CN102604860B - Burkholderia multivorans WS FJ9 and application thereof to growth promotion of pine - Google Patents
Burkholderia multivorans WS FJ9 and application thereof to growth promotion of pine Download PDFInfo
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- CN102604860B CN102604860B CN 201210026711 CN201210026711A CN102604860B CN 102604860 B CN102604860 B CN 102604860B CN 201210026711 CN201210026711 CN 201210026711 CN 201210026711 A CN201210026711 A CN 201210026711A CN 102604860 B CN102604860 B CN 102604860B
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Abstract
The invention discloses burkholderia multivorans WS FJ9 which is separated from the soil at the root of the pine and can dissolve the slightly-soluble inorganic phosphorus and organic phosphorus efficiently and the application of the burkholderia multivorans. The WS FJ9 burkholderia multivorans is collected in the CCTCC (China Center for Type Culture Collection) on December 1, 2011 and has a collection number of No. M2011435. The burkholderia multivorans WS FJ9 has strong tricalcium phosphate, aluminum phosphate and lecithin dissolving effect under the condition of submerged culture, is very different from the contrast, has strong sphaeropsissapinea inhibiting activity and can be prepared into a microbial inoculum inoculated to the wetland pine to promote the growth of the pine. The invention provided an excellent strain resource for the development of the biological fertilizer for the pine.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of pine tree rhizosphere high-efficient phosphate-solubilizing bacterium and bite bulkholderia cepasea and the application in promoting the pine tree growth thereof more.
Background technology
The phosphorus element is one of plant nutrition three large key elements, it is the important composition composition of plant nucleic acid in vivo and plurality of enzymes, coenzyme etc., various physiological and biochemical procedures in the involved in plant body in many ways again simultaneously, to promoting growing of plant to play an important role with metabolism, scarce phosphorus can cause growth and development of plants to be stagnated, root system development is bad, and plant is short and small, the biomass degradation.Therefore, phosphoric is that plant must obligato nutritive element.
Contain a large amount of deposit phosphorus in soil, can be divided into Inorganic phosphorus and organic phosphorus, but mainly exist with non-molten attitude.Phosphorus in soil is mainly fixed by absorption, precipitation, three kinds of modes of biological fixing, Ca in the phosphorus element in soil more than 95% and soil
2+, Fe
2+, Fe
3+, Al
3+Deng metal ion in conjunction with and lose its validity.Phosphate fertilizer in being manured into soil is most of obligate absorption can occur and chemical precipitation is fixed, and the phos-phate forms of the insoluble that is difficult to absorb with plant accumulates in soil, and utilization ratio is very low; And use in a large number phosphate fertilizer and not only exhausted limited phosphate rock resource but also the eutrophication of aggravating environment.Therefore, solving this problem by the biotechnology approach more and more is subject to people's attention.
Phosphate-solubilizing bacteria (Phosphobacteria) is the Main microbial strains in the soil phosphorus circulation, and it has vital role to improving the absorption of plant phosphorus element and growth.Phosphate-solubilizing bacteria can promote plant to various nutrient uptakes, promote the growth of plant root, improve plant to the utilization ratio of phosphorus, improve the nutritional condition of plant, improve the output of plant, increase the plant resistance against diseases, can also improve Soil structure in addition, improve organic content, the improvement saltings is to cultivating and give full play to the soil ecology fertility, keeping the balance etc. of agroforestry ecotope all to have extremely important effect.
Pine tree distributes very wide, is China mountain region, desert afforestation, gull regulation, and one of Major Tree Species Planted of flower garden greening has huge ecology and economic worth.But the general plantation of pine tree is in poor soil, and the artificial breeding surviving rate is not high, poor growth, and biomass is low.Wherein, the phosphorus element is under-supply is outstanding and universal problem, has caused the pine tree poor growth of China's most area, and resistance descends.Pine tree rhizosphere high-efficient phosphate-solubilizing bacterium can be improved plant availability and the phosphate fertilizer utilization efficiency of insoluble phosphorus in soil, improves the under-supply contradiction of phosphorus element in Larix gmelini plantations production, promotes growing of pine tree.At present, screening and the rarely seen report of applied research of relevant pine tree rhizosphere high-efficient phosphate-solubilizing bacterium.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the purpose of this invention is to provide a kind of bulkholderia cepasea of biting for pine tree rhizosphere high-efficiency dissolution insoluble inorganic phosphorus and organophosphorus more.Another object of the present invention is to provide the above-mentioned bulkholderia cepasea purposes in promoting the pine tree growth of biting more.
Technical scheme: in order to realize the foregoing invention purpose, the technical solution used in the present invention is:
A kind of bulkholderia cepasea of biting more, its Classification And Nomenclature for biting bulkholderia cepasea (Burkholderia multivorans) WS-FJ9 more, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCCNO:M2011435, preservation date: on December 1st, 2011.
The above-mentioned genotype of biting bulkholderia cepasea WS-FJ9 is genotype II more.
Bite bulkholderia cepasea WS-FJ9 more, screening obtains from the 28 years living slash pine pure forest rhizosphere soils in Sha County, Fujian Province government's bank forest farm, and main biological property comprises: on the beef-protein medium flat board, bacterium colony is less, color is yellowish-white, circular, neat in edge; The thalline rod-short, Gram-negative is without gemma, aerobic, oxydase reaction is negative, and catalase test is positive, the Starch Hydrolysis negative, M.R. negative, V.P. are tested positive, the gelatin hydrolysis negative, the nitrate reductase test is positive, the product ammonia test is positive, and hydrogen sulfide production test is negative, and the Citrate trianion growth test is positive.
Adopt the methods such as inoculation onion, tobacco (be used for judgement bacterial strain to plant pathogenic) and inoculation clover (be used for judgement bacterial strain to animal pathogenic) to carry out Security test to bulkholderia cepasea (Burkholderia multivorans) the WS-FJ9 bacterial strain of biting that belongs to Bcc genotype II more, result shows that this bacterial strain does not have pathogenic to onion and clover, tobacco there is faint toxicity, but no pathogenicity.
The 16SrDNA gene order of WS-FJ9 bacterial strain is seen amino acid or nucleotides sequence list 1.Sequence in survey 16SrDNA sequence and GenBank database is carried out BLAST to be compared.Result shows, it is all very high that WS-FJ9 bacterial strain and bulkholderia cepasea belong to (Burkholderia) homology, and wherein the similarity with Burkholderia multivorans is 98%.Identify by utilizing Biolog system automatic analysis, Biolog similarity PROB 99% with Burkholderia multivorans, similar value SIM0.551, combining form, physiological and biochemical property and 16SrDNA sequential analysis are accredited as and bite bulkholderia cepasea (Burkholderia multivorans) more.
The above-mentioned application of bulkholderia cepasea WS-FJ9 in promoting the pine tree growth of biting more.
Above-mentioned bite the application of bulkholderia cepasea WS-FJ9 in phosphorus decomposing more.
A kind of phosphorus decomposing microbial inoculum contains and bites bulkholderia cepasea WS-FJ9 bacterial strain more, and cell concentration is 7～8 * 10
8Cfu/mL.
Above-mentioned bite the application of bulkholderia cepasea in suppressing the former bacterium of sphaeropsis sapinea (Sphaeropsis sapinea) more.
Beneficial effect: bite bulkholderia cepasea WS-FJ9 bacterial strain in the situation that liquid shakes training of the present invention, to tricalcium phosphate, aluminum phosphate and Yelkin TTS have stronger solute effect more, compared with the control, and significant difference; WS-FJ9 is made microbial inoculum inoculation slash pine seedling, and result shows, this microbial inoculum can obviously promote growing of slash pine nursery stock, and therefore, the present invention provides good strain resource for the special-purpose phosphate-solubilizing bacteria fertilizer of exploitation pine tree.
Description of drawings
Fig. 1 is the phosphorus decomposing capability result figure of WS-FJ9 bacterial strain, and wherein, the phosphorus element is Ca
3(PO
4)
2
Fig. 2 is the phosphorus decomposing capability result figure of WS-FJ9 bacterial strain, and wherein, the phosphorus element is AlPO
4
Fig. 3 is inoculation WS-FJ9 microbial inoculum slash pine seedling growth situation figure as a result;
Fig. 4 is WS-FJ9 and the dull and stereotyped figure as a result that stands facing each other of the withered slightly pathogenic bacteria of pine;
Fig. 5 is W-S-FJ9 inoculation onion bulb stem figure as a result;
Fig. 6 is WS-FJ9 inoculation tobacco figure as a result;
Embodiment
The present invention will be further explained below in conjunction with specific embodiment.
The phosphorus decomposing ability test of embodiment 1WS-FJ9 bacterial strain:
Phosphorus decomposing culture medium A: glucose 10g, Ca
3(PO
4)
25g, MgCl
25g, KCl 0.2g, MgSO
4.7H
2O0.25g, (NH
4)
2SO
40.1g, distilled water 1000mL, pH 7.0.
Phosphorus decomposing substratum B: use AlPO
4Replace the Ca in the phosphorus decomposing culture medium A
3(PO
4)
2, other composition is identical with content.
Meng Jinna organophosphorus substratum, the liquid culture phosphorus decomposing ability mensuration that is used for separating the organophosphorus bacterium: glucose 10g, (NH
4)
2SO
40.5g, NaCl 0.3g, KCl 0.3g, CaCO
35g, MgSO
47H
2O 0.3g, FeSO
47H
2O 0.03g, MnSO
44H
2O 0.03g, Yelkin TTS 0.2g, distilled water 1000mL, pH7.0～7.5.
WS-FJ9 inoculation NB substratum (extractum carnis 3g with activation, 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, take the phosphorus decomposing substratum that connects the blank seed liquor of equal volume as contrast (CK), each processes 3 repetitions, 30 ℃, after 180r/min shaking culture 72h, 4 ℃ of fermented liquids, the centrifugal 10min of 10000r/min, molybdenum antimony resistance colorimetric method is measured the fermented liquid available phosphorus content.
The Meng Jinna organophosphorus liquid nutrient medium that the prepares loading amount by every bottle of 50mL is added in the triangular flask of 100mL, sterilization, cooling rear every bottle of Yelkin TTS and 0.5mL bacterial suspension that adds 0.5mL, contrast connects the inactivated bacteria suspension of equivalent, and all are processed and all do 3 repetitions.Inoculate rear 30 ℃, 180r/min shaking culture 3d.
Result as shown in Figure 1, as can be seen from Figure 1, with Ca
3(PO
4)
2During for unique phosphorus source, the fermented liquid available phosphorus content reaches 711.29mgL
-1, be contrast (CK) 18.61 times; As shown in Figure 2, with AlPO
4During for unique phosphorus source, the fermented liquid available phosphorus content reaches 274.4mgL
-1, be contrast (CK) 3.5 times.Take Yelkin TTS as sole carbon source, the fermented liquid available phosphorus content reaches 3.22mgL
-1, to sum up showing, the WS-FJ9 bacterial strain is to tricalcium phosphate, and aluminum phosphate and Yelkin TTS have stronger dissolving power.
The suitableeest phosphorus decomposing condition test of embodiment 2WS-FJ9 bacterial strain:
After the WS-FJ9 activation, 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, get the 0.5mL seed liquor and be inoculated in respectively that 50mL is housed is in the 100mL triangular flask of minimum medium with NBRIP (Plant Res Internat B. V.'s phosphoric acid salt growth medium, formula with the phosphorus decomposing culture medium A), the NBRIP culture medium carbon source is made as respectively sucrose, fructose, maltose, lactose and N.F,USP MANNITOL; Nitrogenous source is made as respectively peptone, extractum carnis, saltpetre, ammonium nitrate, nitrocalcite, take the phosphorus decomposing substratum that connects the blank seed liquor of equal volume as contrast (CK), each processes 3 repetitions, 30 ℃, after 180r/min shaking culture 72h, 4 ℃ of fermented liquids, the centrifugal 10min of 10000r/min, molybdenum antimony resistance colorimetric method is measured the fermented liquid available phosphorus content.Result shows, the suitableeest phosphorus decomposing condition is, in the 100mL triangular flask, and take glucose as carbon source, ammonium sulfate is nitrogenous source, 28 ℃～30 ℃ of liquid amount 30～50mL, initial pH value 7.0～7.2, inoculum size 4%～5%, temperature.
The test of embodiment 3WS-FJ9 bacterial strain greenhouse pot culture:
After the WS-FJ9 activation, be inoculated in a small amount of thalline of transfering loop picking in the 100mL triangular flask that 50mLNB substratum (formula is with embodiment 1) is housed, 29 ℃, 180r/min shaking culture 72h.(4 ℃, 6000r/min) centrifugal 5min after 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 the WS-FJ9 microbial inoculum.Inoculation slash pine seedling (seedling age 120d), take the equivalent stroke-physiological saline solution as contrast (CK), inoculum size is the 15mL/ strain.20 repetitions of every processing are placed in the greenhouse unified management, and illumination is 12h/d, waters in good time.
Slash pine seedling inoculation 180d growing state, result such as table 1 and shown in Figure 3, inoculation WS-FJ9 microbial inoculum has obvious growth-promoting functions to the slash pine seedling.The height of seedling make the slash pine seedling, stem are processed in inoculation slightly has in various degree increase than contrast.Wherein, height of seedling and stem have slightly increased respectively 20.52% and 8.4% than contrast.
Growth and the biomass of table 1WS-FJ9 to the slash pine seedling
Annotate: P＜0.05, the not identical significant difference that represents of same column different rows lowercase.
Embodiment 4WS-FJ9 bacterial strain has the stronger activity resistent of picking up to the sphaeropsis sapinea fungal pathogens
The former bacterium of cultured sphaeropsis sapinea is beaten conglobate aperture at Bechtop equably with the punch tool (diameter 5mm) of sterilization.Adopt the method for dull and stereotyped face-off, pathogenic bacteria bacterium cake is moved the center of receiving each culture dish, line after the activation of WS-FJ9 bacterial strain is connected on plate edge.Apart from 30mm place, center, only to connect pathogenic bacteria as blank, each processes 3 repetitions, observe after cultivating 3～7d for 28 ℃, result as shown in Figure 4, the WS-FJ9 bacterial strain has obvious fungistatic effect to the withered slightly pathogenic bacteria of pine.
The safety testing of embodiment 5WS-FJ9 bacterial strain:
The present invention adopts inoculation onion and tobacco to judge pathogenic to plant of bacterial strain, adopts the inoculation clover to judge pathogenic to animal of bacterial strain.The tobacco (Nicotiana benthniciana) and alfalfa (Medicago sativa) seed that supply the examination material to comprise onion (Allium cepa), do not bloom.
Onion Pathogenicity: get Burkholderia WS-FJ9 bacteria suspension (1 * 10 with syringe
7-10
8Cfu/mL) on onion bulb stem 2 to lining injection (degree of depth 1-2cm), every some injection volume is 1mL.Take the LMG1222 bacterial strain (CK1) that contains virulence gene (BCESM and cblA) and aqua sterilisa (CK2) as dual contrast.All processing all repeat 3 times, are placed in 26 ℃ of cultivations, check result after 4-5d.Wherein, the LMG1222 bacterium source is in BCCM (Belgian microbial preservation center)/Ghent University.
Onion inoculation test result is inoculated the onion of WS-FJ9 bacterial strain as shown in Figure 5 only at the less irritated spot of vaccination generation, continues to cultivate, spot does not continue expansion, and after onion was cut, internal color did not change, be destitute of smell, comparing with the aqua sterilisa contrast does not have obvious difference yet.Infer that accordingly this bacterial strain is not the pathogenic bacterium of onion, the speckle of generation is only the anaphylaxis of onion.And inoculation contains the onion of the LMG1222 bacterial strain of virulence gene and not only produces irritated spot in vaccination, and along with the prolongation of incubation time, spot constantly enlarges, after cutting onion, inner jaundice, tissue is soft rotten shape and dense stink is arranged, infer accordingly, the LMG1222 bacterial strain is the onion pathogenic bacterium.Bulkholderia cepasea (Burkholderia multivorans) the WS-FJ9 bacterial strain of biting that above explanation is under the jurisdiction of Bcc genotype II does not have pathogenicly more to onion, be not the onion pathogenic bacteria, and onion is had security.
The tobacco Pathogenicity: the bacteria containing amount of getting 0.5 μ L with sterilizing syringe is 10
7-10
8The bacteria suspension of cfu/mL punctures to tobacco leaf, and bacterium liquid is injected the wound.Take the LMG1222 bacterial strain (CK1) that contains the virulence gene and aqua sterilisa (CK2) as dual contrast.All processing all repeat 3 times, be placed in 25 ℃, relative humidity 85%, sunshine 16h growth cabinet cultivate.In the 24-48h withered spot of performance or the positive reaction of water stain spot, the negative reaction of macula lutea appears about 3d.
Tobacco inoculation test result as shown in Figure 6, the tobacco leaf of inoculation WS-FJ9 bacterial strain has produced slighter anaphylaxis, has produced less water stain spot, but do not cause withered spot, not obvious with the aqua sterilisa contrast difference, though this explanation WS-FJ9 has slighter hypotoxicity to tobacco, and no pathogenicity.The LMG1222 bacterial strain that contains the virulence gene makes the larger water stain spot of tobacco, namely produces withered spot after 24h, illustrates that this bacterium is the pathogenic bacterium of tobacco.
The clover Pathogenicity: bacteria tested is inoculated in the NA liquid nutrient medium, and 24h is cultivated in 30 ℃ of concussions (180r/min), regulates bacteria suspension to 10 with aqua sterilisa
7-10
8Cfu/mL is used for inoculation.Alfalfa seed is soaked 20min (approximately 10mL soaks 300 seeds) with 98% vitriol oil, clean 4 times with the 500mL aqua sterilisa, then steep in the 60mL aqua sterilisa, 32 ℃ of concussions (150r/min) seed soaking 6-8h, clean again 2 times, change similarity condition overnight incubation after water.After cleaning 2 times with aqua sterilisa again next day (each 60mL water), put with aseptic nipper on 1% water agar plate, every ware is put 30 seeds, removes abnormal seed that do not germinate and germinate after germination, the normal seed that germinates is counted, waited for inoculation.Alfalfa seed on the water agar plate is stung an aperture with inoculating needle on cotyledon, get the 20 ready bacteria suspension points of μ L in the wound, every inoculation repeats 3 times, if 2 kinds of CK process, wherein 1 inoculation contains the LMG1222 bacterial strain (CK1) of virulence gene, another 1 inoculation sterilized water (CK2).Cultivate in the growth cabinet of postvaccinal seed under 37 ℃, 70% relative humidity, regularly illumination (12h illumination, 12h dark) condition, check sickness rate after 7d.
The clover inoculation test, inoculation LMG1222 bacterial strain sickness rate (50.6%) utmost point is significantly higher than the clover (seeing Table 2) that inoculates WS-FJ9 bacterial strain (8.7%) and inoculation aqua sterilisa (7.8%).Whether clover is to replace mouse as measuring the model plant of Bcc bacterial strain to mammalian toxicity, can detect quickly and easily the Bcc bacterial strain with the clover model and have pathogenic to Mammals.(Burkholderia multivorans) WS-FJ9 bacterial strain that above-mentioned test-results explanation belongs to Burkholderia genotype II does not have toxic to Mammals.
The present invention adopts the methods such as inoculation onion, tobacco (be used for judgement bacterial strain to plant pathogenic) and inoculation clover (be used for judgement bacterial strain to animal pathogenic) to carry out Security test to bulkholderia cepasea (Burkholderia multivorans) the WS-FJ9 bacterial strain of biting that belongs to Bcc genotype II of separating from the pine tree rhizosphere soil more, result shows that this bacterial strain does not have pathogenic to onion and clover, tobacco there is faint toxicity, but and no pathogenicity.As seen, bulkholderia cepasea (Burkholderia multivorans) the WS-FJ9 bacterial strain of biting that belongs to Bcc genotype II is the very potential safe biological and ecological methods to prevent plant disease, pests, and erosion of a strain and short bacterial strain of giving birth to pine tree more.
The significance of difference analysis of seedling of alfalfa sickness rate after table 2 inoculation WS-FJ9 and LMG1222
SEQUENCE LISTING
＜110〉Nanjing Forestry University
＜120〉a kind of bite bulkholderia cepasea and the application in promoting the pine tree growth thereof more
<130> 100
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1293
<212> DNA
<213> Burkholderia multivorans
<400> 1
tcgaacggca gcacgggtgc ttgcacctgg tggcgagtgg cgaacgggtg agtaatacat 60
cggaacatgt cctgtagtgg gggatagccc ggcgaaagcc ggattaatac cgcatacgat 120
ctacggatga aagcggggga tcttcggacc tcgcgctata gggttggccg atggctgatt 180
agctagttgg tggggtaaag gcctaccaag gcgacgatca gtagctggtc tgagaggacg 240
atcagccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 300
tttggacaat gggggaaacc ctgatccagc aatgccgcgt gtgtgaagaa ggccttcggg 360
ttgtaaagca cttttgtccg gaaagaaatc ctttgggtta atacctcgga gggatgacgg 420
taccggaaga ataagcaccg gctaactacg tgccagcagc cgcggtaata cgtagggtgc 480
gagcgttaat cggaattact gggcgtaaag cgtgcgcagg cggtttgtta agacagatgt 540
gaaatccccg ggcttaacct gggaactgca tttgtgactg gcaagctaga gtatggcaga 600
ggggggtaga attccacgtg tagcagtgaa atgcgtagag atgtggagga ataccgatgg 660
cgaaggcagc cccctgggcc aatactgacg ctcatgcacg aaagcgtggg gagcaaacag 720
gattagatac cctggtagtc cacgccctaa acgatgtcaa ctagttgttg gggattcatt 780
tccttagtaa cgtagctaac gcgtgaagtt gaccgcctgg ggagtacggt cgcaagatta 840
aaactcaaag gaattgacgg ggacccgcac aagcggtgga tgatgtggat taattcgatg 900
caacgcgaaa aaccttacct acccttgaca tggtcggaat cctgaagaga tttgggagtg 960
ctcgaaagag aaccgataca caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg 1020
ttgggttaag tcccgcaacg agcgcaaccc ttgtccttag ttgctacgca agagcactct 1080
aaggagactg ccggtgacaa accggaggaa ggtggggatg acgtcaagtc ctcatggccc 1140
ttatgggtag ggcttcacac gtcatacaat ggtcggaaca gagggttgcc aacccgcgag 1200
ggggagctaa tcccagaaaa ccgatcgtag tccggattgc actctgcaac tcgagtgcat 1260
gaagctggaa tcgctagtaa tcgcggatca gca 1293
Claims (4)
1. bite bulkholderia cepasea one kind more, its Classification And Nomenclature for biting bulkholderia cepasea (Burkholderia multivorans) WS-FJ9 more, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:M2011435, preservation date: on December 1st, 2011.
2. the application of bulkholderia cepasea in dissolving phosphoric acid DFP, aluminum phosphate and Yelkin TTS of biting claimed in claim 1 more.
3. the application of bulkholderia cepasea in promoting the slash pine seedling growth of biting claimed in claim 1 more.
4. the application of bulkholderia cepasea in suppressing the former bacterium of sphaeropsis sapinea of biting claimed in claim 1 more.
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|---|---|---|---|---|
| CN104017745A (en) * | 2013-12-27 | 2014-09-03 | 井冈山大学 | Bacterial strain resistant to acid-aluminum |
| CN104017745B (en) * | 2013-12-27 | 2017-02-01 | 井冈山大学 | Bacterial strain resistant to acid-aluminum |
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