CN102604860A - 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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- CN102604860A CN102604860A CN2012100267117A CN201210026711A CN102604860A CN 102604860 A CN102604860 A CN 102604860A CN 2012100267117 A CN2012100267117 A CN 2012100267117A CN 201210026711 A CN201210026711 A CN 201210026711A CN 102604860 A CN102604860 A CN 102604860A
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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 the efficient phosphate-solubilizing bacteria of a kind of pine tree rhizosphere 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 big key elements; Be the important composition composition of plant nucleic acid in vivo and plurality of enzymes, coenzyme etc., simultaneously intravital various physiological and biochemical procedures of involved in plant in many ways again play an important role to promoting growth and development of plant and metabolism; Scarce phosphorus can cause growth and development of plants to be stagnated; Root system development is bad, and plant is short and small, and living weight seriously descends.Therefore, phosphoric is that plant must obligato nutritive element.
Contain a large amount of deposit phosphorus in the soil, can be divided into inorganic states phosphorus and organic phosphorus, but mainly exist with non-molten attitude.Phosphorus in the soil is mainly fixed through absorption, deposition, three kinds of modes of biological fixing, Ca in phosphorus element in the soil more than 95% and the soil
2+, Fe
2+, Fe
3+, Al
3+Combine and lose its validity Deng metals ion.Phosphate fertilizer in being manured into soil is most of obligate absorption can to take place 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 a large amount of application of P fertilizer had not only been exhausted limited phosphate rock resource but also the eutrophication of aggravating environment.Therefore, solve the attention that this problem more and more receives people through the biotechnology approach.
Phosphate-solubilizing bacteria (Phosphobacteria) is the major microorganisms monoid in the soil phosphorus circulation, and it has vital role to improving plain absorption of plant phosphorus and growth.Phosphate-solubilizing bacteria can promote the absorption of plant to various nutritive elements, promotes the growth of plant root, improves the utilization ratio of plant to 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 eagroforestry ecotope all to have important role.
Pine tree distributes very wide, is China mountain region, desert afforestation, gully improvement, and one of main reproducting tree species of flower garden greening have 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 living weight is low.Wherein, the phosphorus element is under-supply to be outstanding and universal problem, has caused the pine tree poor growth of China's most of areas, and resistance descends.The efficient phosphate-solubilizing bacteria of pine tree rhizosphere can improve the plant availability and the phosphate fertilizer utilization efficiency of insoluble phosphorus in the soil, improves the phosphorus plain under-supply contradiction of pine tree artificial forest in producing, promotes growing of pine tree.At present, the screening and the rarely seen report of applied research of the relevant efficient phosphate-solubilizing bacteria of pine tree rhizosphere.
Summary of the invention
Goal of the invention: the deficiency to existing in the prior art the purpose of this invention is to provide a kind of bulkholderia cepasea of biting that the pine tree rhizosphere efficiently dissolves insoluble inorganic phosphorus and organophosphorus that is used for more.Another object of the present invention provides 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 scheme that the present invention adopts is:
A kind of bulkholderia cepasea of biting more; Its classification called after is bitten bulkholderia cepasea (Burkholderia multivorans) WS-FJ9 more; Be preserved in Chinese typical 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; Screened acquisition the living slash pine pure forest rhizosphere soil in 28 years from Sha County, Fujian Province government's bank forest farm, 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, no gemma, aerobic; Oxydase reaction is negative, and catalase test is positive, and the starch hydrolysis experiment is negative, and M.R. tests negative; V.P. test is positive, and the gelatin hydrolysis test is negative, and 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 methods such as inoculation onion, tobacco (be used to judge bacterial strain to plant pathogenic) and inoculation clover (be used to judge bacterial strain to animal pathogenic) that bulkholderia cepasea (Burkholderia multivorans) the WS-FJ9 bacterial strain of biting that belongs to Bcc genotype II is carried out security and measures more; The 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 tabulation 1.Sequence in survey 16SrDNA sequence and the GenBank DB is carried out the BLAST comparison.The result shows that 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%.Through utilizing the automatic Analysis and Identification of Biolog system; 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 of the present invention more and shake at liquid under the situation of training, to tricalcium phosphate, phosphagel phosphaljel and Yelkin TTS have stronger solute effect, compare significant difference with contrast; WS-FJ9 is processed microbial inoculum inoculation slash pine seedling, and the result shows that 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 growing state 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 pathogenic bacteria slightly 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
Below in conjunction with specific embodiment the present invention is done further explanation.
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, zero(ppm) 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 to separate 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, zero(ppm) water 1000mL, pH7.0～7.5.
With activatory WS-FJ9 inoculation NB substratum (Carnis Bovis seu Bubali cream 3g, peptone 10g, sodium-chlor 5g; Zero(ppm) water 1000mL, pH 7.2～7.4) in, 30 ℃ of shaking culture 18～24h process seed liquor; Getting the 0.5mL seed liquor and be inoculated in respectively in the 100mL triangular flask that contains 50mL phosphorus decomposing culture medium A and 50mL phosphorus decomposing substratum B, is contrast (CK) with the phosphorus decomposing substratum that connects the blank seed liquor of equal volume, 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 is measured the fermented liquid available phosphorus content.
The loading amount of the Meng Jinna organophosphorus liquid nutrient medium for preparing by every bottle of 50mL added in the triangular flask of 100mL; Sterilization; Every bottle of Yelkin TTS and the 0.5mL bacterial suspension that adds 0.5mL in cooling back, contrast connects the inactivated bacteria suspension of equivalent, and all are handled and all do 3 repetitions.Inoculate back 30 ℃, 180r/min shaking culture 3d.
The result is as shown in Figure 1, as can beappreciated from fig. 1, and 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.With Yelkin TTS is sole carbon source, and the fermented liquid available phosphorus content reaches 3.22mgL
-1, showing that to sum up the WS-FJ9 bacterial strain is to tricalcium phosphate, phosphagel phosphaljel and Yelkin TTS have stronger dissolving power.
The righttest phosphorus decomposing condition test of embodiment 2WS-FJ9 bacterial strain:
After the WS-FJ9 activation, inoculation NB substratum (Carnis Bovis seu Bubali cream 3g, peptone 10g; Sodium-chlor 5g, zero(ppm) water 1000mL, pH 7.2～7.4) in; 30 ℃ of shaking culture 18～24h process 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, prescription with the phosphorus decomposing culture medium A), the NBRIP culture medium carbon source is made as sucrose, fructose, SANMALT-S, lactose and N.F,USP MANNITOL respectively; Nitrogenous source is made as peptone, Carnis Bovis seu Bubali cream, saltpetre, an ammonium nitrate, nitrocalcite respectively; Phosphorus decomposing substratum to connect 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 is measured the fermented liquid available phosphorus content.The result shows that the righttest phosphorus decomposing condition does, in the 100mL triangular flask, is that carbon source, ammonium sulfate are nitrogenous source with glucose, 28 ℃～30 ℃ of liquid amount 30～50mL, original ph 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 the 100mL triangular flask that 50mLNB substratum (prescription is with embodiment 1) is housed with a small amount of thalline of transfering loop picking, 29 ℃, 180r/min shaking culture 72h.(4 ℃, 6000r/min) centrifugal 5min behind SPSS rinse thalline 2～3 times, regulates bacteria suspension (7～8 * 10 with SPSS to fermented liquid
8Cfu/mL) process the WS-FJ9 microbial inoculum.Inoculation slash pine seedling (seedling age 120d) is contrast (CK) with the equivalent SPSS, and inoculum size is the 15mL/ strain.20 repetitions of every processing place 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 are with shown in Figure 3, and inoculation WS-FJ9 microbial inoculum has tangible growth-promoting functions to the slash pine seedling.Inoculation is handled the height of seedling, the stem that make the slash pine seedling and is slightly compared according to increase is in various degree arranged.Wherein, height of seedling and stem are slightly compared according to having increased by 20.52% and 8.4% respectively.
Table 1WS-FJ9 is to the growth and the living weight of slash pine seedling
Annotate: P＜0.05, same column different rows lowercase representative significant difference inequality.
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 broken into equably the aperture of circle with the punch tool (diameter 5mm) of sterilization at Bechtop.Adopt the method for dull and stereotyped face-off, pathogenic bacteria bacterium cake is moved the center of receiving each petridish, line after the activation of WS-FJ9 bacterial strain is connected on plate edge.The 30mm place apart from the center is a blank only to connect pathogenic bacteria, and each handles 3 repetitions, observes behind 28 ℃ of cultivation 3～7d, and the result is as shown in Figure 4, and the WS-FJ9 bacterial strain has obvious fungistatic effect to the withered pathogenic bacteria slightly of pine.
The safety testing of embodiment 5WS-FJ9 bacterial strain:
The present invention adopts inoculation onion and tobacco to judge bacterial strain pathogenic to plant, adopts to inoculate clover and judge bacterial strain pathogenic to animal.The tobacco (Nicotiana benthniciana) and alfalfa (Medicago sativa) seed that supply the examination material to comprise onion (Allium cepa), do not bloom.
The pathogenic mensuration of onion: get onion bulkholderia cepasea 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.LMG1222 bacterial strain (CK1) and aqua sterilisa (CK2) to contain virulence gene (BCESM and cblA) are dual contrast.All processing all repeat 3 times, place 26 ℃ of cultivations, check result behind the 4-5d.Wherein, the LMG1222 bacterium source is in BCCM (Belgian microbial preservation center)/Ghent University.
The result is as shown in Figure 5 in the onion inoculation test, and the onion of inoculation WS-FJ9 bacterial strain only produces less irritated spot at the inoculation point, 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 evident difference yet.Infer that in view of the above this bacterial strain is not the pathogenic bacterium of onion, the speckle of generation only is the anaphylaxis of onion.And inoculation contains the onion of the LMG1222 bacterial strain of virulence gene and not only produces an irritated spot at inoculation point, 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 that in view of the above the LMG1222 bacterial strain is the onion pathogenic bacterium.More than explanation bulkholderia cepasea (Burkholderia multivorans) the WS-FJ9 bacterial strain of biting that is under the jurisdiction of Bcc genotype II does not have pathogenicly more to onion, is not the onion pathogenic bacteria, and onion is had security.
The pathogenic mensuration of tobacco: the bacteria containing amount that uses sterilizing syringe to get 0.5 μ L is 10
7-10
8The bacteria suspension of cfu/mL punctures to tobacco leaf, and bacterium liquid is injected the wound.LMG1222 bacterial strain (CK1) and aqua sterilisa (CK2) to contain the virulence gene are dual contrast.All processing all repeat 3 times, place 25 ℃, relative humidity 85%, sunshine 16h growth cabinet cultivate.Show withered spot or the positive reaction of water stain spot at 24-48h, occur the negative reaction of macula lutea about 3d.
The result is as shown in Figure 6 in the tobacco inoculation test, and 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 bigger water stain spot of tobacco, promptly produces withered spot behind the 24h, explains that this bacterium is the pathogenic bacterium of tobacco.
The pathogenic mensuration of clover: 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 (about 10mL soaks 300 seeds) with 98% vitriol oil, with 500mL aqua sterilisa cleaning 4 times, steep then in the 60mL aqua sterilisa, 32 ℃ of concussions (150r/min) 6-8h that soaks seed cleans 2 times again, changes similarity condition overnight cultures behind the water.After cleaning 2 times with aqua sterilisa again next day (each 60mL water), put with aseptic nipper on 1% the water agar plate, every ware is put 30 seeds, removes abnormal seed that do not germinate and germinate after the germination, and the normal seed that germinates is counted, and waits for inoculation.Alfalfa seed on the water agar plate is stung an aperture with inoculating needle on cotyledon; Get the ready bacteria suspension point of 20 μ L in the wound, every inoculation repeats 3 times, establishes 2 kinds of CK and handles; Wherein 1 inoculation contains the LMG1222 bacterial strain (CK1) of virulence gene, in addition 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 behind the 7d.
The clover inoculation test, inoculation LMG1222 bacterial strain sickness rate (50.6%) utmost point is higher than the clover (seeing table 2) of inoculation WS-FJ9 bacterial strain (8.7%) and inoculation aqua sterilisa (7.8%) significantly.Whether clover is to replace mouse as measuring the model plant of Bcc bacterial strain to mammalian toxicity, can detect the Bcc bacterial strain quickly and easily with the clover model and have pathogenic to Mammals.(Burkholderia multivorans) WS-FJ9 bacterial strain that above-mentioned test-results explanation belongs to onion bulkholderia cepasea genotype II does not have toxic to Mammals.
The present invention adopts methods such as inoculation onion, tobacco (be used for judging bacterial strain to plant pathogenic) and inoculation clover (be used to judge bacterial strain to animal pathogenic) that bulkholderia cepasea (Burkholderia multivorans) the WS-FJ9 bacterial strain of separating from the pine tree rhizosphere soil of biting that belongs to Bcc genotype II is carried out security and measures more; The result shows that this bacterial strain does not have pathogenic to onion and clover; Tobacco there is faint toxicity, but and no pathogenicity.It is thus clear that bulkholderia cepasea (Burkholderia multivorans) the WS-FJ9 bacterial strain of biting that belongs to Bcc genotype II is 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 clover seedling sickness rate behind table 2 inoculation WS-FJ9 and the 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
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Claims (4)
1. bite bulkholderia cepasea one kind more, its classification called after bite more bulkholderia cepasea (
Burkholderia multivorans) WS-FJ9, being preserved in Chinese typical culture collection center, deposit number is CCTCC NO:M2011435, preservation date: on December 1st, 2011.
2. the described application of bulkholderia cepasea in phosphorus decomposing of biting of claim 1 more.
3. the described application of bulkholderia cepasea in promoting the pine tree growth of biting of claim 1 more.
4. the described application of bulkholderia cepasea in suppressing the former bacterium of sphaeropsis sapinea of biting of claim 1 more.
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