CN101928680A - Bacillus simplex RJGP41 and application thereof - Google Patents
Bacillus simplex RJGP41 and application thereof Download PDFInfo
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- CN101928680A CN101928680A CN2009102233560A CN200910223356A CN101928680A CN 101928680 A CN101928680 A CN 101928680A CN 2009102233560 A CN2009102233560 A CN 2009102233560A CN 200910223356 A CN200910223356 A CN 200910223356A CN 101928680 A CN101928680 A CN 101928680A
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Images
Abstract
The invention relates to bacillus simplex RJGP41 which was collected in China General Microbiological Culture Collection Center (CGMCC) in November 2, 2009, with collection number of CGMCC No.3381. The bacillus simplex RJGP41 with collection number of CGMCC No.3381 is biocontrol bacillus isolated from surrounding oil of dandelion roots in Guo'an village, Jiangdang town, Shigatse city, Tibet, can grow at 4 DEG C, grows well under low temperature, is beneficial to giving play to growth-promoting and biocontrol effects, has rate of controlling soft rot of Chinese cabbage as high as 83% and simultaneously increases the output of the Chinese cabbage by 50%.
Description
Technical field
The present invention relates to a kind of simple pure bacillus, specifically, is a kind of be located away from short suitable simple pure genus bacillus (Bacillussimplex) RJGP41 of giving birth to of low temperature living and prophylaxis effect of Tibet tool and application in control soft rot of Chinese cabbage evil thereof.
Background technology
Soft rot of Chinese cabbage is one of Chinese cabbage three big diseases, is caused that by Erwinia this bacterium is prevalent in the soil, propagate by rainwater and irrigation water, germ just can be infected morbidity as long as contact with the Chinese cabbage wound, as untimely control, will directly influence the yield and quality of Chinese cabbage.Comprise that for crop Micobial Disease soft rot of Chinese cabbage lacks the effective chemical medicament at present, and a large amount of uses of chemical pesticide have not only caused the pollution of air, water source, soil and food, the more important thing is that the orthoselection through chemical pesticide makes pathogenic bacteria produce resistance, the urgent hope of people can search out low toxicity, efficient, eco-friendly novel agrochemical at biological field.Plant rhizosphere is urged endophytic bacteria (Plant Growth-PromotingRhizobacteria, 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 becoming a class biocontrol bacteria that attracts tremendous attention, the biocontrol bacteria that development and utilization is specifically designed to anti-soft rot of Chinese cabbage has broad application prospects.
Summary of the invention
The object of the invention is to provide a kind of to be had better prevention effect, and can effectively promote the simple pure genus bacillus of plant growth for the soft rot of Chinese cabbage evil.
Above-mentioned purpose is achieved by the following technical solution:
A kind of simple pure genus bacillus, classification called after bacillus pumilus (Bacillussimplex) RJGP41, 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.3381.
Simple pure genus bacillus (Bacillus simplex) RJGP41 CGMCC No.3381 separates from the taraxacum rhizosphere soil in Guo An village, Jiang Dang township, Shigatse City, Tibet, obtains through flat board face-off screening.
Simple pure genus bacillus (Bacilluss implex) RJGP41 CGMCC No.3381 is a gram positive bacterium, shaft-like, can be 4 ℃ of growths, but can not be 50 ℃ of growths, can not tolerate the cultivation of pH5.7 and 7%NaCl, catalase test, starch hydrolysis experiment, Citrate trianion utilization test, nitrate reduction test, glucose fermentation and casein test are all positive, and V-P test, anaerobic growth test and propionic salt utilization test are all negative.
Utilize the increase 16S rDNA sequence of simple pure genus bacillus (Bacillus simplex) RJGP41CGMCC No.3381 of round pcr, obtaining length is the fragment of 481bp, 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 simplex FJ455076.1, 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, (Cai Miaoying etc. translate with reference to " bacillus ", agricultural press, 1988) and " microbial taxonomy " (Zhang Jizhong, Fudan University in Shanghai press, nineteen ninety-five) key is retrieved, and is simple pure genus bacillus (Bacillus simplex) with this identification of strains, called after RJGP41.
The microbial inoculum that contains simple pure genus bacillus (Bacillus simplex) RJGP41C GMCC No.3381 also belongs to protection scope of the present invention.
The application method of simple pure genus bacillus (Bacillus simplex) RJGP41 CGMCC No.3381 is fermentation culture → dilution → sprinkling → effect measuring.
The fermention medium of simple pure genus bacillus (Bacillus simplex) RJGP41 CGMCC No.3381 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.
Simple pure genus bacillus (Bacillus simplex) RJGP41 CGMCC No.3381 has significant fungistatic effect to Erwinia, and Arabidopis thaliana, corn and Chinese cabbage are had the significant short fruit of coming into force.
Description of drawings
Fig. 1 is brood cell's picture of embodiment 2 simple pure genus bacillus (Bacillus simplex) RJGP41CGMCC No.3381;
Fig. 2 is the fungistatic effect pictures of embodiment 6 simple pure genus bacillus (Bacillus simplex) RJGP41CGMCC No.3381 for the Chinese cabbage pine root fungus.
Embodiment
Below further describe the present invention by embodiment, as known by the technical knowledge, the present invention also can describe by other the scheme that does not break away from the technology of the present invention feature, thus all within the scope of the present invention or the change that is equal in the scope of the invention all be included in the 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 the 5g soil sample, put into 45ml physiological saline, concussion is 30 minutes on the shaking table, leaves standstill and makes clarification.Draw supernatant liquor 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 simple pure genus bacillus (Bacillus simplex) RJGP41 CGMCCNo.3381.
Embodiment 2:
Simple pure genus bacillus (Bacillus simplex) RJGP41CGMCC No.3381 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 be 4 ℃ of growths, but can not be 50 ℃ of growths, can not tolerate the cultivation of pH5.7 and 7%NaCl, catalase test, starch hydrolysis experiment, Citrate trianion utilization test, nitrate reduction test, glucose fermentation and casein test are all positive, and V-P test, anaerobic growth test and propionic salt utilization test are all negative.
Embodiment 3:
Simple pure genus bacillus (Bacillus simplex) the RJGP41CGMCC No.3381 of embodiment 1 is inoculated 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 P CR amplification.The PCR condition is: 95 ℃, and 4min; 94 ℃, 1min; 50 ℃, 1min; 72 ℃, 2min; 34cycle; 72 ℃, 10min, 4 ℃ of preservations.The PCR product checks order after reclaiming, and is 481bp through confirming its length, and sequencing result is shown in sequence table, and the similarity with Bacillus simplexFJ455076.1 in GenB ank is 94%.
Embodiment 4:
Substratum:
The 1C substratum
| Moiety | 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
| Moiety | Final concentration | Volume (ml) |
| 5×1C? | 1×? | 50? |
| 20% glucose | 0.1％? | 1.25? |
| Soil extract | 10％? | 25? |
| Water | ? | 173.75? |
Simple pure genus bacillus (Bacillus simplex) the RJGP41CGMCC No.3381 of embodiment 1 is inoculated in the aforesaid liquid substratum, 37 ℃ of overnight incubation, get 5ul bacterium liquid and put right-angled intersection point apart from the dull and stereotyped center 2.5cm of solid medium, 5 repetitions of each bacterial strain, be 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, simple genus bacillus of the present invention (Bacillus simplex) RJGP41 CGMCC No.3381 can grow in the environment of temperature≤10 ℃, to be designated as the low-temperature epitaxy bacterial strain at the bacterial strain that can in a week, manifest bacterium colony under this temperature, obtain the suitable living bacillus pumilus Bacillussimplex NMCC46C GMCC No.3378 of low temperature, can under 4 ℃ condition, grow through further testing this bacterial strain.
Embodiment 5:
Simple pure genus bacillus (Bacillus simplex) the RJGP41CGMCC No.3381 of embodiment 1 is seeded on the LB substratum, incubated overnight, then the simple pure genus bacillus of activatory (Bacillus simplex) RJGP41 CGMCC No.3381 is transferred to and carries out shake flask fermentation in the Landy substratum, temperature is 30 ℃, shaking speed is 200rpm, fermentation time is 38 hours, gets the fermented liquid of simple pure genus bacillus (Bacillus simplex) RJGP41 CGMCC No.3381.
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, complementing to cumulative volume with filtered water is 1L, is adjusted to pH7.0.
Embodiment 6:
Adopt dull and stereotyped face-off method, in the NA of 150ml substratum, add cultured soft rot of Chinese cabbage germ 5ml, the flat board of falling NA, each flat board is put the filter paper of four sterilizations, drawing the bacteria suspension of simple pure genus bacillus (Bacillus simplex) the RJGP41 CGMCC No.3381 of the embodiment 1 of 5 μ l then beats on filter paper, repeat 3 times, to observe the fungistatic effect of simple pure genus bacillus (Bacillus simplex) RJGP41 CGMCC No.3381 Chinese cabbage soft rot bacteria.The result as shown in Figure 2, colony diameter is 1.0cm, antibacterial circle diameter is 1.6cm, simple pure genus bacillus promptly of the present invention (Bacillussimplex) RJGP41 CGMCC No.3381 has significant fungistatic effect to Chinese cabbage soft rot bacteria.
Embodiment 7:
The Arabidopis thaliana seed is seeded on the MS culture medium flat plate (9cm) after clorox (effectively Cl content is 5%) surface sterilization, and is standby after one week of hot-house culture.
Suitable simple pure genus bacillus (Bacillus simplex) the RJGP41 CGMCC No.3381 that gives birth to is inoculated into overnight incubation in the LB liquid nutrient medium with the low temperature of embodiment 4, after the mensuration OD value, quantitatively is 10 with its concentration
5After cfu/ml, Arabidopis thaliana seedling soak root 5min with bacterium liquid, plant the MS flat board (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: with concentration is 10
5The average fresh weight of Arabidopis thaliana that the bacteria suspension of cfu/ml is handled is 0.018386g, the average fresh weight of the Arabidopis thaliana of negative control group is 0.0097g, the average fresh weight of the Arabidopis thaliana of positive controls is 0.033789g, and simple pure genus bacillus promptly of the present invention (Bacillus simplex) RJGP41 CGMCC No.3381 has the well short fruit of coming into force to Arabidopis thaliana.
Embodiment 8:
The suitable life of low temperature simple pure genus bacillus (Bacillus simplex) the RJGP41 CGMCC No.3381 of embodiment 4 is inoculated into overnight incubation in the LB liquid nutrient medium, after the mensuration OD value, concentration is distinguished quantitatively 10
7Cfu/ml, 10
6Cfu/ml, 10
5Cfu/ml and 10
4Cfu/ml gets the cell suspending liquid 10ml of different concns and 1% carboxymethyl cellulose 30ml respectively and is mixed with mixed solution, and is standby; Will be through surface sterilization (75% alcohol disinfecting 5min, clorox (effectively Cl content is 5%) sterilization 5min, aqua sterilisa cleaning 3～5 times) corn seed is immersed in the mixed solution of aforementioned different concns, 10 seeds of each concentration, three repetitions, the taking-up seed spends the night and dries up behind the 2h, is placed on then in the culture dish that is covered with moistening filter paper, and cultivate 30 ℃ of dark places; Record germinating seed number is measured seed fresh weight behind the 2d behind the 6d, and bud is long, and root is long.Control group is the mixed solution seed soaking with fresh culture and adhesive agent.The result shows: with concentration is 10
5When the bacteria suspension of cfu/ml was handled corn seed, the bud mean length was 2.829cm, and the bud mean length of control group is 2.457cm; With concentration is 10
4When the bacteria suspension of cfu/ml was handled corn seed, the mean length of root was 7.838cm, and average fresh weight is 0.885g; And the root mean length of control group is 4.485cm, and average fresh weight is 0.762g.Be that simple pure genus bacillus of the present invention (Bacillus simplex) RJGP41 CGMCC No.3381 is good to the growth-promoting functions of corn seed sprouting.
Embodiment 9:
Chinese cabbage seed soaks 2h with clear water, under 28 ℃ of wet conditions, be seeded in the flowerpot that aseptic soil is housed after the vernalization, when treating Chinese cabbage seedling length to 3 slice true leaf, irritate root with bacteria suspension, the big bean seedlings of average every strain water the bacterium liquid of simple pure genus bacillus (Bacillussimplex) RJGP41 CGMCC No.3381 of 20ml embodiment 5, and (concentration is 10
8Cfu/ml), water this bacterium liquid after three days more once, 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 Chinese cabbage seedling of handling through bacterium liquid is obviously compared the photograph growing way.After measured, the plant height increment rate reaches 56.63%, the long increment rate of root reaches 4.94%, and the fresh weight increment rate reaches 112.8%, and simple pure genus bacillus promptly of the present invention (Bacillus simplex) RJGP41 CGMCC No.3381 has tangible growth-promoting functions to Chinese cabbage.
Embodiment 10:
With 100 times of the fermented liquid dilutions of simple pure genus bacillus (Bacillus simplex) the RJGP41CGMCC No.3381 of embodiment 5, find that at the field of serious morbidity throughout the year piece the sickness rate of soft rot of Chinese cabbage reaches at 1% o'clock and begins to spray fermented liquid, the spray in 7 days of later every interval is once sprayed 3 times continuously.Prevention effect for soft rot of Chinese cabbage can reach 83%, increase Chinese cabbage output and reach 50%, be that simple pure genus bacillus of the present invention (Bacillussimplex) RJGP41 CGMCC No.3381 can obviously suppress soft rot of Chinese cabbage, and can effectively promote Chinese cabbage growth, increase Chinese cabbage output.
Sequence table
TGAGGCGTAGTTGCCGTGGCTTTCTGGTTAGGTACCGTCAGGTA
CCAACGTTTACTCTGGTACTTGTTCTTCCCTAACAACAGAATTTA
CGACCCGAGCCTTCTTCGTTCACGCGGCGTTGCTCCGTCAGACT
TTCGTCCATTGCGGAAAATTCCCTACTGCTGCCTCCCGTAGGAG
TCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAG
GTCGGCTACGCATCGTCGCCTTGGTGAGCCATTACCTCACCAAC
TAGCTAATGCGCCGCGGGCCCATCTATAAGTGACAGCGTAAACC
GTCTTTCCATCTTCTCTCATGCGAGAAAAGAACGTATCCGGTATT
AGCTCCGGTTTCCCGAAGTTATCCCAGTCTTATAGGCAGGTTGC
CCACGTGTTACTCACCCGTCCGCCGCTAATCTCAGGAGCAAGCT
CCCATCGATCCGCTCGACTTGCATGTATTCAGATCCAAC
Claims (7)
1. a simple pure genus bacillus (Bacillus simplex) RJGP41 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.3381.
2. simple pure genus bacillus according to claim 1 (Bacillus simplex) RJGP41C GMCC No.3381 is characterized in that, is to separate from the taraxacum rhizosphere soil in Guo An village, Jiang Dang township, Shigatse City, Tibet, obtains through flat board face-off screening.
3. simple pure genus bacillus according to claim 1 (Bacillus simplex) RJGP41 CGMCC No.3381, it is characterized in that, be gram positive bacterium, shaft-like, can produce gemma, can be 4 ℃ of growths, but can not be 50 ℃ of growths, can not tolerate the cultivation of pH 5.7 and 7%NaCl, catalase test, starch hydrolysis experiment, Citrate trianion utilization test, nitrate reduction test, glucose fermentation and casein test are all positive, and V-P test, anaerobic growth test and propionic salt utilization test are all negative.
4. simple pure genus bacillus according to claim 1 (Bacillus simplex) RJGP41C GMCC No.3381 is characterized in that the partial nucleotide sequence of its 16S rDNA is shown in sequence table.
5. the application method of the described simple pure genus bacillus of claim 1 (Bacillus simplex) RJGP41C GMCC No.3381 is characterized in that, comprises fermentation culture → dilution → four steps of sprinkling → effect measuring.
6. the application method of simple pure genus bacillus according to claim 5 (Bacillus simplex) RJGP41C GMCC No.3381, it is characterized in that, fermention medium in the described fermentation culture process 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
4 6H
2O 0.15mg/L, MnSO
4 H
2O 5.0mg/L, CuSO
4 5H
2O 0.16mg/L, all the other are filtered water, and are adjusted to pH7.0.
7. any one simple pure genus bacillus (Bacillussimplex) the RJGP41C GMCC No.3381 of claim 1～4 is suppressing soft rot of Chinese cabbage and is promoting Chinese cabbage to grow, increase the application in the Chinese cabbage output.
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| CN103250741A (en) * | 2013-03-05 | 2013-08-21 | 吉林农业大学 | Preparation method of ginseng endophytic Paenibacillus polymyxa biological control microbial inoculum |
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| CN103250741A (en) * | 2013-03-05 | 2013-08-21 | 吉林农业大学 | Preparation method of ginseng endophytic Paenibacillus polymyxa biological control microbial inoculum |
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| CN105002121A (en) * | 2015-08-14 | 2015-10-28 | 山东泰诺药业有限公司 | Bacillus simplex and application thereof |
| CN105670958A (en) * | 2015-11-10 | 2016-06-15 | 黑龙江省科学院微生物研究所 | Low-temperature biocontrol bacterial strain and application thereof |
| CN105670958B (en) * | 2015-11-10 | 2018-12-25 | 黑龙江省科学院微生物研究所 | One plant of low temperature biocontrol bacterial strain and its application |
| CN105695376A (en) * | 2016-04-25 | 2016-06-22 | 北京市农林科学院 | Bacillus simplex for poisoning plant root-knot nematodes and application of bacillus simplex |
| CN105695376B (en) * | 2016-04-25 | 2019-03-19 | 北京市农林科学院 | One plant of simple bacillus for killing plant root-knot nematode and its application |
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| CN101928680B (en) | 2012-11-14 |
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