CN101955903B - Bacillus licheniformis bacterial strain and application thereof - Google Patents
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Abstract
The invention discloses a bacillus licheniformis bacterial strain and application thereof. The bacterial strain 1*2-4 was collected in General Microbiology Centre of China Committee for Culture Collection of Microorganisms on June 4th, 2010, wherein the collection number is CGMCC No.3911. The bacterial strain of the invention has the characteristics of nitrogen fixation and high temperature resistance. The invention also provides a separation and purification method for the bacterial strain and application of the bacterial strain in preparing a microbial inoculum or a biofertilizer for promoting plant growth and improving disease resistance, can effectively promote the growth and the disease resistance of clausena lansium, lettuces and other crops.
Description
Technical field
The invention belongs to biological fertilizer field, relate to a kind of novel lichem bacillus strain and the application in the production in farm crop.
Background technology
Bacillus is one type of aerobic or amphimicrobian, the degeneration-resistant endosporic rod-shaped bacterium of generation; With battalion's saprogenesis; Be widespread in nature and mainly be distributed in soil, plant materials surface and the water body; Growth is fast, nutrition simply, very easily separate and cultivate, high-output stress-resistance, heat-resistingly, anti-do, Ginkgo Biloba Leaf Extract, anti-electromagnetic-radiation, reproduction speed is fast, easy grows at plant surface surely.Genus bacillus is as a kind of important Microbial resources; It is rich in enzyme and other abundant meta-bolitess such as proteolytic enzyme, glycase, lipase; The part bacterium can also be decomposed the insoluble phosphate in the soil, plays an important role in soil with other mikrobes.
Bacillus licheniformis not only is widely used at industry, medicine, and controlling plant diseases has certain utility value equally in agriculture prodn, is a very potential biocontrol strain.But (Regitex FA, antibacterial protein, polypeptide class, toluylic acid have reported that obviously pressing down sick effect has gaeumannomyces graminis (Gaeumannomyces graminis var.tritici), early blight of tomato (Alternariasolani), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) late blight of potato (Phytophthora infestans) etc. for Bacillus licheniformis metabolism generation antimicrobial protein and microbiotic.
Calusena lansium (formal name used at school: Clausena lansium), be rutaceae, be evergreen dungarunga, high 5 to 10 meters.Calusena lansium blooms spring, and white Xiao Hua has fragrance.Autumn in fruit summer is ripe, and is avette, long 1 to 3 centimetre, 1 to 2 centimetre of transverse diameter, the fruit succulence, sweet and sour, in the seed of three to four greens is arranged.Calusena lansium is the torrid zone, subtropical fruit, originates in SOUTHERN CHINA, mainly is ground such as Yunnan, Guangdong, Guangxi and Hainan, and is nutritious, of many uses, and economic worth is high.In recent years, Calusena lansium plant husbandry develops rapidly, and planting scale constantly enlarges.Yet at present the fertilizer used of Calusena lansium plantation is main with nitrogen, phosphorus, potash fertilizer, causes easily that fruit quality is poor, economic benefit is low, and causes environmental pollution.Be badly in need of the development research environmental protection, efficiently, bio-fertilizer cheaply, develop in a healthy way to promote Calusena lansium plant husbandry.
Root separation and cultivation and the symbiotic soil microorganisms of root from plant; Then itself and plant inoculation perhaps are prepared into the soil that microbial inoculum or bio-fertilizer are granted plant-growth; Promote the growth of root system of plant, thereby the growth of promotion plant and solid is the focus of studying at present.
Still belong to blank but be applied to the biological bacterial strain of Calusena lansium or other plant plantation aspect or the technology of bacteria agent at present, do not see any technology report.
Summary of the invention
The objective of the invention is to remedy the deficiency of prior art, a kind of have fixed nitrogen, resistant to elevated temperatures novel lichem bacillus strain are provided.
Another object of the present invention provides the cultural method of said bacterial strain.
The present invention also has a purpose to provide the application of said bacterial strain in farm crop produce.
The object of the invention is achieved through following technical scheme:
A kind of novel lichem bacillus strain (Bacillus licheniformis lx2-4) is provided; Be kept at No. 3 China Committee for Culture Collection of Microorganisms in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City common micro-organisms center (CGMCC) on June 4th, 2010, preserving number is CGMCCNo.3911.
The novel Bacillus licheniformis of the present invention has following form and physiology, biochemical characteristic:
A, thalli morphology characteristic
The cell of novel Bacillus licheniformis is Gram-negative bacteria, and is shaft-like, and size is (0.8-1.2) * (1.2-2.5) μ m.
B, colonial morphology characteristic
After cultivating 72h on the Nfb solid medium flat board, colonial morphology is circular or coniform, and smooth surface is the mucus shape, and neat in edge is transparent, and the bacterium colony size is 2～3mm.
C, major physiological biochemical characteristic
Amphimicrobian, methyl red test, urase is measured negative; Produce indoles, voges-Proskauer test (V.P. test), nitrate reduction, hydrogen peroxide is active, produces ammonia test, gelatin hydrolysis, the starch hydrolysis, cellulose hydrolysis all is positive.
Lichem bacillus strain according to the invention (Bacillus licheniformis lx2-4) has 16S rDNA sequence shown in the SEQ ID NO.1.
Confirming of the molecular classification status of the novel lichem bacillus strain of the present invention (Bacillus licheniformis):
Adopt bacterial 16 S rDNA gene universal primer 25f and 1492r to increase; The PCR product is directly carried out sequencing; The dna sequence dna input GenBank that obtains is compared; The result finds that novel lichem bacillus strain of the present invention (Bacillus licheniformis) belongs to bacillus, with lichem bacillus strain (Bacillus licheniformis)) formula bacterial strain IAM13417 has 98% similarity.SDS-PAGE whole-cell protein electrophoresis and IS-PCR fingerprint map analyzing show; Sequential analysis shows that lichem bacillus strain of the present invention (Bacilluslicheniformis lx2-4) and lichens gemma bar type strain IAM 13417 exist than big-difference, should be a new subspecies in the Bacillus licheniformis.
In conjunction with above-mentioned physio-biochemical characteristics, 16S rDNA The sequencing results, novel Bacillus licheniformis of the present invention should belong to bacillus (Bacillus sp.), is a new subspecies of lichem bacillus strain (Bacillus licheniformis).The following document of its determination methods reference: Wei et al, Appl Biochem Biotechnol, 2010,160 (5): 1332-1340; Bezawada et al, Environ Technol, 2010,31 (1): 63-72; Xiong et al, ApplEnviron Microbiol, 2010; Liu et al, Bioresour Technol, 2010,101 (14): 5528-5533; Songsiriritthigul et al, Bioresour Technol, 2010,101 (11): 4096-4103; Manocha and Margaritis, Biotechnol Prog, 2010; Zhang et al, Prep Biochem Biotechnol, 2010,40 (1): 65-75; Tourney et al, J ColloidInterface Sci, 2009,337 (2): 381-389; Li et al, Bioresour Technol, 2009,100 (14): 3650-3656; Banyko and Vyletelova, Lett Appl Microbiol, 2009,48 (3): 318-323; Emptage et al, Biochem Pharmacol, 2009,77 (1): 21-29.
The invention provides the cultural method of said bacterial strain; Through adopting selective medium under the anaerobic and aerobic culture condition; The interior growing nitrogen-fixing bacterium that obtains separates, purifying to separating in the Calusena lansium roots of plants that grows in Fanyu, Guangdong soil; Obtain the novel species that novel Bacillus licheniformis belongs to,, can be used as Inoculant and promote Calusena lansium, naked oats dish and other plant growth and resistance against diseases through discovering that it has fixed nitrogen, high temperature resistant.
Specifically, the separation purification method of bacterial classification of the present invention is following:
The fresh sample of Calusena lansium roots of plants that grows in Fanyu soil is cleaned with zero(ppm) water, cut root then and place sterilized three petridish, with the ydrogen peroxide 50 (H of 3% (concentration of volume percent)
2O
2) soak 3～5min, to remove the rotten material on root surface, sterilized water washs once, uses the mercuric chloride (HgCl of 0.1% (mass percent concentration) again
2) soak, the vibration washing is 5～7 times in the sterilized water, each 5～10min, and last washings coated stop substratum admittedly, whether thorough to detect sterilization.
With surface sterilization completely root be inoculated into respectively in three test tubes that the 5mlNfb semisolid medium is housed with sterilizing scalpel chopping, after the plug sealing, place in 28 ℃ the artificial culture case and cultivate.Entire operation is all accomplished under aseptic condition.
After waiting to grow thalline; In pipe, inject the gas mixture of 1/10 volume; Gas mixture is the acetylene gas of volume percent 10% and 90% air; (acetylene gas volumeter per-cent final concentration is 1%), (28 ℃ artificial culture casees) are cultivated 24h more under the same conditions, measure its nitrogenase activity (acetylene reduction method).
The bacterial strain that will have nitrogenase activity is connected to from the semisolid pipe on the corresponding solid medium, and plate streaking separates, and 28 ℃, humidity is under 85% condition, carries out anaerobism and aerobic cultivation.Picking list bacterium colony is seeded to respectively in the semisolid medium test tube again, to detect nitrogenase activity, is coated with flat board again up to obtaining pure bacterial strain, at last through microscopy, further observes its form and whether confirms purifying.
The Nfb substratum constitutes: malic acid (oxysuccinic acid) 5.0g/L, K
2HPO
40.5g/L, MgSO
47H
2O 0.2g/L, NaCl 0.1g/L, CaCl
20.02g/L; 0.5%bromthymolblue 2.0mL/L (being dissolved in earlier in the 0.2N KOH solution); Vitamin solution (VITAMINs liquid) 1.0mL/L, micronutrient solution (liquid microelement) 2.0mL/L, 1.64%Fe-EDTA solution 4.0mL/L; KOH 4.5g/L, pH=6.8.Wherein vitamin solution (VITAMINs liquid) consists of: biotin (vitamin H) 100.0mg/L, pyridoxol-HCl (pyridoxine hydrochloride, Y factor) 200.0mg/L; Micronutrient solution (liquid microelement) consists of: CuSO
40.4g/L, ZnSO
47H
2O 0.12g/L, H
2BO
31.4g/L, Na
2MoO
42H
2O 1.0g/L, MnSO
41.5g/L.Add 1.7% agar in the solid medium, add 0.15～0.25% agar in the semisolid medium.
The present invention provides the application of said bacterial strain simultaneously.
Bacterial classification of the present invention can be used as Inoculant or bio-feritlizer, for example can single bacterium colony that separation and purification obtains adopted the LB liquid culture cultivated 48 hours based on 28 ℃, and the bacterium number reaches 2.5 * 10
9, CFU.mL
-1, make carrier with vermiculite.Bacillus licheniformis bacterium liquid is mixed with vermiculite (2: 1), be prepared into the bacterium number and reach 1～2 * 10
9CFU.g
-1Microbial inoculum, Inoculant or bio-bacterial manure are applied in the soil of kind of plant, can effectively promote Calusena lansium, naked oats dish and other plant growth and resistance against diseases.
The invention has the beneficial effects as follows:
The invention provides a kind of new lichem bacillus strain, have good fixed nitrogen performance.
The present invention is that tropical fruit Calusena lansium, vegetables crops such as (especially naked oats dishes) provide a kind of good Inoculant with the said microbial inoculum for preparing for new lichem bacillus strain; It derives from the Calusena lansium plant tissue; Can obviously promote the growth, solid and improve the resistance of Calusena lansium of Calusena lansium or vegetables, can overcome and use chemical fertilizer to make soil acidification, be unfavorable for the defective of plant-growth the pathogenic soil mikrobe.
Description of drawings
The sequence alignment result of Fig. 1 two strain bacterium
Fig. 2 the present invention is applied to the design sketch that the naked oats colza is planted test
Fig. 3 the present invention is applied to the design sketch of Calusena lansium planting experiment
Embodiment
Below in conjunction with accompanying drawing and specific embodiment further explain the present invention.Employed TP is ordinary method like no specified otherwise among the following embodiment; Employed material, reagent etc. like no specified otherwise, are the reagent and the material that can obtain from commercial sources.
The fresh sample of Calusena lansium roots of plants of gathering Fanyu soil is cleaned with zero(ppm) water, cut root then and place sterilized three petridish, ydrogen peroxide 50 (H with 3%
2O
2) soaking 4min to remove the rotten material on root surface, sterilized water washs once, uses 0.1% mercuric chloride (HgCl again
2) soaking 5min, the vibration washing is 5 times in the sterilized water, each 8min.With surface sterilization completely root be inoculated into respectively in three test tubes that the 5mlNfb semisolid medium is housed with sterilizing scalpel chopping, after the plug sealing, place in 28 ℃ the artificial culture case and cultivate.Entire operation is all accomplished under aseptic condition.After waiting to grow thalline, in pipe, inject 1/10 volume, 10% acetylene gas (final concentration is 1%), cultivate 24h under the same conditions again, measure its nitrogenase activity (acetylene reduction method).The bacterial strain that will have nitrogenase activity is connected to from the semisolid pipe on the corresponding solid medium, and plate streaking separates, and 28 ℃, humidity is under 85% condition, carries out anaerobism and aerobic cultivation.Picking list bacterium colony is seeded to respectively in the semisolid medium test tube again, detects nitrogenase activity, is coated with flat board again up to obtaining pure bacterial strain, through microscopy, further observes its form at last, and confirms purifying.
The Nfb substratum constitutes: malic acid (oxysuccinic acid) 5.0g/L, K
2HPO
40.5g/L, MgSO
47H
2O 0.2g/L, NaCl 0.1g/L, CaCl
20.02g/L; 0.5%bromthymolblue 2.0mL/L (being dissolved in earlier in the 0.2N KOH solution); Vitamin solution (VITAMINs liquid) 1.0mL/L, micronutrient solution (liquid microelement) 2.0mL/L, 1.64%Fe-EDTA solution 4.0mL/L; KOH 4.5g/L, pH=6.8.Wherein vitamin solution (VITAMINs liquid) consists of: biotin (vitamin H) 100.0mg/L, pyridoxol-HCl (pyridoxine hydrochloride, Y factor) 200.0mg/L; Micronutrient solution (liquid microelement) consists of: CuSO
40.4g/L, ZnSO
47H
2O 0.12g/L, H
2BO
31.4g/L, Na
2MoO
42H
2O 1.0g/L, MnSO
41.5g/L.Add 1.7% agar in the solid medium, add 0.15～0.25% agar in the semisolid medium.
Said bacterial strain has following form and physiology, biochemical characteristic:
A, thalli morphology characteristic
The cell of novel Bacillus licheniformis is Gram-negative bacteria, and is shaft-like, and size is (0.8-1.2) * (1.2-2.5) μ m.
B, colonial morphology characteristic
After cultivating 72h on the Nfb solid medium flat board, colonial morphology is circular or coniform, and smooth surface is the mucus shape, and neat in edge is transparent, and the bacterium colony size is 2～3mm.
C, major physiological biochemical characteristic
Amphimicrobian, methyl red test, urase is measured negative; Produce indoles, voges-Proskauer test (V.P. test), nitrate reduction, hydrogen peroxide is active, produces ammonia test, gelatin hydrolysis, the starch hydrolysis, cellulose hydrolysis all is positive.
The 16S rDNA sequence of said lichem bacillus strain (Bacillus licheniformis lx2-4) is shown in SEQ ID NO.1.Confirming of the molecular classification status of the novel lichem bacillus strain of the present invention (Bacilluslicheniformis):
Adopt bacterial 16 S rDNA gene universal primer 25f and 1492r to increase with reference to laboratory, this area ordinary method; The PCR product is directly carried out sequencing; The dna sequence dna input GenBank that obtains is compared; The result finds that novel lichem bacillus strain of the present invention (Bacillus licheniformis) belongs to bacillus, with lichem bacillus strain (Bacillus licheniformis)) formula bacterial strain IAM 13417 has 98% similarity, and comparison figure sees shown in the accompanying drawing 1.Sequence alignment shows that lichem bacillus strain of the present invention (Bacilluslicheniformis lx2-4) and lichens gemma bar type strain IAM 13417 exist than big-difference, should be a new subspecies in the Bacillus licheniformis.
Primer 2 5f and 1492r sequence are following:
25f：5’-AAC?T(G/T)A?AGA?GTT?TGA?TCC?TGG?CTC-3’；
1492r：5’-TAC?GG(C/T)TAC?CTT?GTT?ACG?ACT?T-3’。
In conjunction with above-mentioned physio-biochemical characteristics, 16S rDNA The sequencing results, novel Bacillus licheniformis of the present invention should belong to bacillus (Bacillus sp.), is a new subspecies of lichem bacillus strain (Bacillus licheniformis).
Separation and purification is obtained lichem bacillus strain called after Bacillus licheniformis (Bacillus licheniformis lx2-4); Be stored in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), preserving number is CGMCC No.3911.
The application experiment of embodiment 2 growing vegetables aspects
Single bacterium colony that activation is obtained adopts the LB liquid culture to cultivate 48 hours based on 28 ℃, mixes (1 liter of nutrient solution mixes 2 kilograms of vermiculites) with vermiculite with 1: 2 weight ratio and processes novel Bacillus licheniformis-vermiculite Inoculant.The LB liquid nutrient medium is formed: Yeast extact (yeast extract paste) 5g, Peptone (peptone) 10g, NaCl (sodium-chlor) 10g, deionized water 1000mL.
In advance soil pH value is adjusted into 5.0,6.0,7.0 respectively; With lichengermium-vermiculite Inoculant; Be applied to 15 days naked oats dish seedling, those skilled in the art are with reference to specifically needing to confirm application dosage, and present embodiment inserts 10g vermiculite Inoculant by per 10 kilograms of mixing with soil and uses.Compare with nonvaccinated contrast (inserting the 10g vermiculite) after 75 days by per 10 kilograms of mixing with soil, the heavy average increase by 20% of naked oats dried vegetable in soil pH value 5.0,6.0,7.0 potted plant, the interior nitrogen of plant on average increases by 20%.In the soil of pH7.0, nonvaccinated naked oats dish leaf appears withered and yellow, and the naked oats dish leaf of inoculation presents normal green; Dry weight increases by 25%; Nitrogen on average increases by 23% in the plant, shows that Bacillus licheniformis has significantly improved the yield and quality of naked oats dish, sees shown in the accompanying drawing 2; Bacterial manure is used in the perpendicular behavior in the left side in the accompanying drawing 2, and the right is a control group.
The application experiment of embodiment 3 Calusena lansium plantation aspects
The single bacterium colony that activation is obtained adopts the LB liquid culture to cultivate 48 hours based on 28 ℃, is that the bio-fertilizer of matrix mixes (20 kilograms of cavings matrix bio-fertilizers of 1 liter of nutrient solution mixing) with 1: 20 weight ratio and processes Bacillus licheniformis-bio-fertilizer with commercial cavings.Bacillus licheniformis-bio-fertilizer is used the Calusena lansium plant in vegetative period, with the conventional chemical fertilizer group contrast of using of Calusena lansium plantation.Apply the Calusena lansium that 1 kilogram of bio-fertilizer is inoculated bacterial manure by every wampee rhizosphere, completely filled fruit, size is even, and leaf look strong green is seen shown in the accompanying drawing 3 left figure; Nonvaccinated contrast (applying 1 kilogram of cavings matrix) group by every wampee rhizosphere, fruit is not of uniform size to be caused, and the leaf look is a yellow-green colour, sees shown in the accompanying drawing 3 right figure.The setting percentage of the Calusena lansium of inoculation bacterial manure improves 7%, and the fruit weight in average increases by 10%.
Claims (4)
1. a lichem bacillus strain (Bacillus licheniformis) 1x2-4; Be kept at No. 3 China Committee for Culture Collection of Microorganisms common micro-organisms centers, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on June 4th, 2010, preserving number is CGMCC No.3911.
2. the described Bacillus licheniformis of claim 1 is characterized in that having 16S rDNA sequence shown in the SEQ ID NO.1.
3. the application of claim 1 or 2 described lichem bacillus strains is characterized in that being applied to prepare bacteria agent or the bio-feritlizer that is applicable to wampee or naked oats dish.
4. application according to claim 3 is characterized in that it being that claim 1 or 2 said strain cultured solutions are mixed with vermiculite, is prepared into the bacterium number and reaches 1～2 * 10
9CFU.g
-1Microbial inoculum or bio-feritlizer, microbial inoculum or bio-feritlizer are applied in the soil of plantation wampee or naked oats dish.
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| CN201010297391XA CN101955903B (en) | 2010-09-28 | 2010-09-28 | Bacillus licheniformis bacterial strain and application thereof |
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| CN101955903B true CN101955903B (en) | 2012-06-27 |
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| CN102765809B (en) * | 2012-07-05 | 2013-10-16 | 昆明豪原特自控有限公司 | Biological wetland forming growth promoter and preparation method thereof |
| CN103555624B (en) * | 2013-10-25 | 2015-08-19 | 王天喜 | One strain fixed nitrogen Bacillus licheniformis and uses thereof |
| UY36334A (en) * | 2014-12-29 | 2017-04-28 | Fmc Corp | COMPOSITIONS OF BACILLUS LICHENIFORMIS RT1184 AND USES METHODS TO BENEFIT THE GROWTH OF PLANTS |
| AR102123A1 (en) * | 2014-12-29 | 2017-02-08 | Fmc Corp | SEED MATRICES THAT INCLUDE MICROORGANISMS OF BACILLUS SPP. TO BENEFIT THE GROWTH OF THE PLANT |
| CN106180175B (en) * | 2016-07-11 | 2019-04-16 | 绿艺园林建设有限公司 | A method of plantation rape restoration of soil polluted by heavy metal |
| CN110791456B (en) * | 2019-11-26 | 2022-03-04 | 黄河三角洲京博化工研究院有限公司 | Salt-tolerant growth-promoting bacillus and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1110708A (en) * | 1994-04-21 | 1995-10-25 | 长濑生化学工业株式会社 | Bacterial preparation for agricultural use no priority right |
| CN101050424A (en) * | 2007-03-19 | 2007-10-10 | 中国科学院昆明植物研究所 | Method for preparing endophyte of plant |
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2010
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1110708A (en) * | 1994-04-21 | 1995-10-25 | 长濑生化学工业株式会社 | Bacterial preparation for agricultural use no priority right |
| CN101050424A (en) * | 2007-03-19 | 2007-10-10 | 中国科学院昆明植物研究所 | Method for preparing endophyte of plant |
Non-Patent Citations (1)
| Title |
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| 张毅民等.微生物菌群在生物有机肥制备中研究进展.《化学工业与工程》.2003,第20卷(第6期),第523－527页. * |
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Address after: 510000, Guangzhou, Guangdong, Panyu District, South Village, seven star feet Patentee after: GUANGZHOU YONGXIONG BIOLOGICAL SCIENCE & TECHNOLOGY CO.,LTD. Address before: 511442, Guangdong, Guangzhou province Panyu District town south village near the Jinshan Road Patentee before: GUANGZHOU YONGXIONG ORGANIC FERTILIZER Co.,Ltd. |
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Effective date of registration: 20220909 Address after: No. 02-12-818, Dongyuan Street, Hangban Industrial Park, Xilinhot City, Xilin Gol League, Inner Mongolia Autonomous Region, 010000 Patentee after: Inner Mongolia sijifengying agriculture and animal husbandry technology Co.,Ltd. Address before: Qixinggangjiao, Nancun Town, Panyu District, Guangzhou City, Guangdong Province, 510000 Patentee before: GUANGZHOU YONGXIONG BIOLOGICAL SCIENCE & TECHNOLOGY CO.,LTD. |
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