CN101760439B - Bacillus pumilus NMCC 46 and application thereof - Google Patents
Bacillus pumilus NMCC 46 and application thereof Download PDFInfo
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- CN101760439B CN101760439B CN200910223358XA CN200910223358A CN101760439B CN 101760439 B CN101760439 B CN 101760439B CN 200910223358X A CN200910223358X A CN 200910223358XA CN 200910223358 A CN200910223358 A CN 200910223358A CN 101760439 B CN101760439 B CN 101760439B
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Images
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
Abstract
The invention relates to Bacillus pumilus NMCC 46, which is preserved in CGMCC with preservation date as November 2, 2009, and preservation number as CGMCC No.3378. The Bacillus pumilus NMCC 46 CGMCC No.3378 in the invention is a biological control bacillus separated from grass root surrounding soil near Namco Lake in Tibet, which can grow at 10 DEG C, is favorable for exerting growth promotion and biological control effects, can restrain soybean root pythium myriotylum and saccharomycetes, can boost growth of Arabidopsis and soybean, has a prevention and control effects on soybean root pythium myriotylum as high as 85%, can increase yield of soybean to 24%, and has good application prospect.
Description
Technical field
The present invention relates to a kind of bacillus pumilus, specifically, is that a kind of short low temperature living and prophylaxis effect of Tibet tool that is located away from is fitted living bacillus pumilus (Bacillus pumilus) NMCC46 and the application in control soybean root rot disease thereof.
Background technology
Root rot all has generation in China soybean producing region at present, and for example in the Northeast, China soybean main producing region, the field diseased plant rate is 30%～40%, and bottom land and second crop soil diseased plant rate reach more than 70%, and the highest death rate reaches more than 15%; And in the east soil humid area, general time soybean fertility early stage (before flowering period) diseased plant rate is about 75%, disease index is 35%～50%, rainy time disease index can reach more than 60%, the safety of soybean in China in serious threat.For a long time; Chemical pesticide is the important means of preventing and treating the phytopathy Chinese caterpillar fungus plague of rats always; But a series of problems such as soil fertility decline, environmental pollution and pathogenic bacteria that the agricultural chemicals abuse is brought develop immunity to drugs are constantly impelling people to go biological field to seek low toxicity, efficient, eco-friendly novel agrochemical.Plant rhizosphere is urged endophytic bacteria (PlantGrowth-Promoting Rhizobacteria; Be called for short PGPR) because and have good mutualism relation between the plant; Not only can produce disease resistance by inducing plant; And can promote plant-growth, raising crop yield, thereby become the one type of biocontrol bacteria that attracts tremendous attention, but do not see the report of the biocontrol bacteria that is specifically designed to control soybean root rot disease at present.
Summary of the invention
The object of the invention is to provide a kind of to be had better control effect, and can effectively promote the bacillus pumilus of plant growth for the soybean root rot disease.
Above-mentioned purpose realizes through following technical scheme:
A kind of bacillus pumilus; Classification called after bacillus pumilus (Bacilluspumilus) NMCC46; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC), preservation day: on November 2nd, 2009, preserving number: CGMCC No.3378.
Bacillus pumilus (Bacillus pumilus) NMCC46 CGMCC No.3378 is that grass roots encloses the soil and separates from the Nam Co Basin in Tibet lake, obtains through dull and stereotyped face-off screening.
Bacillus pumilus (Bacillus pumilus) NMCC46 CGMCC No.3378 is gram positive bacterium, and is shaft-like, can be 10 ℃ of growths; Be that a kind of low temperature is fitted living bacterium; Also can grow at 50 ℃, can tolerate the cultivation of 7%NaCl, but be impatient at the cultivation of pH5.7; Catalase test, Citrate trianion utilization test, V-P test, glucose fermentation and casein test are all positive, and starch hydrolysis experiment, nitrate reduction test, anaerobic growth test and propionic salt utilization test are all negative.
Utilize the 16S rDNA sequence of round pcr amplification bacillus pumilus (Bacillus pumilus) NMCC46CGMCC No.3378; Obtaining length is the fragment of 482bp; Known 16S rDNA among test result (shown in sequence table) and the GenBank is carried out homology relatively; Discovery is the highest with the similarity of Bacillus pumilus EF157741.1, is 94%.
According to the gramstaining of thalline, the morphological specificity of bacterium colony, the Physiology and biochemistry character of bacterial strain and the result of 16S rDNA sequential analysis; With reference to " bacillus " (Cai Miaoying etc. translate, agriculture press, 1988) and " microbial taxonomy " (Zhang Jizhong; Fudan University in Shanghai press; Nineteen ninety-five) key is retrieved, and is bacillus pumilus (Bacillus pumilus) with this identification of strains, called after NMCC46.
The microbial inoculum that contains bacillus pumilus (Bacillus pumilus) NMCC46 CGMCCNo.3378 also belongs to protection scope of the present invention.
The application method of bacillus pumilus (Bacillus pumilus) NMCC46 CGMCC No.3378 bacterial strain is fermentation culture → dilution → sprinkling → effect measuring.
The fermention medium of bacillus pumilus (Bacillus pumilus) NMCC46 CGMCC No.3378 consists of: glucose 20g/L, L-L-glutamic acid 5g/L, MgSO
40.5g/L, KCl 0.5g/L, KH
2PO
41g/L, Fe
2SO
46H
2O 0.15mg/L, MnSO
4H
2O 5.0mg/L, CuSO
45H
2O 0.16mg/L, all the other are filtered water, and are adjusted to pH7.0.
Bacillus pumilus (Bacillus pumilus) NMCC46 CGMCC No.3378 has significant fungistatic effect to pathogen of soybean root rot and yeast, and Arabidopis thaliana and soybean are had the significant short fruit of coming into force.
Description of drawings
Fig. 1 is brood cell's photo of embodiment 2 bacillus pumilus (Bacillus pumilus) NMCC46CGMCC No.3378;
Fig. 2 is the fungistatic effect photo of embodiment 6 bacillus pumilus (Bacillus pumilus) NMCC46CGMCC No.3378 for pathogen of soybean root rot;
Fig. 3 is that embodiment 8 bacillus pumilus (Bacillus pumilus) NMCC46CGMCC No.3378 is for the saccharomycetic fungistatic effect photo of fungi.
Embodiment
Below further describe the present invention through embodiment; Can know by technological general knowledge; The present invention also can describe through other the scheme that does not break away from technical characterictic of the present invention, thus all within the scope of the present invention or the change that is equal in the scope of the invention all comprised by the present invention.
Embodiment 1:
Adopt five point samplings, remove table soil, take soil sample tens grams in 5～25mm place with little scoop, in the sampler bag of packing into, good sampling time of mark, place, vegetation.Take by weighing in 5g soil sample and the 45ml saline water, concussion is 30 minutes on the shaking table, leaves standstill and makes clarification.Draw supernatant respectively with sterilized water dilution 10
-1, 10
-2, 10
-3, 10
-4, 10
-5Five concentration gradients, 80 ℃ of water-bath 10min.Draw 200 μ l and be coated with the LB flat board, 37 ℃ of incubated overnight.After treating to grow bacterium colony on the flat board, picking list bacterium colony, numbering is preserved behind the line purifying, and screening obtains bacillus pumilus (Bacillus pumilus) NMCC46C GMCC No.3378.
Embodiment 2:
Bacillus pumilus (Bacillus pumilus) NMCC46CGMCC No.3378 to embodiment 1 carries out morphological specificity observation and physiological and biochemical property evaluation, and the result shows that this bacterium is Gram-positive, and is shaft-like, produces gemma (as shown in Figure 1).Can not only be 10 ℃ of growths; And can be 50 ℃ of growths; Can tolerate the cultivation of 7%NaCl; But be impatient at the cultivation of pH5.7, catalase test, Citrate trianion utilization test, V-P test, glucose fermentation and casein test are all positive, and starch hydrolysis experiment, nitrate reduction test, anaerobic growth test and propionic salt utilization test are all negative.
Embodiment 3:
With bacillus pumilus (Bacillus pumilus) the NMCC46CGMCC No.3378 inoculation of embodiment 1 in liquid LB substratum; 180rpm, centrifugal collection thalline was cultivated in 37 ℃ of concussions 10 hours; Add N,O-Diacetylmuramidase and SDS broken wall after suspending, adopt phenol-chloroform method to extract genomic dna.With the total DNA that extracts is template, with forward primer PRB1601 (5 '-GGATCCTAATACATGCAAGTCGAGCGG-3 ') and reverse primer PRB1602 (5 '-GGATCCACGTATTACCGCGGCTGCTGGC-3 ') the 16S rDNA gene of bacterial strain is carried out pcr amplification.The PCR condition is: 95 ℃, and 4min; 94 ℃, 1min; 50 ℃, 1min; 72 ℃, 2min; 34cycle; 72 ℃, 10min, 4 ℃ of preservations.Checks order after the PCR product reclaims, through confirming that its length is 482bp, sequencing result is 94% with Bacillus pumilusEF157741.1 similarity in GenB ank shown in sequence table.
Embodiment 4:
Substratum:
The 1C substratum
| Moity | Concentration | 1×1C | 5×1C |
| Tryptones | 0.7 | 4.2g | 21g |
| Sodium-chlor | 0.5 | 3g | 15g |
| Soybean papain hydrolyzate | 0.3 | 1.8g | 9g |
| Final volume | 600ml | 600ml |
Soil extract
Main medium
| Moity | Final concentration | Volume (ml) |
| 5×1C | 1× | 50 |
| 20% glucose | 0.1％ | 1.25 |
| Soil extract | 10％ | 25 |
| Water | 173.75 |
Bacillus pumilus (Bacillus pumilus) the NMCC46CGMCC No.3378 of embodiment 1 is inoculated in the aforesaid liquid substratum; 37 ℃ of overnight cultures; Get 5ul bacterium liquid and put the right-angled intersection point apart from the dull and stereotyped center 2.5cm of solid medium, 5 repetitions of each bacterial strain are placed on respectively in 4 ℃, 10 ℃, 14 ℃, 18 ℃, 30 ℃ the incubator and cultivate; Write down the growing state of bacterial strain every day, write down altogether 15 days.The result shows; Bacillus pumilus of the present invention (Bacillus pumilus) NMCC46 CGMCC No.3378 can grow in the environment of temperature≤10 ℃; We will be under this temperature; The bacterial strain that can in a week, manifest bacterium colony is designated as the low-temperature epitaxy bacterial strain, obtains low temperature and fits living bacillus pumilus (Bacillus pumilus) NMCC46 CGMCC No.3378.
Embodiment 5:
Bacillus pumilus (Bacillus pumilus) the NMCC46CGMCC No.3378 of embodiment 1 is seeded on the LB substratum; Incubated overnight; Then activatory bacillus pumilus (Bacillus pumilus) NMCC46 CGMCC No.3378 is transferred to and carries out shake flask fermentation in the Landy substratum, temperature is 30 ℃, and shaking speed is 200rpm; Fermentation time is 38 hours, gets the fermented liquid of bacillus pumilus (Bacillus pumilus) NMCC46 CGMCC No.3378.
Consisting of of fermention medium wherein: 20g glucose, 5g L-L-glutamic acid, 0.5gMgSO
4, 0.5g KCl, 1g KH
2PO
4, 0.15mg Fe
2SO
46H
2O, 5.0mgMnSO
4H
2O, 0.16mg CuSO
45H
2O uses filtered water to complement to TV and is 1L, is adjusted to pH7.0.
Embodiment 6:
Adopt dull and stereotyped face-off method; Good PDA is dull and stereotyped, at the pathogen of soybean root rot bacterium piece that diameter 1cm places in dull and stereotyped central authorities, around it, places four filter papers apart from 2cm place symmetry; On filter paper, make a call to the bacteria suspension of bacillus pumilus (Bacillus pumilus) the NMCC46 CGMCC No.3378 of 5 μ l embodiment 1; Triplicate was cultivated 5 days in 25 ℃ of illumination boxs, and the result is as shown in Figure 2; Average diameter of inhibition zone is 18mm, and bacillus pumilus promptly of the present invention (Bacillus pumilus) NMCC46 CGMCC No.3378 is remarkable for the fungistatic effect of pathogen of soybean root rot.
Embodiment 7:
The Arabidopis thaliana seed is seeded on the MS culture medium flat plate (9cm) after Youxiaolin (effectively Cl content is 5%) surface sterilization, and is subsequent use after one week of hot-house culture.
The low temperature of embodiment 4 being fitted living bacillus pumilus (Bacillus pumilus) NMCC46 CGMCC No.3378 be inoculated into overnight cultures in the LB liquid nutrient medium, after the mensuration OD value, quantitatively is 10 with its concentration
5After cfu/ml, Arabidopis thaliana seedling soak root 5min with bacterium liquid, plant MS dull and stereotyped (on 20 * 20cm), each dull and stereotyped 4 seedling, each bacterial strain is provided with 3 repetitions.The fresh weight of every Arabidopis thaliana of dull and stereotyped weighing is taken out in hot-house culture 21 days.Negative control group is soaked the Arabidopis thaliana of root for the fresh liquid substratum, and positive controls is the Arabidopis thaliana that FZB42 soaks root.Test-results: using concentration is 10
5The average fresh weight of Arabidopis thaliana that the bacteria suspension of cfu/ml is handled is 0.079017g, and the average fresh weight of the Arabidopis thaliana of negative control group is 0.03944g, and the average fresh weight of the Arabidopis thaliana of positive controls is 0.05393g.Be that bacillus pumilus of the present invention (Bacillus pumilus) NMCC46 CGMCC No.3378 has the well short fruit of coming into force to Arabidopis thaliana.
Embodiment 8:
The low temperature of embodiment 4 is fitted livings bacillus pumilus (Bacillus pumilus) NMCC46 CGMCC No.3378 to be inoculated in the 1C substratum behind the cultivation 8h; Be transferred to 1: 100 ratio in the Landy substratum of 10ml; Fermented about 40 hours, centrifugal collection supernatant, 4 ℃ of storages are subsequent use.
Fungal bacterial strain yeast to be measured is inoculated into overnight cultures in the appropriate media, after the mensuration OD value bacterium liquid is diluted to 10
7Cfu/ml, mixed culture medium process and contain the bacterium flat board.To be cooled fully after, right-angled intersection punching (anomaly plate center 2.5cm), inoculation bacillus pumilus (Bacillus pumilus) NMCC46 CGMCC No.3378 is in Landy substratum fermented supernatant fluid.Place under the room temperature and cultivate 2d, observe fungistatic effect.The result is as shown in Figure 3, and antibacterial circle diameter is 28mm, and bacillus pumilus promptly of the present invention (Bacilluspumilus) NMCC46 CGMCC No.3378 has significant inhibitory effect to the fungi yeast.
Embodiment 9:
Soybean seeds soaks 2h with clear water; Vernalization under 28 ℃ of wet conditions; Be seeded in the flowerpot that aseptic soil is housed; When treating big bean seedlings length to 3 slice true leaf, irritate root with bacteria suspension, the big bean seedlings of average every strain water bacillus pumilus (Bacilluspumilus) the NMCC46 CGMCC No.3378 bacterium liquid of 20ml embodiment 5, and (concentration is 10
8Cfu/ml), watered this bacterium liquid once at a distance from 3 days later, totally 4 times, do contrast with clear water.Two weeks back extraction, 10 young plants are measured plant height, root length and plant fresh weight.The result shows, it is good that the big bean seedlings of handling through bacterium liquid are obviously compared the photograph growing way.Through measuring, the plant height increment rate reaches 60.2%, and the long increment rate of root reaches 5.2%, and the fresh weight increment rate reaches 150.3%.Be that bacillus pumilus of the present invention (Bacillus pumilus) NMCC46 CGMCC No.3378 has tangible growth-promoting functions to soybean.
Embodiment 10:
With 100 times of the fermented liquid dilutions of bacillus pumilus (Bacillus pumilus) the NMCC46CGMCC No.3378 of embodiment 5, then soybean seeds is soaked wherein, take out and dry the back sowing; After soybean planting is emerged, begin to irritate root, continuous irrigation 3 times was irritated once in 7 days in every interval.Control effect for root rot can reach 85%; Increase soybean yields and reach 24%; Be that bacillus pumilus of the present invention (Bacillus pumilus) NMCC46CGMCC No.3378 can obviously suppress root rot, and can effectively promote soybeans they grow, increase soybean yields.
Sequence table
AGGGCGTGGGAAGAGAACGGCTGGGTGTCGACGGCGGAGGGA
GATAAAAGCGGGAAACCAGCTGGTAAGAGGGGGAGAACACTG
GGAAAAAGGGGCAAATACCGGATAGTACCTAGAACCGCATGGT
TCAAGGAAGAAAGACGGTTTCGGCTGTCACTTGCAGATGGACC
CGCGGCGCATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCG
ACGATGCGTACCCGACCTGAGAGGGTGATCGGCCACACTGGGA
CTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAA
TCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGA
GTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGA
ACAAGTGCGAGAGTAACTGCTCGCACCTTGACGGTACCTAACC
AGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACG
GGGAATCCC
Claims (4)
1. a bacillus pumilus (Bacillus pumilus) NMCC46 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and preservation date is on November 2nd, 2009, and preserving number is CGMCC No.3378.
The described bacillus pumilus of claim 1 (Bacillus pumilus) NMCC46CGMCC No.3378 for soybean root rot disease control, promote the application method of plant growth; It is characterized in that, comprise fermentation culture → dilution → four steps of sprinkling → effect measuring.
3. the application method of bacillus pumilus according to claim 2 (Bacillus pumilus) NMCC46C GMCC No.3378 for the control of soybean root rot disease, promotion plant growth; It is characterized in that; Fermention medium in the said fermentation culture process consists of: glucose 20g/L; L-L-glutamic acid 5g/L, MgSO
40.5g/L, KCl0.5g/L, KH
2PO
41g/L, Fe
2SO
46H
2O0.15mg/L, MnSO
4H
2O5.0mg/L, CuSO
45H
2O0.16mg/L, all the other are filtered water, and are adjusted to pH7.0.
4. the bacillus pumilus of claim 1 (Bacillus pumilus) NMCC46CGMCC No.3378 is suppressing pathogen of soybean root rot and the application that promotes in soybeans they grow, the raising soybean yields.
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| CN102443553B (en) * | 2011-11-09 | 2013-06-19 | 扬州大学 | Wide spectrum diseases resistance and growth promotion bacillus strain |
| CN102994419B (en) * | 2012-11-01 | 2015-05-06 | 无锡亚克生物科技有限公司 | Bacillus pumilus LNXM12 and application thereof |
| CN102978130B (en) * | 2012-11-01 | 2015-05-06 | 无锡亚克生物科技有限公司 | Bacillus pumilus GBSC66 and its application |
| CN103409348B (en) * | 2013-07-25 | 2014-07-23 | 南京厚稷生物科技有限公司 | Bacillus pumilus with efficient alga-lysing activity and application thereof |
| CN103395892B (en) * | 2013-07-25 | 2014-10-15 | 南京农业大学 | Application of dipeptide compound bacilysin in inhibition of growth and propagation of water bloom algae |
| UY36332A (en) * | 2014-12-29 | 2017-04-28 | Fmc Corp | COMPOSITIONS OF BACILLUS PUMILUS RT1279 AND METHODS OF USE TO BENEFIT THE GROWTH OF PLANTS |
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| CN106119155B (en) * | 2016-06-24 | 2019-08-13 | 中国科学院南京土壤研究所 | For preventing and treating the bacterial strain WXX-2 and microbial inoculum of peanut root rot |
| CN107058458B (en) * | 2017-05-12 | 2020-11-24 | 河南农业大学 | Method for detecting colonization of plant rhizosphere growth-promoting bacteria in root system |
| CN110157640B (en) * | 2019-05-20 | 2020-07-31 | 江苏师范大学 | Burdock endophytic bacterium WY035, Burdock special microbial agent containing endophytic bacterium and application |
| CN113913339A (en) * | 2021-11-08 | 2022-01-11 | 南昌航空大学 | Soybean microbial fertilizer and preparation method and use method thereof |
| CN117305163A (en) * | 2023-09-26 | 2023-12-29 | 中国科学院兰州化学物理研究所 | Bacillus pumilus with plant growth regulating activity, and preparation and application thereof |
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| 崔云龙等.短小芽孢杆菌D82对小麦根腐病原菌拮抗的研究.《中国生物防治》.1995,第11卷(第3期),114-118. * |
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