD is used first600Value, Then bacteria suspension centrifuges 10min with relative centrifugal force Fr, c=8497, takes supernatant to add isometric Salkowski color solutions, Lucifuge stands 30min, measures its OD530Value.Standard curve is drawn using analytically pure IAA gradient dilutions assay method, is calculated single IAA contents in the volume zymotic fluid of position.
(3) purify
According to national standard, by production IAA up to 5mgL-1Bacterial strain purification storage, preserve 13 plants of bacterial strains, and strain X 3 altogether IAA yield highests, up to 35.05mgL-1, by the X3 bacterium filtered out using plate streak after purification, it is stored in nutrient agar On slant medium.As a result such as Fig. 4.
2 CHARACTERISTICS IDENTIFICATION of embodiment
(1) thalli morphology characteristic
Gram-positive bacteria, there is gemma.Thalline is rod-shaped, there is motility, obligate aerobic.The micro- photograph of thalline Gram's staining Piece is shown in Fig. 1, and spore staining photo is shown in Fig. 2.
(2) colonial morphology characteristic
It is circular or irregular that the bacterium colony formed after 24h is cultivated on beef-protein medium, and bacterium colony is complete after 48h Portion is circle, and milky, diameter is about 2~3mm, and edge is irregular, flat moistening.
(3) growth characteristics
In beef extract 5g, peptone 5g, sodium chloride 5g, the fluid nutrient medium of water 1L, rotating speed 113rpm, 30 DEG C of temperature, Under conditions of initial ph value is 7.0,18h is cultivated, measures viable count as 2.91 ± 0.60 × 108cfu·mL-1
(4) molecular biological characteristic
X3 bacterium STb genes are extracted using RNA isolation kit.Using bacterial 16 S rDNA universal primers 27f:(AGA GTT TGA TCC TGG CTC AG) and 1492r:(TAC GGC TAC CTT GTT ACG ACT T), the 16S rDNA of PCR amplification bacterium, Occur obvious band near 1000bp, sequencing is carried out after pcr amplification product is recycled, the DNA sequence dna of acquisition is defeated Enter GenBank, analysis is compared to all sequences in database with Blast programs, it turns out that bacterial strain of the present invention 16S rDNA sequences and there is higher homology with bacillus megaterium in GenBank, its similarity is 99%.With reference to upper The flat-plate bacterial colony feature stated, Biolog Ecoplate microplates cultivation results, physio-biochemical characteristics, the knot of 16S rDNA sequences Fruit, the Preliminary Identification bacterial strain is bacillus megaterium (Bacillus megaterium strain), is named as Bacillus megaterium strain X3。
The production siderophore experiment of 3 bacterial strain of embodiment
It is qualitative:Bacterial strain is transferred to after cultivating 24-48h on LB tablets, using an inocalation method, with the toothpick of sterilizing by strain It is connected on CAS solids detection tablet, puts 3 repetitions, 28 DEG C of culture 48h, the CAS by culture is detected on tablet, and secretion iron carries Obvious orange siderophore haloing occurs in the surrounding bacterial colonies of body.The orange haloing of results strain X3 is maximum, illustrates the bacterium Strain production siderophore ability is most strong (Fig. 5).
It is quantitative:By inoculation in limit iron SA fluid nutrient mediums (sucrose 20.0g, L-Aspartic acid 2.0g, K2HPO4 0.5g, MgSO4·7H2O 0.5g, distilled water 1000mL, pH7.0) in, 28 DEG C of shaking table culture (150rpm) 48h;Bacteria suspension passes through 10000rpm centrifuges 15min, takes supernatant, bacterium solution and CAS detection liquid 1:1 volume fully mixes, after static 1h, measure Light absorption value (As) at 630nm wavelength, takes distilled water to return to zero as control, with the nonvaccinated SA fluid nutrient mediums being measured in the same method Light absorption value as reference value (Ar), and calculate siderophore active unit ([(Ar-As)/Ar] × 100).Test result indicates that (Fig. 6), the active unit of strain X 3 is maximum, up to 38.25%.
4 X3 bacterium salt tolerance of embodiment is tested
Bacterial strain is scoring to 0%, 1%, 3%, 5%, 7%, 9%, 11%, on 13% nutrient agar panel, culture 24h, observes the growing state of bacterial strain.As a result the salt resistance ability of different strains is different, and the salt resistance ability of strain X 3 can reach 13g 100mL-1.Such as Fig. 7.
5 X3 bacterium of embodiment produce ammonia ability test
By bacterial strain be transferred to 10mL peptone waters solution (peptone 10.0g, NaCl 5g, distilled water 1000mL, pH7.0~ 7.2) in test tube, 28 ± 2 DEG C are cultivated 48-72h, and often pipe adds 0.5mL Nessler's reagents, and color switchs to yellow by brown Show there is NH3Produce.The result shows that X3 bacterium can produce ammonia, such as Fig. 8.
6 X3 bacterium bacteriostasis of embodiment is tested
Fusarium oxysporum (Fusarium oxysporum) is inoculated in nutrient agar center, while in the tablet Each streak inoculation bacterial strain in both sides, cultivate, observation.The result shows that X3 bacterium have banana blight certain inhibitory action, such as Fig. 9.
The effect experiment of production water-soluble phosphorus of the 7 X3 bacterium of embodiment under slightly solubility Phos medium culture
Load 50mL sterilized indissoluble Phos culture medium (glucose 10.0g, ammonium sulfate 0.5g, chlorination in triangular flask Sodium 0.3g, potassium chloride 0.3g, ferrous sulfate heptahydrate 0.03g, manganese sulfate 0.03g, yeast extract 0.4g, calcium phosphate 10g, distilled water 1000mL), (concentration of calcium phosphate is 5.0gL for pH7.0~7.5-1), access X3 bacterium are made after bacterium solution at 28 DEG C, and 150rpm, shakes Bed culture 7d.By nutrient solution in 10000rpm, 4 DEG C centrifuge 15min, take supernatant molybdenum antimony resistance colorimetric method measure available phosphorus to contain Amount.If not connecing X3 bacterium as control, each processing is repeated 3 times.Water-soluble phosphorus changes of contents (unit mgL-1, that is, represent every liter The milligram number of contained water-soluble phosphorus in sample) result of the test is shown in Figure 10.
Fig. 9's the result shows that:The water-soluble phosphorus content highest of X3 fermented liquids, reaches 279.46mgL-1
8 X3 bacterium of embodiment produce catalase test
After X3 bacterium are activated 24h on beef-protein medium, appropriate strains tested is chosen on glass slide, is added dropwise 3% hydrogen peroxide is observed immediately on strains tested, has a large amount of bubbles to produce, and is the positive, otherwise is feminine gender.Using HL-1 as Positive control, result of the test is as shown in figure 11, and the catalase test for showing X3 bacterium is the positive.
9 X3 fungi degradation starch tests of embodiment
X3 bacterium point is connected on starch culture-medium tablet, 48h is cultivated in 30 DEG C of constant incubators, forms obvious bacterium colony Afterwards, iodine solution is added dropwise on tablet, tablet is in black-and-blue, and periphery of bacterial colonies represents that Starch Hydrolysis is positive if any non-discolouring transparent circle;Still Be it is black-and-blue for feminine gender.Using HL-1 as positive control, result of the test is as shown in figure 12, and the solution starch test for showing X3 bacterium is sun Property.
10 X3 bacterium of embodiment produce Protease assays
Protease detection culture medium (g.L-1):Peptone 10g, sodium chloride 5g, calcium chloride 0.1g, skimmed milk power 10g, agar 18g, 115 DEG C of sterilizings 30min, pH 7.2-7.4.
By in inoculation to protease detection culture medium, 30 DEG C of culture 2d, the transparent circle size for observing periphery of bacterial colonies is come Judge the protein hydrolysis ability of bacterial strain, as a result as shown in figure 13, compared with the control, protein in bacterial strain flat board nearly all by Using complete, illustrate the bacterial strain using the very capable of protein.
11 X3 bacterium of embodiment are to germination Biological control
Pakchoi seed is soaked into 2min with 75% ethanol 1min+5% liquor natrii hypochloritises, then with aseptic water washing 3-4 It is secondary, then ensure sterile working as far as possible with sterile filter paper dry seed, all operations.The seed handled well is placed on dry nothing It is spare in the container of bacterium.Setting two treatment groups, (first group of addition fermented liquid, is represented with X3, the 2nd group of non-inoculating strain of addition Culture medium as blank control, represented with CK) pakchoi seed is soaked into 30min in one or two groups respectively.By the training of sterilizing Support and complete two layers of aseptic filter paper in advance in ware, the seed of seed soaking is placed in culture dish (per 10 seeds of ware), is being planted One layer of filter paper is covered on son, is cultivated (30 DEG C).Add 2mL sterile waters per ware daily, add water time unification.Each strain processing sets 4 A repetition.Measure germination percentage and the bud length of the 3,4,5th day.The result shows that (table 1):Adding bacterium can promote pakchoi to germinate.
1 X3 bacterium of table are to pakchoi seed germination experiment
12 X3 bacterium of embodiment promote the experiment of Growth of Cabbage pot test effect
Piglet s colibacillosis is carried out using X3 bacterium and blank control, is divided into 2 treatment groups (the 1st group of addition X3 fermented liquid 30mL is resuspended through 0.8% physiological saline of centrifugation, is represented with X3, the 2nd group of 0.8% physiological saline 30mL of addition is as blank pair According to being represented with CK), each treatment group is repeated 7 times.Each treatment group soil weight 1.0kg, specific experimental design are shown in Table 2.Pakchoi water Vernalization is washed, soaks into preservation moisture with filter paper, is placed in 30 DEG C of incubators, exposes bud two days later and sows into seedbed.When seedling is grown When going out two one leaves of the heart, transplant seedlings into basin, per 1 seedling of basin.Appropriate amount of water is poured daily, the water per basin is identical, uniformly, is careful not to Make water outflow basin bottom in order to avoid fertilizer loss and error.On January 17th, 2013 measures pakchoi plant height, the number of blade, root long.2013 January 19 measured pakchoi plant root dry weight and leaf dry weight, determined pakchoi above-ground plant parts and under ground portion nitrogen afterwards The content of phosphorus potassium.The result shows that (table 3 and table 4):The growth of pakchoi can be remarkably promoted by adding X3 bacterium.
2 experimental design of table
Treatment group Soil (kg) Urea (mg) Calcium superphosphate (mg) Potassium chloride (mg) Bacterium solution
1 1.0 100 70 103 Re-suspension liquid 30mL
2 1.0 100 70 103 Physiological saline 30mL
Growth-promoting Contrast on effect table of the 3 X3 microbial inoculums of table to pakchoi
Nutritional ingredient contrast table of the 4 X3 microbial inoculums of table to pakchoi
13 X3 bacterium of embodiment promote the experiment of corn growth pot test effect
Piglet s colibacillosis is carried out using X3 bacterium and blank control, is divided into 2 treatment groups (the 1st group of addition X3 fermented liquid 30mL is resuspended through 0.8% physiological saline of centrifugation, is represented with X3, the 2nd group of 0.8% physiological saline 30mL of addition is as blank pair According to being represented with CK), each treatment group is repeated 4 times, and repeats 4 seedlings every time.Per basin soil weight 3.0kg, specific experimental design is shown in Table 5.Corn seed washes vernalization, soaks into preservation moisture with filter paper, is placed in 30 DEG C of incubators, exposes bud two days later and sow to seedling In bed.When seedling grows to about 15cm high, transplant seedlings into basin, per 2 seedlings of basin.Appropriate amount of water is poured daily, the water per basin is identical, It is even, it is careful not to make water outflow basin bottom in order to avoid fertilizer loss and error.On May 13rd, 2014 measures corn plant height, the number of blade, leaf Green element, root long, leaf length, leaf width, stem is thick, aerial part and under ground portion fresh weight.Measurement on May 15th, 2014 plant is on the ground Part and under ground portion dry weight.The result shows that (table 6 and table 7):The growth of corn can be remarkably promoted by adding X3 bacterium.
5 experimental design of table
Growth-promoting Contrast on effect table of the 6 X3 microbial inoculums of table to corn
Growth-promoting Contrast on effect table of the 7 X3 microbial inoculums of table to corn
Note:Data are the average value ± standard error of 4 repetitions, and the different letter persons of same column, represent in 0.05 level error in table Different notable (DMRT methods).
14 X3 bacterium of embodiment test the improved effect of edaphon functional diversity
Concrete operations are as follows:The fresh soil weighed equivalent to 10g drying soil samples is added to equipped with 100mL sterile physiological salt In the 250mL triangular flasks of water (0.85%);After shaking 1min on vortex oscillator, 1min is stood in ice-water bath;Repeat 3 It is secondary;2min is stood, takes the above-mentioned soil extractions of 5mL to add in 45mL sterile salines (0.85%), after mixing, repeats this step Suddenly, the X3 bacterium solutions after 1000 times will be diluted to add in Biolog Eco plates, adds 150 μ L per hole;By the microplate of inoculation at 30 DEG C Incubator culture, respectively at 0,24,48,72,96h light absorption value under 590nm is read with microplate reader.Calculate soil microbial community Multifarious formula such as table 8.
Table 8 calculates the formula of Microbial Community Diversity index
Experimental result is shown in Table 9, shows that each processing AWCD values increase with the increase of time, and adds the processing soil of X3 strains (edaphon metabolic activity AWCD, diversity of soil microorganism refer to earth (X3 bacterium group) Microbial Community Diversity indices Number Shannon and edaphon species richness R ichness) it is all remarkably higher than soil (the i.e. blank control for not adding X3 strains Group, is represented with CK, reaches significant difference (P<0.05).Illustrate that X3 bacterium considerably improve diversity of soil microorganism.
9 different disposal soil microbial community diversity indices (48h) of table
Processing AWCD Shannon(H) Richness
CK groups 0.15±0.05b 15.3±3.7b 0.97±0.08b
X3 bacterium groups 0.57±0.08a 27.7±0.9a 1.29±0.03a
Note:Data are the average value ± standard error of 3 repetitions, and the different letter persons of same column, represent in 0.05 level error in table Different notable (DMRT methods).
15 X3 bacterium of example promote the experiment of Growth of Lettuce field efficacy
Piglet s colibacillosis is carried out using X3 bacterium and blank control, be divided into 2 treatment groups (the 1st group of addition X3 fermented liquid, Represented with X3;The culture medium of the 2nd group of non-inoculating strain of addition is represented as blank control with CK), each 3 cells for the treatment of group, 2.5 × 1.4m of each plot area2, 15 × 8=120 of planting density young plants (removing edge effect 1 plant of row of protection, actual strain number 13 × 6=78 plants).Romaine lettuce washes vernalization, soaks into preservation moisture with filter paper, exposes bud two days later and sow into seedbed.When seedling is grown When going out two one leaves of the heart, crop field is moved into.On April 15th, 2013 measures romaine lettuce plant height, root long, leaf area and chlorophyll.In April, 2013 The dry weight of measurement romaine lettuce aerial parts on the 17th and under ground portion.The result shows that (table 10):Romaine lettuce can be remarkably promoted by adding X3 bacterium Growth.
Growth-promoting Contrast on effect table of the 10 X3 microbial inoculums of table to romaine lettuce
16 X3 bacterium of example promote the experiment of corn growth field efficacy
Piglet s colibacillosis is carried out using X3 bacterium and blank control, be divided into 2 treatment groups (the 1st group of addition X3 fermented liquid, Represented with X3;The culture medium of the 2nd group of non-inoculating strain of addition is represented as blank control with CK), each 3 cells for the treatment of group, Each plot area 5.3m2, planting density is 34 plants/cell, and 6 cells take randomized complete-block design.Corn washing is urged Bud, soaks into preservation moisture with filter paper, exposes bud two days later and sow into seedbed.When maize seedling length is to 10cm, crop field is moved into.Survey Determine thick florescence and jointing stage corn plant height, root long, the number of blade, leaf length, leaf width, stem, fresh weight and dry weight, maturity period spike length, fringe Slightly, grain number per spike, 100-grain weight and economic flow rate etc..The result shows that (table 11,12,13):Corn can be remarkably promoted by adding X3 bacterium Growth, corn yield is compared with CK increases by 29.8%.
Growth-promoting Contrast on effect table of the 11 X3 microbial inoculums of table to corn
Growth-promoting Contrast on effect table of the 12 X3 microbial inoculums of table to corn
Growth-promoting Contrast on effect table of the 13 X3 microbial inoculums of table to corncob

Claims (9)

1. one plant of bacillus megaterium (Bacillus megaterium) X3, deposit number is CGMCC No.10803, in 2015 On May 11, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. bacillus megaterium X3 according to claim 1, it is characterised in that its 16S rDNA nucleotide sequence such as SEQ ID NO:Shown in 1.
3. applications of the bacillus megaterium X3 of claim 1 or 2 in terms of production auxin or/and siderophore or/and production ammonia.
4. applications of the bacillus megaterium X3 of claim 1 or 2 in terms of insoluble phosphorus or salt tolerant is decomposed.
5. applications of the bacillus megaterium X3 of claim 1 or 2 in terms of banana blight is prevented.
6. applications of the bacillus megaterium X3 of claim 1 or 2 in terms of production catalase, amylase and protease.
7. applications of the bacillus megaterium X3 of claim 1 or 2 in terms of plant seed germination is promoted.
8. applications of the bacillus megaterium X3 of claim 1 or 2 in terms of promoting plant growth or/and improving yield.
9. the bacillus megaterium X3 of claim 1 or 2 is in increase soil microbial community diversity or/and increase microorganism The application of abundance.
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