CN101575583B - Pseudomonas fluorescens and application thereof in promoting growth of poplar - Google Patents
Pseudomonas fluorescens and application thereof in promoting growth of poplar Download PDFInfo
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- CN101575583B CN101575583B CN2009100258811A CN200910025881A CN101575583B CN 101575583 B CN101575583 B CN 101575583B CN 2009100258811 A CN2009100258811 A CN 2009100258811A CN 200910025881 A CN200910025881 A CN 200910025881A CN 101575583 B CN101575583 B CN 101575583B
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Abstract
The invention discloses pseudomonas fluorescens separated from rooting zone soil and capable of dissolving difficultly soluble inorganic phosphorus in high efficiency. The pseudomonas fluorescens is classification-named as pseudomonas fluorescens JW-JS1 and has been stored in the China Center for Type Culture Collection, the storage No. is M 209027, and the storage data is Feb. 18, 2009. The invention also discloses application of the pseudomonas fluorescens in promoting growth of poplar. Under the condition of liquid shaking culture, JW-JS1 strains have stronger effect of dissolving tricalcium phosphate and hydroxyapatite; and the pseudomonas fluorescens JW-JS1 can be inoculated to cuttage seedlings of NL-895poplar and tree seedlings of eastern cottonwood. Results show that the pseudomonas fluorescens obviously promotes the growth of the poplar. Therefore, the invention provides a desirable strain resource for developing a phosphorus-dissolving bacteria fertilizer special for the poplar.
Description
Technical field
The invention belongs to the microbial fertilizer technical field in the biological fertilizer field, be specifically related to a kind of Pseudomonas fluorescens and application in promoting growth of poplar thereof of willow rhizosphere high-efficient phosphate-solubilizing.
Background technology
Phosphorus is one of limiting plant growth essential a large amount of mineral nutrient elements of growing, but Ca in the plain and soil of the phosphorus 95% or more in the soil
2+, Fe
2+, Fe
3+, Al
3+Deng metal ion in conjunction with and the forfeiture plant availability.Soluble phosphoric manure major part very fast generation obligate absorption in being manured into soil and chemical precipitation and be fixed, the this season utilization ratio has only 5%～10%, add the aftereffect of crop, utilization ratio does not generally exceed 25%, most of phosphate fertilizer accumulates in soil with the phosphoric acid salt of insoluble, and plant is difficult to absorb; On the other hand, use phosphate fertilizer in a large number and not only exhaust limited phosphate rock resource but also the eutrophication of aggravating environment, cause problems such as a series of energy dilemma and environmental pollution.Therefore, solving this problem by the biotechnology approach is more and more accepted by people.
Studies show that, have a large amount of phosphate solubilizing microorganisms in the soil, they can be converted into the insoluble phosphate in the soil effective form that plant can absorb.If these microbial hosts produce organic acid in metabolic process, the release of the pH value by reducing edatope and the complexing action promotion phosphorus of organic anion.Plant availability and phosphate fertilizer utilization efficiency that the inoculation phosphate solubilizing microorganism improves the soil insoluble phosphorus receive much concern always.Compare with fungi, actinomycetes, the plant rhizosphere bacterium is considered to the most effective phosphate solubilizing microorganism, and they can discharge insoluble phosphate in the soil efficiently, for the plant absorbing utilization.But this quasi-microorganism, is difficult to effectively play a role to compete with other microorganism at the quantity not sufficient of plant rhizosphere usually.Therefore, be necessary under field conditions (factors), screening, efficiently phosphate-solubilizing bacteria good from the root face of specified plant, rhizosphere soil with this plant affinity, make after the microbial inoculum tieback in the soil to promote the growth of plant.Many documents are pointed out both at home and abroad, to soil or crop root inoculation phosphate solubilizing bacteria, can promote the output of wheat, green vegetables, corn, naked oats, wild cabbage, peanut, rape, khuskhus, clover etc.But have not yet to see research report at perennial crop inoculation phosphate solubilizing microorganism.
Willow is that the highest speed of middle latitude plains region, world cultivated area maximum, timber yield is given birth to one of commerical tree species, is the important operation seeds of industrial timberland in short felling cycle, also is the important composition seeds of China's agricultural ecosystem.Willow is fast because of growing, and felling cycle, short and frequency was big to soil nutrient consumption, was prone to the soil fertility decay.Wherein, the phosphorus element is under-supply to be outstanding and universal problem, causes the most of geographic growth of poplar of China bad, degeneration-resistant decline.Willow rhizosphere high-efficient phosphate-solubilizing bacterium can be improved the plant availability and the phosphate fertilizer utilization efficiency of insoluble phosphorus in the soil, and the plain under-supply contradiction of phosphorus, the promotion growth of poplar that improve in the Poplar Plantation production are grown.At present, screening and the applied research of relevant willow rhizosphere high-efficient phosphate-solubilizing bacterium do not appear in the newspapers.
Summary of the invention
Technical problem to be solved by this invention provides the Pseudomonas fluorescens that a kind of willow rhizosphere efficiently dissolves the insoluble inorganic phosphorus;
The technical problem that the present invention also will solve provides the application of above-mentioned Pseudomonas fluorescens in promoting growth of poplar.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is as follows:
From the 2 years life 351 poplar rhizosphere soils in Chen Xu forest farm, Sihong County, Xiuqian City, Jiangsu Province, screening has obtained Pseudomonas fluorescens (Pseudomonas fluorescens) JW-JS1 that a plant height is imitated phosphorus decomposing.Be preserved in Chinese typical culture collection center C CTCC, deposit number: M 209027, preservation date: on February 18th, 2009.
The main biological property of JW-JS1 bacterial strain: on the beef-protein medium flat board, bacterium colony is less, and color is pale yellow, circular, neat in edge; The thalline direct rod shape, Gram-negative, about 0.45 * 0.74 μ m of size～1.91 * 2.06 μ m, no gemma is extremely given birth to one or three flagellums; Aerobic, the oxydase reaction feminine gender, the catalase test positive, starch hydrolysis experiment feminine gender, glucose fermentation, M.R. and V.P. test are negative, and the gelatin hydrolysis test is positive, and the nitrate reductase test is negative, produces ammonia and hydrogen sulfide production test feminine gender, the Citrate trianion growth test positive; Behind the dull and stereotyped cultivation of King ' s B (peptone 20g, glycerine 10g, dipotassium hydrogen phosphate 1.5g, sal epsom 1.5g, agar 15g, distilled water 1000ml, pH7.2) 24h, periphery of bacterial colonies produces diffusible cyanine, bacterium colony green-emitting fluorescence under 366nm wavelength UV-light, periphery of bacterial colonies show blue or green to blue; Thalline is autofluorescence under fluorescent microscope.
JW-JS1 bacterial strain 16SrDNA gene order is seen shown in the SEQ ID No.1.
Sequence in survey 16SrDNA sequence and the GenBank database is carried out the BLAST comparison.The result shows that JW-JS1 bacterial strain and Rhodopseudomonas (Pseudomonas) homology is all very high, and wherein the similarity with P.fluorescens is 98%.Combining form, physiological and biochemical property and 16SrDNA sequential analysis are accredited as Pseudomonas fluorescens (Pseudomonas fluorescens).
The potted plant inoculation test of JW-JS1 microbial inoculum shows that this microbial inoculum has obviously promoted growing of willow.
Beneficial effect: the JW-JS1 bacterial strain shakes at liquid under the situation of training, to tricalcium phosphate (Ca
3(PO
4)
3) and hydroxyapatite (Ca
10(PO
4)
6(OH)
2) have stronger solute effect, compare significant difference with contrast (CK); JW-JS1 makes microbial inoculum inoculation NL-895 poplar cuttage seeding and eastern cottonwood seedling, and the result shows that this microbial inoculum obviously promotes growing of poplar seedlings.Therefore, the present invention provides good strain resource for the special-purpose phosphate-solubilizing bacteria fertilizer of exploitation willow.
Description of drawings
Pseudomonas fluorescens (Pseudomonas fluorescens) JW-JS1 has been preserved in Chinese typical culture collection center C CTCC, deposit number: M 209027, preservation date: on February 18th, 2009.
Fig. 1 is the dissolving power of JW-JS1 bacterial strain to tricalcium phosphate and hydroxyapatite.
Fig. 2 is inoculation JW-JS1 microbial inoculum eastern cottonwood seedling growing state.
Embodiment:
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that embodiment describes concrete material proportion, processing condition and result thereof and only is used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1:JW-JS1 phosphorus decomposing ability test:
Phosphorus decomposing culture medium A: glucose 10g, Ca
3(PO
4)
35g, MgCL
25g, KCL 0.2g, MgSO
4.7H
2O 0.25g, (NH
4)
2SO
40.1g, distilled water 1000mL, pH 7.0.
Phosphorus decomposing substratum B: use Ca
10(PO
4)
6(OH)
2Replace the Ca in the phosphorus decomposing culture medium A
3(PO
4)
3, other composition is identical with content.
With activatory JW-JS1 inoculation NB substratum (extractum carnis 3g, peptone 10g, sodium-chlor 5g, distilled water 1000mL, pH 7.2～7.4) in, 30 ℃ of shaking culture 18～24h make seed liquor, getting the 0.5ml seed liquor is inoculated in respectively in the 100mL triangular flask that contains 50mL phosphorus decomposing culture medium A and 50mL phosphorus decomposing substratum B, with the phosphorus decomposing substratum that connects the blank seed liquor of equal volume is contrast (CK), and each handles 3 repetitions, 30 ℃, behind the 180r/min shaking culture 72h, 4 ℃ of fermented liquids, the centrifugal 10min of 10000r/min, molybdenum antimony resistance colorimetric method measure fermented liquid available phosphorus content (see figure 1).
As can be seen from Figure 1, with Ca
3(PO
4)
3During for unique phosphorus source, the fermented liquid available phosphorus content reaches 669.56mgL
-1, be contrast (CK) 18.61 times; With Ca
10(PO
4)
6(OH)
2During for unique phosphorus source, the fermented liquid available phosphorus content is 546.24mgL
-1, be contrast (CK) 113 times.Show that to sum up the JW-JS1 bacterial strain has stronger dissolving power to tricalcium phosphate and hydroxyapatite.
The suitableeest phosphorus decomposing condition test of embodiment 2:JW-JS1:
With NBRIP (Plant Res Internat B. V.'s phosphoric acid salt growth medium, prescription is with the phosphorus decomposing culture medium A) is minimum medium, changes C source, N source, different C/N and envrionment conditions.The result shows that the suitableeest phosphorus decomposing condition is, in the 100mL triangular flask, is that carbon source, ammonium sulfate are nitrogenous source with glucose, and C/N is 80/1～100/1,28 ℃～30 ℃ of liquid amount 20～30mL, initial pH value 7.0～7.2, inoculum size 3%～4%, temperature.
The test of embodiment 3:JW-JS1 greenhouse pot culture:
With after the JW-JS1 activation, be inoculated in the 100mL triangular flask that 50mLNB substratum (sodium-chlor 5g, distilled water 1000mL, pH 7.2～7.4 for extractum carnis 3g, peptone 10g) are housed with a small amount of thalline of transfering loop picking, 29 ℃, 180r/min shaking culture 72h.(4 ℃, 6000r/min) centrifugal 5min behind stroke-physiological saline solution rinse thalline 2～3 times, regulates bacteria suspension (7～8 * 10 with stroke-physiological saline solution to fermented liquid
8Cfu/mL) make microbial inoculum.Inoculating NL-895 cuttage seeding and eastern cottonwood seedling (seedling age 60 days) respectively, is contrast with the equivalent stroke-physiological saline solution, and inoculum size is cuttage seeding 15mL/ strain, seedling 5mL/ strain.6 repetitions of every processing place the greenhouse unified management, and illumination is 12h/ days, waters in good time.
150 days growth situations (seeing Table 1) of NL-895 poplar cuttage seeding.Can find out that from table 1 inoculation JW-JS1 microbial inoculum is to the tangible growth-promoting functions of having of the potted plant cuttage seeding of NL-895.Inoculation handles that height of seedling, the stem make the NL-895 poplar are thick, fresh weight and dry weight comparison be according to the increase that has in various degree.Wherein, height of seedling and stem are slightly compared according to having increased by 21.96% and 5.04% respectively; Fresh weight and dry weight comparison are according to having increased by 57.33% and 54.36% respectively.
Table 1JW-JS1 is to the growth and the biomass of NL-895 poplar cuttage seeding
Annotate: P＜0.05, same column different rows lowercase representative significant difference inequality
90 days growth situations of eastern cottonwood seedling (see Table 2 and Fig. 2).Can find out that from table 2 inoculation JW-JS1 microbial inoculum is to the tangible growth-promoting functions of having of the potted plant seedling of eastern cottonwood.Inoculation is handled the height of seedling, leading thread, fresh weight and the dry weight comparison that make the eastern cottonwood seedling and is shone the increase that has in various degree.Wherein, the comparison of height of seedling and leading thread is according to having increased by 51.66% and 1.98% respectively; Fresh weight and dry weight comparison are according to having increased by 150.18% and 168.49% respectively.
Table 2JW-JS1 is to the growth and the biomass of eastern cottonwood seedling
Annotate: P＜0.05, same column different rows lowercase representative significant difference inequality
SEQUENCE?LISTING
＜110〉Nanjing Forestry University
＜120〉a kind of Pseudomonas fluorescens and the application in promoting growth of poplar thereof
<130>njfu090122
<160>1
<170>PatentIn?version?3.3
<210>1
<211>1323
<212>DNA
<213>Pseudomonas?fluorescens
<400>1
acacatgcaa?gtcgagcggc?agcacgggta?cttgtacctg?gtggcgagcg?gcggacgggt 60
gagtaatgcc?taggaatctg?cctggtagtg?ggggataacg?ctcggaaacg?gacgctaata 120
ccgcatacgt?cctacgggag?aaagccaggg?gaaccttcgg?gccttgcgct?atcagatgag 180
cctaggtcgg?attagctagt?tggtggaggt?aatggctcac?caaggcgacg?attccgtaac 240
tggtctgaga?ggatgatcca?gtcacactgg?aactgaggac?acggtccaga?ctcctacggg 300
aggcagcagt?ggggaatatt?ggacaatggg?cgaaagcctg?atccagccat?gccgcgtgtg 360
tgaagaaggt?cttcggattg?taaagcactt?taagttggga?ggaagggcag?ttacctaata 420
cgtaattgtt?ttgacgttac?cgacagaata?agcaccggct?aactctgtgc?cagcagccgc 480
ggtaatacag?agggtgcaag?cgttaatcgg?aattactggg?cgtaaagcgc?gcgtaggtgg 540
tttgttaagt?tggatgtgaa?atccccgggc?tcaacctggg?aactgcattc?aaaactgaca 600
agctagagta?tggtagaggg?tggtggaatt?tcctgtgtag?cggtgaaatg?cgtagatata 660
ggaaggaaca?ccagtggcga?aggcgaccac?ctggactgat?actgacactg?aggtgcgaaa 720
gcgtggggag?caaacaggat?tagataccct?ggtagtccac?gccgtaaacg?atgtcaacta 780
gccgttggga?gccttgagct?cttagtggcg?cagctaacgc?attaagttga?ccgcctgggg 840
agtacggccg?caaggttaaa?actcaaatga?attgacgggg?gcccgcacaa?gcggtggagc 900
atgtggttta?attcgaagca?acgcgaagaa?ccttaccagg?ccttgacatc?caatgaactt 960
tccagagatg?gattggtgcc?ttcgggaaca?ttgagacagg?tgctgcatgg?ctgtcgtcag 1020
ctcgtgtcgt?gagatgttgg?gttaagtccc?gtaacggagc?gcaacccttg?tccttagtta 1080
ccagcacgta?atggtgggca?ctctaaggag?actgccgtgt?gacaaaccgg?aggaaggtgg 1140
ggatgaacgt?caaggtctat?caatggccct?tacggctctg?ggctacacac?gtgctacaat 1200
ggtcggtaca?gagggttgcc?aagccgcgag?gtggagctaa?tcccacaaaa?ccgatcgtag 1260
tccggatcgc?agtctgcaac?tcgactgcgt?gaagtcggaa?tcgctagtaa?tcgcgaatca 1320
gaa 1323
Claims (2)
1. Pseudomonas fluorescens, its classification called after Pseudomonas fluorescens (Pseudomonas fluorescens) JW-JS1, be preserved in Chinese typical culture collection center C CTCC, deposit number is CCTCC No:M 209027, preservation date: on February 18th, 2009.
2. the application of the described Pseudomonas fluorescens of claim 1 in promoting growth of poplar.
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| CN101709217B (en) * | 2009-12-03 | 2011-06-08 | 中国农业大学 | Pseudomonas fluorescens CKD18 and application thereof |
| US8796179B2 (en) | 2009-12-22 | 2014-08-05 | Lantmannen Bioagri Ab | Fluorescent pseudomonad of the species pseudomonas azotoformans for enhancement of plant emergence and growth |
| CN102191199A (en) * | 2011-03-25 | 2011-09-21 | 合肥工业大学 | High-efficiency composite dephosphorizing bacterial agent and preparation method thereof |
| CN102911895B (en) * | 2012-09-27 | 2015-02-18 | 四川农业大学 | Phosphorus-dissolving stain PAN 4 and application of phosphorus-dissolving stain PAN 4 in promoting growth of walnuts |
| CN103131649B (en) * | 2012-12-31 | 2014-11-26 | 浙江工业大学 | Pseudomonas fluorescens and application in preparation of transform-4-aminomethyl-naphthenic acid thereof |
| CN103173361B (en) * | 2013-03-05 | 2014-06-25 | 福建农林大学 | Endophytic fungus promoting casuarina equisetifolia nutrient element absorption |
| CN103396972B (en) * | 2013-08-22 | 2014-12-31 | 牛赡光 | Pseudomonas fluorescens and application thereof |
| CN109022324B (en) * | 2018-08-28 | 2022-01-28 | 西安文理学院 | Pseudomonas fluorescens and application thereof |
| CN110699303A (en) * | 2019-11-19 | 2020-01-17 | 河南工业大学 | High-efficiency phosphate-solubilizing pseudomonas as well as microbial agent and application thereof |
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| CN100390272C (en) * | 2005-10-10 | 2008-05-28 | 中国农业大学 | Fluorescent pseudomonads and its fermenting culture process and application |
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| CN100390272C (en) * | 2005-10-10 | 2008-05-28 | 中国农业大学 | Fluorescent pseudomonads and its fermenting culture process and application |
Non-Patent Citations (1)
| Title |
|---|
| 刘国奇等.韭菜根际荧光假单胞菌株的分离和初步研究.《微生物学通报》.1999,第26卷(第3期),189-192. * |
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Application publication date: 20091111 Assignee: Jiangsu Tianyue Ecological Agriculture Co.,Ltd. Assignor: NANJING FORESTRY University Contract record no.: X2020980008190 Denomination of invention: Pseudomonas fluorescens and its application in promoting poplar growth Granted publication date: 20100825 License type: Common License Record date: 20201118 |
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Application publication date: 20091111 Assignee: NANJING SHENGXING HARMFUL BIOLOGICAL CONTROL TECHNOLOGY Co.,Ltd. Assignor: NANJING FORESTRY University Contract record no.: X2020980008312 Denomination of invention: Pseudomonas fluorescens and its application in promoting poplar growth Granted publication date: 20100825 License type: Common License Record date: 20201120 |