CN102352331A - Nitrogen-fixing Paenibacillus sp. 1-33 and application thereof - Google Patents
Nitrogen-fixing Paenibacillus sp. 1-33 and application thereof Download PDFInfo
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- CN102352331A CN102352331A CN 201110323796 CN201110323796A CN102352331A CN 102352331 A CN102352331 A CN 102352331A CN 201110323796 CN201110323796 CN 201110323796 CN 201110323796 A CN201110323796 A CN 201110323796A CN 102352331 A CN102352331 A CN 102352331A
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Abstract
The invention provides a Paenibacillus sp. strain 1-33 with nitrogen-fixing capability, the collection number of which is CGMCC No.4964. The Paenibacillus sp. 1-33 has a nitrogenase-structured gene nifH, and the acetylene reduction method determines that the nitrogenase activity of the Paenibacillus sp. 1-33 is 565.94nmolC2H4/(mg protein h) negative 1. The nitrogen-fixing Paenibacillus sp. strain 1-33 provided by the invention has a remarkable inhibition effect on a variety of plant pathogenic fungi, and nitrogen-fixing microorganism fungicide or bioorganic fertilizer prepared with the strain 1-33 can remarkably promote the germination and growth of plant seeds, and can be popularized to use in agricultural production to effectively reduced the usage of urea.
Description
Technical field
The present invention relates to biological technical field, particularly, relate to a kind of fixed nitrogen series bacillus 1-33 and application thereof with nitrogen fixing capacity.
Background technology
Gramineous crop such as wheat, corn and paddy rice etc. are the main foods of about 6,500,000,000 populations in the whole world.Along with the rise of chemical fertilizer industry, the output that improves farm crop mainly depends on the amount of application that increases chemical nitrogen fertilizer.China is maximum in the world chemical fertilizers production state and country of consumption, and annual applying quantity of chemical fertilizer is up to 4,800 ten thousand tons, and wherein nitrogenous fertilizer is 2,800 ten thousand tons, 1,200 ten thousand tons of (P of phosphate fertilizer
2O
5), and under the situation of cultivated area decline, the year aggregate demand of chemical fertilizer goes up not down.Yet; Only being less than 30% chemical fertilizer of using can be utilized; (Van Kessel et al.; 2009); The oxynitride per kilogram that excessive applied nitrogen produced approximately is 298 times of carbonic acid gas to the influence that heats; And the production nitrogenous fertilizer power consumption of annual China reaches 100,000,000 tons of standard coals; 500,000,000 tons of CO2 equivalent have been discharged altogether in production and the use; 0.7 hundred million tons of carbonic acid gas of deduction nitrogen application crop yield institute's fixed; The clean quantity discharged of greenhouse gases reaches 4.3 hundred million tons of carbonic acid gas, accounts for 8% of national total release.The excessive of chemical fertilizer used, but causes soil absorbing state micronutrient levels to descend, and causes fluorine and heavy metal contamination in the soil simultaneously, causes soil acidification serious, and heavy metal, nitric nitrogen are accumulated in soil, soil compaction, and fertility descends.Most of nitrogenous fertilizer is through the loss of approach such as seepage, volatilization and nitrification and denitrification, and a part of chemical fertilizer gets into rivers and lakes with rainwater, causes the eutrophication of water body, and China has the lake more than 80% to receive pollution (IV class--bad V class).According to WHO standard, the overall exceeding standard rate of China's groundwater azotate is 29.91%.The vegetable plot nitrate concentration is more than 100ppm, and facilities vegetable surpasses 200ppm, and serious threat is to food safety and national health.
And biological nitrogen fixation not only can effectively reduce the usage quantity of chemical fertilizer; Improve the utilization ratio of chemical fertilizer; But also can improve soil physical property; Increase soil fertility; Increase organic matter, thereby improve the absorption of farm crop, improve the rhizosphere microecosystem nutrition; Thereby the reduction agriculture production cost is realized the Sustainable development of agricultural.
According to the marriage relation between nitrogen-fixing microorganism and the host plant, nitrogen fixation can be divided into symbiotic nitrogen fixation, association nitrogen fixation and from growing nitrogen-fixing (outstanding Chong Shao chief editor, biological nitrogen fixation, Science Press, 1987).The symbiotic nitrogen fixation microorganism is closely to live in other symbiosis, forms the quasi-microorganism that special symbiotic structure carries out fixed nitrogen.They are generally lived in the root nodule, and the photosynthate that provides with host plant is that the energy carries out fixed nitrogen, thereby nitrogen nutrition are provided for host plant growth.Symbiotic nitrogen fixation efficient is high, and amount of nitrogen fixation is big, is the most clearly fixed nitrogen system of studying so far; But its host range and narrow only limits to leguminous plants (rhizobium leguminosarum) and some non-pulse family trees (Fu Shi fixed nitrogen actinomycetes), serious limit its widespread use.The association nitrogen fixation microorganism is meant rhizosphere or the root table that is colonizated in plant, and some can get into the cortical tissue of root but not form root nodule, carries out the microorganism of fixed nitrogen.Because between combination azotobacter and the root system of plant is a kind of loose associating; And do not differentiate tangible structure; And expose at the roots of plants table; The association nitrogen fixation that is is subject to Effect of Environmental; Be difficult for being secreted into external by association nitrogen fixation microorganism fixed nitrogen, be to have caused the low reason of association nitrogen fixation microorganism nitrogen-fixing efficiency in addition.Spontaneous nitrogen-fixing microorganism is when in physical environment or substratum, living on one's own life usually, just molecular nitrogen can be fixed as a quasi-microorganism of ammonia.Spontaneous nitrogen-fixing microorganism does not need to unite just ability fixed nitrogen with other biological, has the advantages that condition is wide, adaptation is wide, and can also secrete some plant hormones, stimulates growing of root system and plant, just contains azotobacter in more existing nitrogenous fertilizer microbial inoculums.Simultaneously, azotobacter can be improved little ecologic community of plant rhizosphere, plays the promotion plant-growth, improves the effect of stress resistance of plant.
The class sporeformer has characteristics such as survival time length and strong stress resistance, extensively is distributed in the different soil and plant rhizosphere, and what have also can the invaded plants organization internal.Many bacterial strains in this genus are with its nitrogen fixing capacity, make them receive investigator's concern widely to the resistances of pathogenic bacterium with to the growth-promoting functions of plant.China is vast in territory, and environment is various, and the soil property situation is widely different, makes the very abundant from the growing nitrogen-fixing resource of China, and Application and Development has a high potential.But, because particularly domestic research to azotobacter both at home and abroad starts late, big limitations the series bacillus bacterial classification as the application of potential microbiobacterial agent on agricultural.
Summary of the invention
The object of the present invention is to provide a kind of series bacillus and application thereof with nitrogen fixing capacity.
Bacterial strain 1-33 provided by the invention is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 21st, 2011 and (is called for short CGMCC; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica; Postcode 100101); Deposit number is CGMCC No.4964, classification series bacillus (Paenibacillus sp.) by name.
Fixed nitrogen series bacillus 1-33 well-grown on the LD solid medium, bacterium colony are circular, and the edge is irregular, and color is milky white, and diameter is 1～2.5mm.
The invention provides the microbial inoculum that contains fixed nitrogen series bacillus 1-33.
The present invention finds that this bacterium contains nitrogenase, and its structure gene is nifH, the nucleotide sequence of this gene of encoding such as SEQ ID No.1.It is 565.94nmolC that the acetylene reduction method is measured its nitrogenase activity
2H
4/ (mg protein h)
-1
The present invention provides fixed nitrogen series bacillus 1-33 or its tunning or contains the application of microbial inoculum on the inhibition pathogenic bacteria of fixed nitrogen series bacillus 1-33.
Described pathogenic bacteria is Fusarium oxysporum (Fusarium oxysporum), cereal rhizoctonia (Rhizoctonia cerealis), sclerotinite (Sclerotinia sclerotiorum) and/or Botrytis cinerea (Botrytis cinerea).
The present invention provides fixed nitrogen series bacillus 1-33 or its tunning or contains the application of microbial inoculum in the promotion plant-growth of fixed nitrogen series bacillus 1-33.
The invention provides the application of fixed nitrogen series bacillus 1-33 in ecological nitrogen-fixing microorganism microbial inoculum of preparation or biological organic fertilizer.
The invention provides with fixed nitrogen series bacillus 1-33 is the application of preparation on agricultural of effective constituent.
Fixed nitrogen series bacillus bacterial strain 1-33 provided by the invention has the obvious suppression effect to the various plants pathogenic bacteria; Simultaneously plant-growth had promoter action; The output of this bacterial strain secretion plant hormone indolylacetic acid is 14.94mmol/mL; Bacterial strain 1-33 of the present invention is processed nitrogen-fixing microorganism microbial inoculum or biological organic fertilizer can produce obvious facilitation the germination and growth of plant seed; Can effectively reduce the usage quantity of urea, can in agriculture production, promote the use of.
Description of drawings
Fig. 1 is the colonial morphology of Paenibacillus sp.1-33 on no nitrogen flat board;
Fig. 2 is the relevant electrophorogram of bacterial strain Paenibacillus sp.1-33, wherein M:ladder; The nitrogenase structure gene nifH band that the 1:PCR amplification obtains; The 16S rDNA band that the 2:PCR amplification obtains;
Fig. 3 is the restraining effect figure of Paenibacillus sp.1-33 to phytopathogen, and wherein a figure is the restraining effect figure of Paenibacillus sp.1-33 to Botrytis cinerea (Botrytis cinerea); B figure is the restraining effect figure of Paenibacillus sp.1-33 sclerotinite (Sclerotinia sclerotiorum); C figure is the restraining effect figure of Paenibacillus sp.1-33 to Fusarium oxysporum (Fusarium oxysporum); D figure is the restraining effect figure of Paenibacillus sp.1-33 to cereal rhizoctonia (Rhizoctonia cerealis);
Fig. 4 is the growth-promoting functions figure of Paenibacillus sp.1-33 to wheat seed, is respectively bacterium liquid growth-promoting functions to wheat seed under 10 times, 20 times, 50 times, 100 times situation of dilution.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting scope of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, used chemical reagent is conventional commercial reagent among the embodiment, the conventional means that used technique means is well known to those skilled in the art among the embodiment.
Used soil sample in the present embodiment is from various places different plant foundation soil.
The cultivation and the evaluation of embodiment 1 fixed nitrogen series bacillus 1-33 bacterial strain
(1) cultivation of bacterial strain
The making of substratum: liquid nutrient medium (1L): 20g sucrose; 12.06g K
2HPO
43.4gKH
2PO
40.2g MgSO
47H
2O; 0.01g NaCl; 0.01g FeCl
30.002gNaMoO
42H
2O; H
2O 1L.Sterilized 25 minutes for 120 ℃.The making of solid medium is in every liter of liquid nutrient medium, to add 14g agar.The fixed nitrogen series bacillus is inoculated in solid or the liquid nutrient medium, and 30 ℃ of incubators were cultivated 3 days.
(2) method of the extraction process for extracting reference literature of bacteria total DNA [Ausubel et al.Current Protocols in Molecular Biology.New York:Wiley.1987].
(3) amplification of the evaluation of bacterial strain and nitrogenase structure gene nifH and 16S rDNA
Extract genomic dna from the pure growth of bacterial strain Paenibacillus sp.1-33 of the present invention, and as template, be the upstream and downstream primer with nifH P1 in the table 1 and nifH P2, pcr amplification nitrogenase structure gene nifH has obtained the big or small band that is about 320bp.After this band reclaimed purifying, the company of delivering checked order.The nucleotide sequence of gene nifH is shown in SEQ ID NO.1.With the online BLAST comparison of sequencing result at the GenBank database, the homology of result and Paenibac illus sp.Bb54 reaches 98%.
With the colibacillary 16S rDNA of procaryotic type strain sequences Design primer, see table 1, be template with the pure growth genomic dna of the bacterial strain Paenibacillus sp.1-33 that extracts, pcr amplification 16S rDNA sequence.The result has obtained then this band being reclaimed purifying, and delivering order-checking for the band about 1.5Kb.16S rDNA sequence is shown in SEQ ID NO.2, and sequencing result carries out online BLAST comparison, and the result shows that this bacterial strain belongs to series bacillus and belongs to.This bacterial strain 1-33 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 21st, 2011 and (is called for short CGMCC; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica; Postcode 100101); Deposit number is CGMCCNo.4964, classification series bacillus (Paenibacillus sp.) by name.
Table 1 is used to clone the primer of nifH and 16S rRNA gene
Amplification system
Amplification condition
The amplification of nifH gene:
The amplification of 16S rDNA:
The amplification of nitrogenase structure gene nifH and 16S rDNA is seen Fig. 2.
The mensuration that embodiment 2 nitrogenases are lived
1, the 1-33 inoculation is surveyed in the substratum that enzyme lives in 5mL, 30 ℃ leave standstill overnight incubation, and the inoculum size by 1% is transferred in the 50mL triangular flask, and 30 ℃ of incubated overnight are once more collected thalline then, and survey substratum that enzyme lives and suspend specifically with an amount of, and adjust OD
600To 1.With 1.5mL bacterium liquid inoculation culture 2h, get 100 μ L gases and measure ethylene content with Gas chromatography.
Surveying enzyme substratum alive is: 26.3g Na
2HPO
412H
2O; 3.4g KH
2PO
410 μ g vitamin Hs; 26mg CaCl
22H
2O; 30mg MgSO
40.33mg MnSO
4H
2O; The 36mg ironic citrate; 7.6mg Na
2MoO
42H
2O; 10 μ g para-amino benzoic acid; 4g glucose.Add separately behind 115 ℃ of sterilizations of glucose 30min, other reagent can prepare back 121 ℃ of sterilization 30min.
2, the measuring method of protein content (Bradford, 1976)
(1) configuration of solution
Bradford storage liquid: 100mL 95% ethanol, 200mL 88% phosphoric acid, 350mg Coomassie brilliant blue G250;
Bradford working fluid: 425mL distilled water, 15mL 95% ethanol, 30mL88% phosphoric acid, 30mL Bradford storage liquid;
Filter paper filtering is stored in the brown bottle, and room temperature is placed.Can preserve several weeks, but need before using to filter.
(2) making of typical curve
Get 5ml Bradford working fluid in test tube, add 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, the 0.8mg/mL of 40 μ L, the BSA solution of 1.0mg/mL respectively, mixing is placed 3～5min, shows blue look.Measure OD
595Value.With the 5mL Bradford working fluid that adds 40 μ L water as contrast.With OD
595Value is ordinate zou, and the BSA strength of solution is an X-coordinate drawing standard curve.Obtain typical curve equation y=0.3973x, R
2Be 0.996.
(3) detect
Centrifugal collection thalline, the NaOH that adds 200 μ L 0.5M boils 5min, and the HCl that adds 200 μ L0.5M again mixes the centrifugal absorption supernatant liquor 40 μ L in back, joins in the 5mL Bradford working fluid, and mixing is placed 3～5min, shows blue look.Measure OD
595Value is 0.097.With OD
595Value is brought in the typical curve equation, and the calculating protein concentration is 0.2441mg.
3, the demarcation of 1nmol ethene
(1) prepares two of 120mL serum bottles, be designated as 1#, 2# bottle;
(2) serum bottle is filled water, and good with the plug plug that is inserted with syringe needle, prevent that bubble from producing, take off plug, pour out 100mL water (measuring) with volumetric flask, change new plug, the volume of air in the bottle is 100mL like this;
(3) in the 1# bottle, inject 2.24mL and (be 2.24mL under the normal conditions; Under the non-standard situation; Calculate injection rate according to PV=nRT) ethene; From the 1# bottle, take out 1mL gas again and inject the 2# bottle; From the 2# bottle, take out 100 μ L gases and squeeze into gas chromatograph (instrument model HP6890); The peak area that is shown is the amount of 1nmol ethene, can calculate on the recording paper per unit peak area in view of the above and represent how much nmol ethene.
(4) nitrogenase (nmol C alive
2H
4/ mg protein hr) calculation formula is:
Acetylene reduction method (ARA) shows that this bacterium can utilize N
2As nitrogenous source, and have higher biological nitrogen fixation activity, under the pure culture condition, its nitrogenase activity is 565.94nmolC
2H
4/ (mgprotein h)
-1
The bacteriostatic activity experiment of embodiment 3 fixed nitrogen series bacillus 1-33 bacterial strains
The PDA substratum (potato 200g, glucose 20g, agar powder 16g, adding distil water is settled to 1L).Inoculate phytopathogen and series bacillus 1-33 provided by the invention respectively at 2 at the dull and stereotyped central 2cm of distance P DA place; Then flat board is placed 30 ℃ of incubators to cultivate 3～7 days; Observe the restraining effect of this series bacillus to phytopathogen, the result sees Fig. 3.The used phytopathogen of present embodiment is respectively: Fusarium oxysporum (Fusarium oxysporum), cereal rhizoctonia (Rhizoctonia cerealis), sclerotinite (Sclerotinia sclerotiorum), Botrytis cinerea (Botrytis cinerea).
Embodiment 4 fixed nitrogen series bacillus 1-33 bacterial strains are to the growth-promoting functions experiment of wheat seed
(1) plant hormone indolylacetic acid (IAA) Determination on content
In 5ml LD liquid nutrient medium (NaCl 2.5g, deionized water is settled to 1l, regulates pH value to 6.8 with 1mol/L HCl or NaOH for peptone 10g, yeast powder 5g), place on 30 ℃ of shaking tables shaking culture to spend the night series bacillus 1-33 bacterial strain list colony inoculation.Be inoculated in freshly prepared LD of 200mL or the LB liquid nutrient medium by 5% inoculum size then, place 30 ℃ of shaking table shaking culture 48h after, measure their IAA output.The result shows that series bacillus 1-33 bacterial strain of the present invention can be secreted IAA, and its output is 14.94mmol/mL, and plant is had growth-promoting functions.
(2) series bacillus 1-33 bacterial strain is to the growth-promoting functions experiment of wheat seed
Cultured this bacterial strain bacterium liquid is carried out gradient dilution * 10, * 20, * 50, * 100, then with the plump-eared wheat seed with 1% clorox sterilization 15min, again with the deionized water wash of sterilization 5 times.Next respectively with the deionized water and gradient dilution * 10 of sterilization, * 20, * 50, * 100 bacterium liquid soaks wheat kind 2min.With wheat seed with the sterilization deionized water through same processing as control group.With the wheat seed after handling neat put in the aseptic plate that is covered with moistening filter paper 100 in every ware.Then plate is placed 25 ℃ of constant incubators, the dark germination.Day by day recording processing group and control group seed germination number.After cultivating 6～7 days, measure length, single-strain fresh weight, total dry weight, seed germination index, rate of emergence and the seedling vitality index of seed bud, root, the result sees table 2.
Germination index (germination index, GI), percentage of germination (germination percentage, GP) with the seed vitality formula of index following:
The total grain of the total grain of percentage of germination (%)=germination number/test number * 100%
Germination index=∑ (number of shoots in the T time/corresponding germination fate T)
Vitality index=seed germination index * average the dry weight of individual plant seedling.
Table 2 bacterial strain Paenibacillus sp.1-33 is to the short fruit of coming into force of wheat seed
As shown in table 2, compare with control group, with bacterium liquid gradient dilution of the present invention * 10, * 20 with * 50 o'clock, to the growth-promoting functions of wheat seed apparently higher than control group; And dilute * 100 o'clock, because bacterial concentration is low excessively, the short fruit of coming into force is not obvious.When bacterium liquid diluted * 20 the time, the germination index of the wheat seed of handling with its has improved 77.61% than control group, the seed vitality index has improved 68.33%.When bacterium liquid diluted * 50, germination index had improved 81.93%, and percentage of germination has improved 3.72%, and the seed vitality index has improved 68.44%.It is thus clear that bacterial strain 1-33 of the present invention can be used as a kind of biological nitrogen-fixing fertilizer with inhibition phytopathogen and promotion plant-growth effect and is applied aborning.Bacterial strain of the present invention is cultivated the back as microbial fertilizer, can be applicable in the field planting industry of gramineous crops such as corn, wheat, can effectively reduce the usage quantity of urea.
Claims (8)
1. fixed nitrogen series bacillus (Paenibacillus sp.) bacterial strain 1-33, its preserving number is CGMCC No.4964.
2. the microbial inoculum that contains the said fixed nitrogen series bacillus of claim 1 1-33.
3. the nitrogenase with nitrogenase activity that produces of the described fixed nitrogen series bacillus of claim 1 1-33, the gene of coding nitrogenase is nifH, the nucleotide sequence of this gene of encoding such as SEQ ID No.1.
4. claim 1 described fixed nitrogen series bacillus 1-33 or its tunning or the described microbial inoculum of claim 2 are in the application that suppresses on the phytopathogen.
5. application as claimed in claim 4; It is characterized in that described phytopathogen is Fusarium oxysporum (Fusarium oxysporum), cereal rhizoctonia (Rhizoctonia cerealis), sclerotinite (Sclerotinia sclerotiorum) and/or Botrytis cinerea (Botrytis cinerea).
6. the described series bacillus 1-33 of claim 1 or its tunning or the described microbial inoculum of claim 2 are in the application that promotes in the plant-growth.
7. the application of the described fixed nitrogen series bacillus of claim 1 1-33 in ecological nitrogen-fixing microorganism microbial inoculum of preparation or biological organic fertilizer.
8. the described fixed nitrogen series bacillus of claim 1 1-33 is the application of preparation on agricultural of effective constituent.
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| CN103451130A (en) * | 2013-07-25 | 2013-12-18 | 中国农业大学 | Nitrogen fixing gene cluster and application thereof |
| CN105985917A (en) * | 2015-02-06 | 2016-10-05 | 中国农业大学 | Method for improving biomass of chlorella in swine wastewater |
| CN106011005A (en) * | 2016-05-20 | 2016-10-12 | 东莞市保得生物工程有限公司 | Bacillus amyloliquefaciens T600 and preparation method and application of microbial agent |
| CN106167770A (en) * | 2016-05-20 | 2016-11-30 | 东莞市保得生物工程有限公司 | A kind of bacillus megaterium B16 and microbial inoculum thereof and bacterial preparation process |
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| CN103451130A (en) * | 2013-07-25 | 2013-12-18 | 中国农业大学 | Nitrogen fixing gene cluster and application thereof |
| CN105985917A (en) * | 2015-02-06 | 2016-10-05 | 中国农业大学 | Method for improving biomass of chlorella in swine wastewater |
| CN105985917B (en) * | 2015-02-06 | 2019-12-13 | 中国农业大学 | Method for increasing biomass of chlorella in pig-raising wastewater |
| CN106011005A (en) * | 2016-05-20 | 2016-10-12 | 东莞市保得生物工程有限公司 | Bacillus amyloliquefaciens T600 and preparation method and application of microbial agent |
| CN106167770A (en) * | 2016-05-20 | 2016-11-30 | 东莞市保得生物工程有限公司 | A kind of bacillus megaterium B16 and microbial inoculum thereof and bacterial preparation process |
| CN106011005B (en) * | 2016-05-20 | 2020-01-17 | 东莞市保得生物工程有限公司 | Bacillus amyloliquefaciens T600 and preparation method and application of microbial inoculum thereof |
| CN106167770B (en) * | 2016-05-20 | 2020-01-17 | 东莞市保得生物工程有限公司 | Bacillus megaterium B16, microbial inoculum thereof and preparation method of microbial inoculum |
| CN110272841A (en) * | 2019-06-17 | 2019-09-24 | 甘肃省科学院生物研究所 | One plant of yellow shortwave monad and its application on informal voucher Radix Codonopsis |
| CN110747142A (en) * | 2019-11-12 | 2020-02-04 | 中国农业大学 | Natural ammonium-resistant nitrogen-fixing microorganism LQ3 and application thereof |
| CN110747142B (en) * | 2019-11-12 | 2021-02-05 | 中国农业大学 | Natural ammonium-resistant nitrogen-fixing microorganism LQ3 and application thereof |
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