CN102352333B - Nitrogen-fixing Paenibacillus sp. 1-49 and application thereof - Google Patents
Nitrogen-fixing Paenibacillus sp. 1-49 and application thereof Download PDFInfo
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Abstract
The invention provides a Paenibacillus sp. strain 1-49 with nitrogen-fixing capability, the collection number of which is CGMCC No.4966. The Paenibacillus sp. 1-49 has a nitrogenase-structured gene nifH, and the acetylene reduction method determines that the nitrogenase activity of the Paenibacillus sp. 1-49 is 969. 49nmolC2H4/(mg protein h) negative 1. The nitrogen-fixing Paenibacillus sp. strain 1-49 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 1-49 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 1-49 and application thereof with nitrogen fixing capacity.
Background technology
Gramineous crop such as wheat, corn and paddy rice etc. are the approximately main food of 6,500,000,000 populations of 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 in 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 produces approximately is 298 times of carbonic acid gas on the impact 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 procedure, 0.7 hundred million tons of carbonic acid gas that deduction nitrogen application crop yield is 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 simultaneously Fluorine in Soils and heavy metal contamination, causes soil acidification serious, and heavy metal, nitric nitrogen are accumulated in soil, soil compaction, and fertility descends.Most of nitrogenous fertilizer is by the loss of the approach such as seepage, volatilization and nitrification and denitrification, and a part of chemical fertilizer enters rivers and lakes with rainwater, causes the eutrophication of water body, and China has the lake more than 80% to be subject to polluting (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 farm crop to the absorption of nutrition, improve rhizosphere microecological system, 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 be living together with 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 take host plant carries out fixed nitrogen as the energy, thereby provide nitrogen nutrition for the host plant growth.Symbiotic nitrogen fixation efficient is high, and amount of nitrogen fixation is large, 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-Tree Legumes (Fu Shi fixed nitrogen actinomycetes), serious restriction its widespread use.The association nitrogen fixation microorganism refers to be colonizated in rhizosphere or the root table of plant, and some can enter the cortical tissue of root but not form root nodule, carries out the microorganism of fixed nitrogen.Owing to being a kind of loose associating between combination azotobacter and the root system of plant, and do not differentiate tangible structure, and expose at the roots of plants table, the association nitrogen fixation that is is subject to the impact of environmental factors, the nitrogen of in addition being fixed by the association nitrogen fixation microorganism is difficult for being secreted into external, is to have caused the low reason of association nitrogen fixation microorganism nitrogen-fixing efficiency.Spontaneous nitrogen-fixing microorganism is when usually living on one's own life in physical environment or substratum, just molecular nitrogen can be fixed as a quasi-microorganism of ammonia.Spontaneous nitrogen-fixing microorganism does not need to unite just energy 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 Promoting plant growth, improves the effect of stress resistance of plant.
The class sporeformer has the characteristics such as survival time length and strong stress resistance, extensively is distributed in the different soil and plant rhizospheres, and what have also can the invaded plants organization internal.Many bacterial strains in this genus with its nitrogen fixing capacity, to the resistances of pathogenic bacterium and to the growth-promoting functions of plant so that they have received investigator's widely concern.China is vast in territory, and environment is various, and the soil property situation is widely different, so that China is very abundant from the growing nitrogen-fixing resource, Application and Development has a high potential.But, because domestic and international particularly domestic research to azotobacter is started late, greatly limited 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.
1-49 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 CGMCCNo.4966, Classification And Nomenclature series bacillus (Paenibacillus sp.).
Fixed nitrogen series bacillus 1-49 well-grown on the LD solid medium, bacterium colony are circular, and the edge is irregular, and color is milky white, and diameter is 2～3mm.
The invention provides the microbial inoculum that contains fixed nitrogen series bacillus 1-49.
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 969.49nmolC that the Acetylene Reduction method is measured its nitrogenase activity
2H
4/ (mg protein h).
The microbial inoculum that the invention provides fixed nitrogen series bacillus 1-49 or its tunning or contain fixed nitrogen series bacillus 1-49 is in the application that suppresses on the pathogenic bacteria.
Described pathogenic bacteria is Fusarium oxysporum (Fusarium oxysporum), Rhizoctonia cerealis (Rhizoctonia cerealis), sclerotinite (Sclerotinia sclerotiorum) and/or Botrytis cinerea (Botrytis cinerea).
The invention provides fixed nitrogen series bacillus 1-49 or its tunning or contain the application of microbial inoculum on Promoting plant growth of fixed nitrogen series bacillus 1-49.
The invention provides the application of fixed nitrogen series bacillus 1-49 in the ecological nitrogen-fixing microorganism microbial inoculum of preparation or biological organic fertilizer.
The invention provides the application of preparation on agricultural take fixed nitrogen series bacillus 1-49 as effective constituent.
Fixed nitrogen series bacillus bacterial strain 1-49 provided by the invention has obvious restraining 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 11.59mmol/mL, bacterial strain 1-49 of the present invention is made the nitrogen-fixing microorganism microbial inoculum or biological organic fertilizer can produce to the germination and growth of plant seed obvious promoter action, can effectively reduce the usage quantity of urea, can in agriculture production, promote the use of.
Description of drawings
Fig. 1 is Paenibacillus sp.1-49 without the colonial morphology on the nitrogen flat board;
Fig. 2 is the relevant electrophorogram of bacterial strain Paenibacillus sp.1-49, 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 be Paenibacillus sp.1-49 to the restraining effect figure of phytopathogen, wherein a figure is that Paenibacillus sp.1-49 is to the restraining effect figure of Botrytis cinerea (Botrytis cinerea); B figure is the restraining effect figure of Paenibacillus sp.1-49 sclerotinite (Sclerotinia sclerotiorum); C figure is that Paenibacillus sp.1-49 is to the restraining effect figure of Fusarium oxysporum (Fusarium oxysporum); D figure is that Paenibacillus sp.1-49 is to the restraining effect figure of Rhizoctonia cerealis (Rhizoctonia cerealis);
Fig. 4 be Paenibacillus sp.1-49 to the growth-promoting functions figure of wheat seed, be respectively bacterium liquid growth-promoting functions to wheat seed in 10 times, 20 times, 50 times, 100 times situations of dilution.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, 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 the different plant root soil in various places.
The culture ﹠ identification of embodiment 1 fixed nitrogen series bacillus 1-49 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 to add 14g agar in every liter of liquid nutrient medium.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 extracting method 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.1-49 of the present invention, and as template, nifH P1 and nifH P2 are as the upstream and downstream primer in the table 1, and pcr amplification nitrogenase structure gene nifH has obtained the band that size 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 Paenibacillus Nz29 reaches 94%.
With the colibacillary 16S rDNA of procaryotic type strain primers, see Table 1, take the pure growth genomic dna of the bacterial strain Paenibacillus sp.1-49 that extracts as template, 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 1-49 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), preserving number is CGMCC No.4966, and Classification And Nomenclature is series bacillus (Paenibacillus sp.).
Table 1 is used for the primer of clone 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 1-49 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 then 30 ℃ of incubated overnight again collect thalline, and with an amount of survey substratum that enzyme lives suspend concrete, and adjustment 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 rear 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 respectively 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, the 0.6mg/mL of 40 μ L, the BSA solution of 0.8mg/mL, 1.0mg/mL, mixing is placed 3～5min, aobvious blue look.Measure OD
595Value.With the 5mL Bradford working fluid that adds 40 μ L water in contrast.With OD
595Value is ordinate zou, and the BSA strength of solution is X-coordinate drawing standard curve.Obtain typical curve equation y=0.3973x, R
20.996.
(3) detect
Centrifugal collection thalline, the NaOH that adds 200 μ L 0.5M boils 5min, and the HCl that adds again 200 μ L0.5M mixes rear centrifugal absorption supernatant liquor 40 μ L, joins in the 5mL Bradford working fluid, and mixing is placed 3～5min, aobvious blue look.Measure OD
595Value is 0.0883.With OD
595Value is brought in the typical curve equation, and the calculating protein concentration is 0.2222mg.
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 Bubble formation, 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) be 2.24mL under the injection 2.24mL(normal conditions in the 1# bottle, under the non-standard working conditions, calculate injection rate according to PV=nRT) ethene, from the 1# bottle, take out again in the 1mL gas inject 2# bottle, from the 2# bottle, take out 100 μ L gases and squeeze into gas chromatograph (instrument model HP6890), shown peak area is the amount of 1nmol ethene, can calculate accordingly how much nmol ethene the per unit peak area represents on the recording paper.
(4) nitrogenase [nmol C alive
2H
4/ (mg protein hr)] calculation formula be:
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 969.49nmolC
2H
4/ (mg proteinh).
The bacteriostatic activity experiment of embodiment 3 fixed nitrogen series bacillus 1-49 bacterial strains
The PDA substratum (potato 200g, glucose 20g, agar powder 16g, adding distil water is settled to 1L).Inoculate respectively phytopathogen and series bacillus 1-49 provided by the invention 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 this series bacillus to the restraining effect of phytopathogen, the results are shown in Figure 3.The used phytopathogen of the present embodiment is respectively: Fusarium oxysporum (Fusarium oxysporum), Rhizoctonia cerealis (Rhizoctonia cerealis), sclerotinite (Sclerotinia sclerotiorum), Botrytis cinerea (Botrytis cinerea).
Embodiment 4 fixed nitrogen series bacillus 1-49 bacterial strains are to the growth-promoting functions experiment of wheat seed
(1) mensuration of plant hormone indolylacetic acid (IAA) content
With series bacillus 1-49 bacterial strain list colony inoculation in 5ml LD liquid nutrient medium (peptone 10g, yeast powder 5g, NaCl 2.5g, deionized water is settled to 1l, regulate pH value to 6.8 with 1mol/L HCl or NaOH) in, place on 30 ℃ of shaking tables shaking culture to spend the night.Then by 5%
Inoculum size be inoculated in the freshly prepared LD of 200mL or the LB liquid nutrient medium, place 30 ℃ of shaking table shaking culture 48h after, measure their IAA output.The result shows, series bacillus 1-49 bacterial strain of the present invention can producing IAA, and its output is 11.59mmol/mL, and plant is had growth-promoting functions.
(2) series bacillus 1-49 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 deionized water and gradient dilution * 10 of sterilization, * 20, * 50, * 100 bacterium liquid soaks Wheat Species 2min.Wheat seed is organized through same processing in contrast with the deionized water of sterilization.With the wheat seed after processing 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 vigor index of seed bud, root, the results are shown in Table 2.
Germination index (germination index, GI), percentage of germination (germination percentage, GP) and seed vitality formula of index are as follows:
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.1-49 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 and * 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 excessively low, the short fruit of coming into force is not obvious.When bacterium liquid be diluted * 20 the time, the germination index of the wheat seed of processing with its has improved 83.77% than control group, percentage of germination has improved 2.00% than control group, the seed vitality index has improved 73.85%.When bacterium liquid diluted * 50, germination index had improved 67.93%, and percentage of germination has improved 3.72%, and the seed vitality index has improved 49.99%.As seen, bacterial strain 1-49 of the present invention can be used as a kind of biological nitrogen-fixing fertilizer with Suppressing phytopathogens and Promoting plant growth effect and is applied aborning.As microbial fertilizer, can be applicable in the field planting industry of the gramineous crops such as corn, wheat after bacterial strain of the present invention cultivated, can effectively reduce the usage quantity of urea.
Sequence table
Claims (6)
1. series bacillus (Paenibacillus sp.) bacterial strain 1-49 with nitrogen fixing capacity, its preserving number is CGMCC No.4966.
2. the microbial inoculum that contains the described series bacillus 1-49 of claim 1.
3. the nitrogenase with nitrogenase activity that produces of series bacillus 1-49 claimed in claim 1, the encoding gene that it is characterized in that this nitrogenase is nifH, the nucleotides sequence of this nifH gene is classified SEQ ID No.1 as.
4. series bacillus 1-49 claimed in claim 1 or its tunning or the application of microbial inoculum claimed in claim 2 on Suppressing phytopathogens, it is characterized in that, described phytopathogen is Fusarium oxysporum (Fusarium oxysporum), Rhizoctonia cerealis (Rhizoctonia cerealis), sclerotinite (Sclerotinia sclerotiorum) and/or Botrytis cinerea (Botrytis cinerea).
5. series bacillus 1-49 claimed in claim 1 or its tunning or the application of microbial inoculum claimed in claim 2 on wheat growth-promoting.
6. the application of series bacillus 1-49 claimed in claim 1 in preparation biological nitrogen fixation microbiobacterial agent or biological organic fertilizer.
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| CN103451130B (en) * | 2013-07-25 | 2014-11-26 | 中国农业大学 | Nitrogen fixing gene cluster and application thereof |
| CN111944832B (en) * | 2020-08-28 | 2022-11-29 | 上海市农业科学院 | Self-generated nitrogen fixation gene for plant, nitrogen fixation enzyme expression cassette, preparation method and application thereof |
| CN113502244B (en) * | 2021-07-07 | 2023-03-28 | 中国水稻研究所 | Paenibacillus Hunan and application thereof |
| CN113493754A (en) * | 2021-07-28 | 2021-10-12 | 微生物肥料技术研究推广中心 | Method for liquid fermentation of nitrogen-fixing Paenibacillus |
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| CN101735966B (en) * | 2009-09-25 | 2012-01-04 | 中国科学院南海海洋研究所 | Strong bacillus G25-1-2 and applications thereof |
| CN102086444B (en) * | 2010-12-09 | 2012-02-29 | 南京师范大学 | Paenibacillus elgii strain and application thereof |
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