CN104593301A - Bacillus muralis G1 as well as preparation method and application thereof - Google Patents
Bacillus muralis G1 as well as preparation method and application thereof Download PDFInfo
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- CN104593301A CN104593301A CN201510021861.2A CN201510021861A CN104593301A CN 104593301 A CN104593301 A CN 104593301A CN 201510021861 A CN201510021861 A CN 201510021861A CN 104593301 A CN104593301 A CN 104593301A
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- genus bacillus
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- bacillus
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Classifications
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- C—CHEMISTRY; METALLURGY
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- 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
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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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
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Abstract
The invention discloses bacillus muralis G1 as well as a preparation method and an application thereof and belongs to the technical field of microbial strains. The bacillus muralis G1 has the collection number of CGMCC No. 10189 and is collected at the CGMCC (China General Microbiological Culture Collection Center) at December 17, 2014. The bacillus muralis G1 disclosed by the invention has the functions of efficiently dephosphorizing, producing auxin and producing ammonia and can be used for effectively promoting seed germination and promoting plant growth.
Description
Technical field
The present invention relates to a kind of microorganism and application thereof, be specifically related to wall genus bacillus (Bacillus muralis) G1 that a strain has soil phosphorus decomposing, produces growth hormone, produces ammonia feature, the invention still further relates to the preparation method of this wall genus bacillus G1, and promotion soil phosphorus validity, Plantula Brassicae chinensis seed germination, the application of the aspects such as romaine lettuce and milpa growth.
Background technology
Phosphorus is one of necessary mineral element of growth and development of plants.Utilize molten phosphorus microorganism to be developed into bio-fertilizer or bacteria agent, to improve the validity of phosphate fertilizer and soil indissoluble phosphorus utilization ratio, reduce chemical phosphatic ferfilizer and to drop into and agricultural sustainable development has great importance.The product gemma characteristic of bacillus (Bacillus), there is the ability that stronger opposing external environment is coerced, can resist due to the injury that dry, hot and ultraviolet radiation cause in survived environment, thus be easy to surely grow soil and play field fertilizer efficiency.Therefore, the genus bacillus having molten phosphorus effect has huge application prospect in bio-fertilizer industry.
Current molten Bacillus phosphorus, is mainly the bacterial strain researchs such as bacillus megaterium (Bacillus megaterium), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus cereus (Bacillus cereus), subtilis (Bacillussubtilis), bacillus thuringiensis (Bacillus thuringiensis).For example, Zhang Weina etc. (2012) report that bacillus megaterium (Bacillus megaterium) JD-2 has the ability of stronger degrading organic phosphor, inorganic phosphorus, and available phosphorus content is respectively 25 times, 22 times of control group.After Xu Renjie etc. (2003) report uses bacillus amyloliquefaciens (Bacillus amyloliquefaciens) PS57, the winter Plantula Brassicae chinensis plant height, root length, fresh weight, dry weight, in phosphorus content and root soil, the content of phosphorus is all significantly higher than control group in blade.Under Wu little Qin etc. (2010) are reported in Yelkin TTS liquid culture, the titanium pigment content of bacillus cereus (Bacillus cereus) JYZ-SD1 is 1.94mg/L, contrast (0.37mg/L) 5.17 times, inoculation JYZ-SD1 bacterial strain after 150 days willow cuttage seeding height of seedling and stem slightly increase 38.1% and 15.9% respectively than CK.After Cao Yu (2010) reports that phosphorus decomposing subtilis (Bacillus subtilis) GU uses, banana production adds 30.8%.After bacillus thuringiensis (Bacillus thuringiensis) GL-1 uses, corn plant height, the number of blade, content of soil available phosphor add 4.8%, 8.2% and 60% (Cai Yanfei etc., 2012) than CK respectively.These achievements show the potentiality of genus bacillus in the molten phosphorus of soil, have laid good working foundation and correlation experience for excavating novel molten phosphorus Microbial resources.In recent years, some investigators are separated to wall genus bacillus (Bacillus muralis) from physical environment.Research finds, the biological corrosion of wall genus bacillus to mural painting has a certain impact (Pepe et al, 2008).In addition, Meng Fanxu (2009) reports that wall genus bacillus has certain halophilism.Lan Xiaojun etc. (2009) research finds that wall genus bacillus A1 has certain resistance to nickel.Baorong Fus etc. (2013) separate the wall genus bacillus that a strain has high flocculation ability.But not yet have the report of wall genus bacillus in molten phosphorus at present, the molten ability of phosphorus therefore studying wall genus bacillus has very important significance for the novel molten phosphorus Microbial resources of excavation.
Phosphate solubilizing microorganism screening is in the past confined to simple function bacterial strain more.Wang Lijing (2008) is separated and obtains phosphorus decomposing bacterial strain from agricultural land soil, and it is applied to wheat seedling.Chen Yang etc. (2008) several typical sand binding plant rhizosphere from desert, Erdos is separated phosphate solubilizing bacteria, and studies its dissolving P capacity.Li Zhendongs etc. (2013) select eastern-Qilian mountain Alpine Grasslands dominant plant Herba Anaphalidis Lacteae to be test materials, raw phosphate-solubilizing bacteria in Isolation and screening, have studied its growth-promoting functions to corn.The hot issue that bio-fertilizer is studied both at home and abroad by the multifunctional combination such as product gemma, phosphorus decomposing and growth-promoting.There are some researches show a part of phosphate solubilizing bacteria except the phosphorus element in release soil insoluble phosphorus, outside utilizing for plant absorption, release plant hormone substance can also be synthesized, as phytokinin, growth hormone, Plant hormones regulators,gibberellins, promote that root system of plant absorbs moisture in soil and nutrient effectively, to promote growing of plant, other vital movements of plant materials are regulated and controled (Yao Tuo, 2004) simultaneously.Some phosphate solubilizing bacteria has growth-promoting abilities (Kang Yijun etc., 2010) such as producing siderophore, ammonia, product prussic acid and product microbiotic.The refined grade (2014) of Marvin's, bacterial strain Y16 is isolated from plant rhizosphere, this bacterial strain has molten phosphorus, fixed nitrogen and secretion growth hormone performance concurrently, and arrow Kuo pea ground biomass, underground biomass can be made after inoculating this bacterial strain significantly to increase by 104.5% and 254.1% respectively.Hariprasad etc. (2009) isolate from Karnataka area tomato plant rhizosphere the phosphorus decomposing bacterial strain P.putida PSRB6 having and produce growth hormone and siderophore, the Seeds Germination Tests of tomato seeds is shown, bacterial strain PSRB6 significantly can promote that tomato root is long, seedling is long and percentage of germination, compares according to improving 17.7%, 10.2%, 11.3% respectively.Under greenhouse experiment, after inoculating strain PSRB6, tomato root length, fresh weight, dry weight, content of soil available phosphor improves 22.2%, 20.0%, 112.9%, 66.7% respectively.And the wall genus bacillus with the several functions such as efficient phosphate-solubilizing and product growth hormone was not reported.
Summary of the invention
For the problems referred to above, the object of the present invention is to provide a kind of combining efficient phosphorus decomposing, produce growth hormone, produce ammonia function, effectively can promote seed germination, the wall genus bacillus G1 of Promoting plant growth, the invention still further relates to the preparation method and application of this wall genus bacillus G1.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions above-mentioned purpose:
This wall genus bacillus (Bacillus muralis) G1, is called for short G1 bacterium.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number No.10189, Classification And Nomenclature on December 17th, 2014: wall genus bacillus, (Bacillus muralis); Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Further, described G1 bacterium is separation and purification gained in the soil of stove mountain, Guangzhou, Guangdong, belongs to gram-positive microorganism, has an endogenous spore; Thalline is direct rod shape, often with paired or catenation, and aerobic or amphimicrobian; Catalase test, oxidase test, Starch Hydrolysis test, Protease assays, cellulose degradation test, indole test, pink pigmentation, the fluorochrome positive; Methyl red test, Citrate trianion utilize test, gelatin liquification test feminine gender; The bacterium colony formed after beef-protein medium is cultivated 24h is for circular or irregular, and after 48h, bacterium colony is all circular, and faint yellow, diameter is about 1-2mm, and edge is irregular, dimpling, flat moistening.
Through sequencing, the 16S rDNA nucleotide sequence of this G1 bacterium is as shown in SEQ ID NO:1.
The method for preserving of G1 bacterium, the composition of its storage medium is glucose 10g, ammonium sulfate 0.5g, sodium-chlor 0.3g, Repone K 0.3g, ferrous sulfate 0.03g, four anhydrous manganese 0.03g, calcium phosphate 10g, yeast extract paste 0.4g, agar 20g, distilled water 1000ml or the substratum become according to this proportions, pH value is pH 7.0-7.5, routinely culture presevation temperature conservation.
Another one technical scheme of the present invention is to provide the preparation method of this G1 bacterium, and the method comprises the steps: successively
(1) be separated
Take the soil 10.0g on stove mountain, Guangzhou, Guangdong, put into the bottle that 80-100ml physiological saline is housed, vibration makes cell fully disperse, leave standstill, get 10ml supernatant liquor and be placed in triangular flask, 88-91 DEG C of heating 10-12 minute, after getting 500 μ L water-baths, supernatant liquor is added to 25-35ml indissoluble inorganic phosphorus substratum, carry out enrichment culture, the enrichment culture cycle is 5-7d, enrichment culture got 500 μ L nutrient solutions after terminating and joined separately in new indissoluble inorganic phosphorus substratum and carry out second time enrichment culture first time, after twice enrichment culture terminates, get supernatant liquor and be placed in centrifuge tube, supernatant liquor is got again after centrifugal, measure water-soluble phosphorus content in supernatant liquor, the nutrient solution that reservation water-soluble phosphorus content reaches more than 30mg/L carries out third time enrichment, adopting after three enrichments terminate uses the same method carries out the 4th enrichment culture, cultivation terminates water-soluble phosphorus content in rear mensuration nutrient solution, G1 bacterium water-soluble phosphorus content 220.4 μ g/mL, glycerol stocks method is adopted to preserve this bacterial strain,
(2) purifying
After the G1 bacterium filtered out is utilized plate streak purifying, be stored in the made inclined-plane of beef-protein medium, be placed in 4 DEG C of refrigerators for subsequent use, after molecular biology identification and Physiology and biochemistry qualification, determine that G1 bacterial strain is wall genus bacillus.
Further, the preparation method of above-mentioned wall genus bacillus G1, described indissoluble inorganic phosphorus substratum is made up of following component: glucose 10.0g, ammonium sulfate 0.5g, sodium-chlor 0.3g, Repone K 0.3g, iron vitriol 0.03g, manganous sulfate 0.03g, magnesium sulfate heptahydrate 0.3g, calcium phosphate 10.0g, distilled water 1000mL; PH 7.0-7.5.
Last technical scheme of the present invention is to provide this wall genus bacillus G1 and is decomposing insoluble phosphorus, produces in growth hormone, produces in ammonia and promoting the application in plant seed germination and in Promoting plant growth
Compared with prior art, the present invention has following beneficial effect:
Contriver, through lot of experiments, filters out strain wall genus bacillus (Bacillusmuralis) G1 from the soil on stove mountain, Guangzhou.This bacterial strain combining efficient phosphorus decomposing, product growth hormone, product ammonia function, effectively can promote seed germination, Promoting plant growth.
After applying this bacterial strain, Plantula Brassicae chinensis rate of emergence, the 7th day seedling length, root length, fresh weight improve 45.8%, 44.7%, 31.8%, 30.4%, 30.0% compared with the control respectively.Romaine lettuce results from pot experiment test shows, and the phosphorus of every basin plant, nitrogen, potassium accumulation are respectively than contrast increase by 39.6%, 42.6%, 23.5%, and soil available phosphorus content is than contrast raising 7.8%.Corn results from pot experiment test shows, in maize leaf, phosphorus, nitrogen, potassium content are respectively than contrast raising 18.5%, 23.0%, 10.8%, and soil available phosphorus content is than contrast raising 10.3%.In field plot trial, romaine lettuce dry matter weight, corn yield add 54.1%, 12.5% than contrast respectively.The above results shows this bacterial strain in raising soil and, while indissoluble P availability and phosphate fertilizer utilization efficiency, also significantly can promote the growth of Plantula Brassicae chinensis seed germination and romaine lettuce and milpa.
Accompanying drawing explanation
Fig. 1 .G1 bacterium gramstaining Photomicrograph, thalline is dyed to purple;
Fig. 2 .G1 bacterium spore staining Photomicrograph, in thalline, oval shaped portion is gemma;
Fig. 3. different strains produces the comparison diagram of water-soluble phosphorus concentration under indissoluble inorganic phosphorus substratum;
Fig. 4. the comparison diagram of different strains producing microbial phosphorus concentration under indissoluble inorganic phosphorus substratum;
Fig. 5. different strains produces growth hormone quantitative experiment comparison diagram;
Fig. 6. different strains produces growth hormone qualitative experiment comparison diagram;
Fig. 7 .G1 bacterial strain produces ammonia experiment photo;
Fig. 8 .G1 microbial inoculum promotes Growth of Lettuce pot experiment photo;
Fig. 9 .G1 microbial inoculum promotes corn growth pot experiment photo;
Figure 10 .G1 microbial inoculum promotes Growth of Lettuce field plot trial photo;
Figure 11 .G1 microbial inoculum promotes corn growth field plot trial photo.
Embodiment
Below in conjunction with embodiment; claim of the present invention is described in further detail; but do not form any limitation of the invention, the amendment of anyone limited number of time made within the claims in the present invention scope, still within claims of the present invention.
In following examples unless otherwise indicated, the normal experiment method in this area and operation steps is.
The separation of embodiment 1 bacterial strain, purifying
(1) be separated
Preparation indissoluble inorganic phosphorus substratum: glucose 10.0g, ammonium sulfate 0.5g, sodium-chlor 0.3g, Repone K 0.3g, iron vitriol 0.03g, manganous sulfate 0.03g, magnesium sulfate heptahydrate 0.3g, calcium phosphate 10.0g, distilled water 1000mL, pH 7.0-7.5.
With the soil on stove mountain, Guangzhou, Guangdong for screening soil sample, accurately take screening soil sample 10.0g, put into the 250ml triangular flask (adding granulated glass sphere 10) that 80-100ml (preferred 90ml) 0.85% physiological saline is housed, 200r/min, shaking table vibration 30min, makes cell fully disperse, leaves standstill 20-30s, get 10ml supernatant liquor and be placed in 50ml sterilizing triangular flask, 10-12 minute (preferably 90 DEG C heating in water bath 10 minutes) of 88-91 DEG C of heating.After getting 500 μ L water-baths, supernatant liquor joins indissoluble inorganic phosphorus substratum (containing 30ml substratum in 150ml triangular flask), and each soil sample establishes two repetitions.Be placed in shaking table and carry out enrichment culture, rotating speed 150r/min, temperature 30 DEG C.The enrichment culture cycle is 5-7d, first time enrichment culture get 500 μ L nutrient solutions after terminating and join separately in new substratum and carry out second time enrichment culture.After twice enrichment culture terminates, in Bechtop, get supernatant liquor with liquid-transfering gun appropriate, be placed in centrifuge tube, centrifugal, get supernatant liquor, adopt molybdenum antimony resistance colorimetric method to measure water-soluble phosphorus content in supernatant liquor, the nutrient solution that reservation water-soluble phosphorus content reaches more than 30mg/L carries out third time enrichment.Adopt to use the same method after third time enrichment terminates and carry out the 4th enrichment culture, cultivation terminates rear employing molybdenum blue colorimetric method and measures water-soluble phosphorus content in nutrient solution, glycerol stocks method (nutrient solution and 50% glycerine ratio 7:3 ,-80 DEG C of preservations) is adopted to preserve the higher bacterial strain of water-soluble phosphorus content.The bacterial isolates filtering out amount of phosphorus dissolved higher is numbered C, G1, G2, Q1, Q2 and S2, and wherein bacterium G1 water-soluble phosphorus content reaches 220.4 μ g/mL.
(2) purifying
Beef-protein medium: extractum carnis 3.0g, peptone 5.0g, sodium-chlor 5.0g, agar 18.0g, distilled water 1000ml, pH value 7.0-7.2.
After the G1 bacterium filtered out is utilized plate streak purifying, be stored in the made inclined-plane of above-mentioned storage medium, be placed in 4 DEG C of refrigerators for subsequent use.After molecular biology identification and Physiology and biochemistry qualification, determine that G1 bacterial strain is wall genus bacillus (Bacillus muralis).
Embodiment 2 CHARACTERISTICS IDENTIFICATION
(1) thalli morphology characteristic
Gram-positive microorganism, has an endogenous spore, and thalline is direct rod shape, and often with paired or catenation, tool nose circle or square end, tool flagellum, has mobility.Fig. 1 is shown in by thalline gramstaining Photomicrograph, and Fig. 2 is shown in by spore staining photo.
(2) colonial morphology characteristic
The bacterium colony formed after beef-protein medium is cultivated 24h is for circular or irregular, and after 48h, bacterium colony is all circular, and faint yellow, diameter is about 1-2mm, and edge is irregular, dimpling, flat moistening.
(3) growth characteristics
At yeast powder 5g, peptone 10g, sodium-chlor 10g, in the liquid nutrient medium of water 1L, rotating speed 150rpm, temperature 30 DEG C, initial ph value is under the condition of 7.0, cultivates 18 hours, and recording viable count is 1.97 ± 0.08 × 10
8cfu/ml.
(4) physiology, biochemical characteristic, in table 1.
(5) molecular biological characteristic
RNA isolation kit (purchased from Shanghai Sheng Gong biotechnology company limited) is adopted to extract G1 bacterium STb gene.Adopt bacterial 16 S rDNA universal primer F27 (AGA GTT TGA TCC TGG CTC AG, SEQ ID NO:2) and R1492 (TAC GGC TACCTT GTT ACG ACT T, SEQ ID NO:3), the 16S rDNA of pcr amplification bacterium, the PCR reaction system of 50 μ L is: DNA profiling 1 μ L, dNTP (2.5mM) 4 μ L, primer 1 (l mM) 1 μ L, primer 2 (l mM) 1 μ L, 10 × PCR Buffer 5 μ L, MgCl2 (25mM) 3 μ L, Taq enzyme (5U/ μ L) 0.5 μ L, ddH2O 34.5 μ L.
Pcr amplification reaction program: 94 DEG C of denaturation 4min, enter thermal cycling; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 1min, totally 30 circulations; 72 DEG C extend 10min.
1.0% agarose gel electrophoresis detects length and the concentration of PCR primer, obvious band is there is near 1000bp, after pcr amplification product is reclaimed, order-checking is completed by Beijing six directions Hua Da Gene Tech. Company Limited, BLAST comparison is carried out by the DNA sequence dna (as shown in SEQ ID NO.1) obtained input US National Bioinformatics Institute NCBI webpage, with Blast program, analysis is compared to all sequences in database, found that the 16S rDNA sequence of bacterial strain of the present invention and with GenBank mesospore genus bacillus, there is higher homology, its similarity is 99%.In conjunction with the result of above-mentioned thalli morphology characteristic, flat-plate bacterial colony feature, physio-biochemical characteristics, 16SrDNA sequential analysis, this bacterial strain of preliminary evaluation is wall genus bacillus (Bacillus muralis), called after wall bacillus muralis G1.
Table 1 G1 bacterium physio-biochemical characteristics
| Characteristic | Result | Characteristic | Result |
| Pink pigmentation | + | Casein hydrolysis | + |
| Oxydase | + | Fluorochrome | + |
| Aerobism | +/w | Cellulose decomposition | + |
| Catalase | + | Indoles | + |
| Starch Hydrolysis | + | Methyl red | － |
| Gelatine liquefication | － | Citrate trianion | － |
| Produce ammonia | + | Siderophore | w |
Note :+be expressed as the positive;-be expressed as feminine gender; W is expressed as the weak positive;
Embodiment 3 G1 bacterium produces the effect experimental of water-soluble phosphorus under insoluble inorganic phosphorus culture medium culturing
(1) substratum preparation:
1. indissoluble inorganic phosphorus substratum: glucose 10.0g, ammonium sulfate 0.5g, sodium-chlor 0.3g, Repone K 0.3g, iron vitriol 0.03g, manganous sulfate 0.03g, magnesium sulfate heptahydrate 0.3g, calcium phosphate 10.0g, distilled water 1000mL, pH 7.0-7.5;
2. LB liquid nutrient medium: yeast extract 5.0g, Tryptones 10.0g, sodium-chlor 10.0g, distilled water 1000mL, pH7.0;
3. LB solid medium: yeast extract 5.0g, Tryptones 10.0g, sodium-chlor 10.0g, agar powder 18.0g, distilled water 1000mL, pH7.0.
(2) liquid preparation to be measured
On picking nutrient agar slant medium, G1 thalline lines on LB solid medium flat board, cultivates 24h at 30 DEG C.By G1 thalline access on flat board containing in the 250mL triangular flask of 50ml LB liquid nutrient medium, 30 DEG C, 150r/min, 24h cultivated by shaking table.By centrifugal for nutrient solution 6000r/min 5min, with the resuspended thalline of sterilized water, on vortex mixed instrument, diffusion evenly, and with liquid-transfering gun by the 250mL triangular flask of bacteria suspension equivalent access containing 50mL indissoluble inorganic phosphorus substratum, 30 DEG C, 150r/min, 7d cultivated by shaking table.
(3) water-soluble phosphorus assay
By nutrient solution at 10000g, 4 DEG C of centrifugal 15min, get supernatant liquor, adopt molybdenum antimony resistance colorimetric method to measure water-soluble phosphorus content.If do not connect G1 bacterium, access equivalent sterilized water is contrast, each process repetition 3 times.Water-soluble phosphorus content (unit is μ g/mL, namely represents the micrograms of contained water-soluble phosphorus in every ml sample) test-results is shown in Fig. 3.
The result of Fig. 3 shows: the water-soluble phosphorus content of G1 bacterium is up to 219.6 μ g/mL.G1 bacterium has stronger dissolving P capacity.Though the water-soluble phosphorus content of G1 bacterium is slightly lower than G2 bacterium, comprehensive other performance of bacterial strain (produce growth hormone, produce ammonia ability, to aspects such as plant effects), selects G1 as published object.
The effect test of the producing microbial phosphorus of embodiment 4 G1 bacterium under insoluble inorganic phosphorus culture medium culturing
Precipitation sterilized water after centrifugal in embodiment 3 is washed, 10000g, 4 DEG C of centrifugal 15min, abandoning supernatant, to eliminate water-soluble phosphorus residual in centrifuge tube.Precipitation lysate process (5mg N,O-Diacetylmuramidase joins in 1mL TEN solution), be placed in digital display thermostat water bath, 37 DEG C of water-bath 1h, period shakes for several times gently, is beneficial to sufficient reacting and carries out, then 4 DEG C, the centrifugal 15min of 10000g, get supernatant liquor, adopt molybdenum antimony resistance colorimetric method to measure microbe-P, each process repeats 3 times.Test-results is shown in Fig. 4.Result shows: the microbe-P content of G1 bacterium is up to 70.7 μ g/mL.
Embodiment 5 G1 bacterium produces growth hormone effect test
(1) growth hormone qualitative test: be inoculated in by G1 bacterium in the 250mL triangular flask of the 50mL LB liquid nutrient medium containing L-Trp (100 μ g/mL), 30 DEG C, 24h cultivated by 150r/min shaking table.Get 50 μ L and cultivate drop on whiteware plate, add 50 μ L Salkowski color solution (35%HClO simultaneously
4+ 1mL0.5mol/LFeCl
3), if the sterile LB medium of 50 μ L is as negative control, the color solution of 10,20,50 (μ g/mL) growth hormone is as positive control.Observe after whiteware plate is placed 30min under room temperature, lucifuge condition, the color person of reddening represents and can produce growth hormone.
(2) growth hormone quantitative assay: be inoculated in by G1 bacterium in the 50mL LB liquid nutrient medium containing L-Trp (μ g/mL), three repetitions, 30 DEG C, 24h cultivated by 150r/min shaking table.By nutrient solution difference centrifugal 10min at 8,000 xg, get 4mL supernatant liquor and add 4mL Salkowski color solution, mixing, 30min is left standstill in room temperature, dark, use ultraviolet spectrophotometer, under 530nm, survey absorbancy, do same treatment in contrast with aseptic culture medium simultaneously, adopt the growth hormone of 0,10,20,30,50 (μ g/mL) to make typical curve.Test-results is shown in Fig. 5.G1 bacterium product growth hormone amount is the highest, reaches 7.1 μ g/mL.
Embodiment 5 G1 bacterium produces ammonia effect test
Substratum: peptone 5g, distilled water 1000ml, pH7.2, packing test tube, 121 DEG C of sterilizing 15min.
Reagent (Nessler): 20g potassiumiodide is dissolved in 50ml water, and add red mercury iodide granule (about 32g) to solution is saturated in this solution and after this add 460mL water and 134g potassium hydroxide again, supernatant liquor is stored in shaded bottle for subsequent use.
G1 bacterium is inoculated in peptone water medium in right amount, puts incubated at room temperature 3d, add reagent number and drip, observe whether occur tan precipitate.Test-results is shown in Fig. 6.Result shows: G1 bacterium has product ammonia function.
Embodiment 6 G1 bacterium promotes the test of plant seed germination efficacy in laboratory
(1) collecting cells:
1. by some access LB liquid nutrient medium after actication of culture, through 24h shaking culture, culture temperature is 30 DEG C, and rotating speed is 150r/min; Described liquid culture based component is: yeast extract 5.0g/L, Tryptones 10.0g/L, sodium-chlor 10.0g/L, pH7.0;
2. bacterium drop is entered blood counting chamber, count under an optical microscope, draw cell concentration in bacterium liquid;
3. bacterium liquid is centrifugal in supercentrifuge, rotating speed 6000r/min, centrifugal 5min;
4. by resuspended for 0.85% physiological saline of thalline sterilizing;
5. be 2.0 × 10 for soaking the bacterial strain concentration of Plantula Brassicae chinensis seed
7the bacteria suspension of cfu/ml.
(2) seed-coat sterilising treatment: seed 75% alcohol immersion 1min, then soak 2min with 2% chlorine bleach liquor, be placed on sterilizing filter paper and naturally dry.
(3) G1 bacterium and blank is adopted to carry out Piglet s colibacillosis, be divided into 2 treatment group (the 1st group adopts 25mLG1 bacterium re-suspension liquid), represent with G1, the 2nd group adopts the process of 25ml 0.85% physiological saline as blank, represents with CK).Each treatment group establishes four repetitions, respectively repeats containing 10 Plantula Brassicae chinensis seeds (after random selecting surface sterilization uniform seed).Treatment group G1 adopts bacteria suspension to soak 30min, CK and adopts the same process of 30ml 0.85% physiological saline do.Get sterilizing culture dish, add two-layer aseptic filter paper, after removing soak solution, put into culture dish with aseptic nipper gripping seed, 10, every ware, then add lid layer aseptic filter paper, to soak into preservation moisture, be placed in 22 DEG C of incubators and cultivate 7 days, water every day in right amount, the water yield of every ware is identical, evenly, results of regular determination percentage of germination, the indexs such as seedling is long, fresh weight.Test-results shows, wall genus bacillus (Bacillus muralis) G1 that the present invention is separated significantly can promote Plantula Brassicae chinensis rate of emergence, the 3rd day seedling length, the 7th day seedling length, root length, fresh weight add 45.8%, 44.7%, 31.8%, 30.4%, 30.0% compared with the control respectively.
Table 2 G1 bacterium is to Plantula Brassicae chinensis seed germination facilitation effect contrast table
Note: data are 4 mean value ± standard errors repeated, in table, the different alphabetical person of same column, represents at 0.05 level difference significantly (DMR method).
Embodiment 7 G1 bacterium promotes the test of potted plant Growth of Lettuce effect
(1) collecting cells:
1. by some access LB liquid nutrient medium after actication of culture, through 24h shaking culture, culture temperature is 30 DEG C, and rotating speed is 150r/min; Described liquid culture based component is: yeast extract 5.0g/L, Tryptones 10.0g/L, sodium-chlor 10.0g/L, pH7.0;
2. bacterium drop is entered blood counting chamber, count under an optical microscope, draw cell concentration in bacterium liquid;
3. bacterium liquid is centrifugal in supercentrifuge, rotating speed 6000r/min, centrifugal 5min;
4. by resuspended for 0.85% physiological saline of thalline sterilizing;
5. be 3.0 × 10 for inoculating the bacterial strain concentration of romaine lettuce
8the bacteria suspension of cfu/ml.
(2) romaine lettuce nursery:
Romaine lettuce washing vernalization, soaks into preservation moisture with filter paper, is placed in 25 DEG C of incubators, move in 50 hole seedling-growing containers, 1, every hole when seminal root reaches 1-2mm.Water every day in right amount, when romaine lettuce seedling grows 3 leaves, for subsequent usely to transplant seedlings.
(3) test design:
Carry out Piglet s colibacillosis with G1 bacterium and blank, (the 1st group adds G1 bacterium 3 × 10 to be divided into 2 treatment group
6cfu/gsoil), represent with G1, the 2nd group adds 0.85% physiological saline 30ml as blank, represents with CK), each treatment group repeats 4 times.The every basin of each treatment group fills native 3.0kg, and use chemical fertilizer urea, calcium phosphate, Repone K, amount of application is respectively: 50mg N kg
-1soil, 25mg P
2o
5kg
-1soil, 50mg K
2o kg
-1soil.
Before transplanting seedlings, fertilizer is mixed with soil, select the uniform romaine lettuce seedling of growing way and move in basin, after transplanting seedlings 2 days, water and execute G1 bacteria suspension, water every day in right amount, the water yield of every basin is identical, evenly, notes not making water to flow out at the bottom of basin in order to avoid fertilizer loss and error.On December 28th, 2013 measures the romaine lettuce number of blade (comprising all blades grown), chlorophyll relative content (SPAD): measure (averaging after measuring plant the 3rd leaf 3-4 point) with portable SPAD-502 transmission-type chlorophyll meter.Measure romaine lettuce main root long (romaine lettuce root is to the length at main root tip) on December 29th, 2013, cauline leaf dry weight, root dry weight (105 DEG C of 30min that complete, dry to constant weight for 75 DEG C), result shows (table 3,4): wall genus bacillus (Bacillus muralis) G1 adding the present invention's separation significantly can promote the release of soil available phosphorus (improving 7.8% compared with the control), improve romaine lettuce to the absorption of soil nitrogen phosphorus potassium and dry-matter accumulation.
Table 3 G1 microbial inoculum is to the growth-promoting effect comparison table of romaine lettuce plant
Note: data are 4 mean value ± standard errors repeated, in table, the different alphabetical person of same column, represents at 0.05 level difference significantly (DMR method).Following table is identical.
The effect of table 4 G1 microbial inoculum to soil phosphorus decomposing and the growth-promoting effect comparison table to romaine lettuce
Embodiment 8 G1 bacterium promotes the test of corn growth pot test effect
(1) collecting cells: with romaine lettuce pot experiment.
(2) corn nursery: corn washing vernalization, soaks into preservation moisture with filter paper, is placed in 30 DEG C of incubators, move in 50 hole seedling-growing containers, 1, every hole when seminal root reaches 2mm.Water every day in right amount, when maize seedling grows to 10cm, prepare to transplant seedlings.
(3) test design:
Adopt G1 bacterium and blank to carry out Piglet s colibacillosis, (the 1st group adds G1 bacterium 3 × 10 to be divided into 2 treatment group
6cfu/gsoil), represent with G1, the 2nd group adds 0.85% physiological saline 80ml as blank, represents with CK), each treatment group repeats 4 times.The every basin of each treatment group fills native 8.0kg, and use chemical fertilizer urea, calcium phosphate, Repone K, amount of application is respectively: 50mg N kg
-1soil, 25mg P
2o
5kg
-1soil, 50mg K
2o kg
-1soil.Before transplanting seedlings, fertilizer is mixed with soil, when seedling grows to about 10cm, select the homogeneous maize seedling of size and move in basin, 4, every basin.After transplanting seedlings 2 days, water and execute G1 bacteria suspension, water every day in right amount, the water yield of every basin was identical, evenly, noted not making water to flow out at the bottom of basin in order to avoid fertilizer loss and error.On March 29th, 2014 completes transplanting, respectively at sampling in the 25th day, 35 days after transplanting.Testing index except with romaine lettuce potted plant identical except, another measurement corn the 3rd true leaf length and width are long and leaf is loose as leaf, formula " leaf area=leaf length × leaf wide × 0.75 " is adopted to calculate Estimating Leaf Area In Maize, thick as footpath from soil surface corn second section by vernier callipers mensuration.Result shows (table 5,6): wall genus bacillus (Bacillus muralis) G1 adding the present invention's separation significantly can promote the release of soil available phosphorus (improving 10.3% compared with the control), improve N-P-K content and promotion corn growth in maize leaf.
Table 5 transplant after the 25th and 35 days time G1 microbial inoculum to the growth-promoting effect comparison table of milpa
Note: data are 4 mean value ± standard errors repeated, in table, the different alphabetical person of same column, represents at 0.05 level difference significantly (DMR method).Following table is identical.
Table 6 transplant after the 35th day G1 microbial inoculum on the impact of milpa nutrition absorption and soil available phosphorus content
Embodiment 9 G1 bacterium promotes Growth of Lettuce field plot effect test
(1) collecting cells: with romaine lettuce pot experiment.
(2) romaine lettuce nursery: with romaine lettuce pot experiment.
(3) test site: Agricultural University Of South China farm, test area 21m
2.
(4) for examination soil: the vegetable garden soil that red earth is grown.
(5) test design:
G1 bacterium and blank is adopted to carry out Piglet s colibacillosis, be divided into 2 treatment group (the 1st Zu Mei community adds G1 bacteria suspension 1.2L), represent with G1,2nd Zu Mei community adds 0.85% physiological saline 1.2L as blank, represent with CK), each treatment group respectively sets 3 communities, and (each plot area is as 3.5m
2), randomized complete-block design is taked in 6 communities.When romaine lettuce seedling grows 3 leaves, select the uniform seedling of growing way and transplant, planting density 120 young plant (get rid of edge effect, if the strain of protection row 1, the strain of actual strain number 78).Transplant to water for latter 2 days and execute G1 bacteria suspension, above-mentioned for 1.2L bacteria suspension is added water to 6L, gives shoot root border, 50mL/ strain (ensures that every young plant rhizosphere G1 bacterium quantity is 3.0 × 10
9cfu).Use composite fertilizer (15-15-15, honest science and technology), transplant Qian Mei community fertilising 0.25kg.Transplant on February 20th, 2014, transplant results sampling in latter 35 days.Testing index is potted plant identical with romaine lettuce.Result shows (table 7,8, Figure 10): under field condition, and wall genus bacillus (Bacillus muralis) G1 adding the present invention's separation significantly can promote the growth of romaine lettuce.
Table 7 G1 microbial inoculum is to the growth-promoting effect comparison table of romaine lettuce plant
Note: data are 3 mean value ± standard errors repeated, in table, the different alphabetical person of same column, represents at 0.05 level difference significantly (DMRT method).Following table is identical.
The impact that table 8 G1 microbial inoculum absorbs romaine lettuce plant nutrition
Embodiment 10 G1 bacterium promotes corn growth field plot effect test
(1) collecting cells: with corn pot experiment.
(2) corn nursery: with corn pot experiment.
(3) test site: Agricultural University Of South China farm, test area 31.8m
2.
(4) for examination soil: the vegetable garden soil that red earth is grown.
(5) test design:
G1 bacterium and blank is adopted to carry out Piglet s colibacillosis, (the 1st Zu Mei community adds G1 bacteria suspension 340ml to be divided into 2 treatment group, represent with G1,2nd Zu Mei community adds 0.85% physiological saline 340ml as blank, represent with CK), each treatment group respectively sets 3 communities, and (each plot area is as 5.3m
2), randomized complete-block design is taked in 6 communities.When maize seedling grows to 10cm, select the uniform seedling of growing way and transplant, planting density is 34 strains/community.Transplant to water for latter 2 days and execute G1 bacteria suspension, above-mentioned for 340mL bacteria suspension is added water to 1.7L, gives maize seedling rhizosphere, 50mL/ strain (ensures that every young plant rhizosphere G1 bacterium quantity is 3.0 × 10
9cfu).Use composite fertilizer (15-15-15, honest science and technology), each community rate of fertilizer application 0.25kg, fertilising 0.15kg before transplanting.Transplant on May 16th, 2014, transplant latter 50 days fertilising 0.1kg.Transplant sampling in latter 80 days.Result shows (table 9,10, Figure 11): under the condition of field plot, and wall genus bacillus (Bacillusmuralis) G1 adding the present invention's separation can significantly promote the growth of corn to improve corn yield 12.6%.
The rear impact that G1 microbial inoculum grew milpa in 80th day transplanted by table 9
Note: data are 3 mean value ± standard errors repeated, in table, the different alphabetical person of same column, represents at 0.05 level difference significantly (DMR method).Following table is identical.
Table 10 transplants rear 80th day G1 microbial inoculum to the impact of corn yield
Claims (10)
1. a strain wall genus bacillus G1, deposit number CGMCC No.10189, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 17th, 2014.
2. wall genus bacillus G1 according to claim 1, it is characterized in that, its 16S rDNA nucleotide sequence is as shown in SEQ IDNO:1.
3. wall genus bacillus G1 according to claim 1, is characterized in that, described wall genus bacillus G1 is separation and purification gained from the soil of stove mountain, Guangzhou, Guangdong.
4. prepare the method for wall genus bacillus G1 according to claim 1, it is characterized in that, the method comprises the steps: successively
(1) be separated
Take the soil 10.0g on stove mountain, Guangzhou, Guangdong, put into the bottle that 80-100ml physiological saline is housed, vibration makes cell fully disperse, leave standstill, get 10ml supernatant liquor and be placed in triangular flask, 88-91 DEG C of heating 10-12 minute, after getting 500 μ L water-baths, supernatant liquor is added to 30-35ml indissoluble inorganic phosphorus substratum, carry out enrichment culture, the enrichment culture cycle is 5-7d, enrichment culture got 500 μ L nutrient solutions after terminating and joined separately in new indissoluble inorganic phosphorus substratum and carry out second time enrichment culture first time, after twice enrichment culture terminates, get supernatant liquor and be placed in centrifuge tube, supernatant liquor is got again after centrifugal, measure water-soluble phosphorus content in supernatant liquor, the nutrient solution that reservation water-soluble phosphorus content reaches more than 30mg/L carries out third time enrichment, adopting after three enrichments terminate uses the same method carries out the 4th enrichment culture, cultivation terminates water-soluble phosphorus content in rear mensuration nutrient solution, G1 bacterium water-soluble phosphorus content 220.4 μ g/mL, glycerol stocks method is adopted to preserve this bacterial strain,
(2) purifying
After the G1 bacterium filtered out is utilized plate streak purifying, be stored in the made inclined-plane of beef-protein medium, be placed in 4 DEG C of refrigerators for subsequent use, after molecular biology identification and Physiology and biochemistry qualification, determine that G1 bacterial strain is wall genus bacillus.
5. the preparation method of wall genus bacillus G1 according to claim 4, it is characterized in that, described indissoluble inorganic phosphorus substratum is made up of following component: glucose 10.0g, ammonium sulfate 0.5g, sodium-chlor 0.3g, Repone K 0.3g, iron vitriol 0.03g, manganous sulfate 0.03g, magnesium sulfate heptahydrate 0.3g, calcium phosphate 10.0g, distilled water 1000mL; PH 7.0-7.5.
6. wall genus bacillus G1 according to claim 1 is decomposing the application in insoluble phosphorus.
7. wall genus bacillus G1 according to claim 1 is producing the application in growth hormone.
8. wall genus bacillus G1 according to claim 1 is producing the application in ammonia.
9. wall genus bacillus G1 according to claim 1 is promoting the application in plant seed germination.
10. the application of wall genus bacillus G1 according to claim 1 in Promoting plant growth.
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| CN108516904A (en) * | 2018-07-05 | 2018-09-11 | 安徽袁粮水稻产业有限公司 | A kind of rice fertilizer of improvement Acid Paddy Soils |
| CN111117919A (en) * | 2020-01-07 | 2020-05-08 | 山东农业大学 | Bacillus thuringiensis producing protease and siderophore and application thereof |
| CN111979159A (en) * | 2020-09-03 | 2020-11-24 | 中南大学 | Phosphate solubilizing bacterium agent and preparation method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108239611A (en) * | 2016-12-26 | 2018-07-03 | 北京有色金属研究总院 | One plant of brevibacterium and its method for heavy metals in farmland pollution in-situ immobilization |
| CN108239611B (en) * | 2016-12-26 | 2021-06-08 | 有研资源环境技术研究院(北京)有限公司 | Brevibacterium strain and method for in-situ remediation of heavy metal pollution of farmland by using same |
| CN108516904A (en) * | 2018-07-05 | 2018-09-11 | 安徽袁粮水稻产业有限公司 | A kind of rice fertilizer of improvement Acid Paddy Soils |
| CN111117919A (en) * | 2020-01-07 | 2020-05-08 | 山东农业大学 | Bacillus thuringiensis producing protease and siderophore and application thereof |
| CN111117919B (en) * | 2020-01-07 | 2021-06-04 | 山东农业大学 | Bacillus thuringiensis producing protease and siderophore and application thereof |
| CN111979159A (en) * | 2020-09-03 | 2020-11-24 | 中南大学 | Phosphate solubilizing bacterium agent and preparation method and application thereof |
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