CN106635922A - Salt-tolerant biocontrol bacterium B268 for bacterial wilt of horsetail beefwood and application of salt-tolerant biocontrol bacterium B268 - Google Patents
Salt-tolerant biocontrol bacterium B268 for bacterial wilt of horsetail beefwood and application of salt-tolerant biocontrol bacterium B268 Download PDFInfo
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Abstract
The invention provides a salt-tolerant biocontrol bacterium B268 for the bacterial wilt of horsetail beefwood. The salt-tolerant biocontrol bacterium B268 for the bacterial wilt of horsetail beefwood is classified and named as Bacillus siamensis, and is preserved in China General Microbiological Collection Center, the preservation number is CGMCC No.13225, and the preservation date is November, 1, 2016. The strain is separated from seawater immersed root soil of horsetail beefwood, has an antagonistic activity for pathogenic bacterial strains of the bacterial wilt of horsetail beefwood and tomatoes, and also has the an activity of dissolving phosphorus, an activity of synthesizing a siderophore and a plant growth hormone and other activities of promoting plant growth. In addition, the tolerance scope of sea salt of the strain is wide, a stable biological film can be formed at high salt content, and the capabilities of growth and colonization are high, and therefore, the strain B268 can be applied to the biological prevention and control of the bacterial wilt of horsetail beefwood, is particularly applied to the preparation of a biological control agent or a pesticide for the bacterial wilt of horsetail beefwood, and is also applied to the preparation of a bacterial manure for promoting the growth and improving the disease resistance of horsetail beefwood.
Description
Technical field
The invention belongs to microbial strains and its application, are related to the horse-tail bacterial wilt biocontrol bacteria of salt tolerant one by one
B268, is one plant of new salt tolerant bacillus siamensis, and bacterial strain preserving number is CGMCC No.13225.This
The bright biological and ecological methods to prevent plant disease, pests, and erosion activity for further relating to the bacterial strain, including bacteriostatic activity and promoting growth of plants activity, and its in the preventing and treating of horse-tail bacterial wilt
In application.
Background technology
Bacterial wilt also known as bacterialo wilt disease (bacterial wilt), be one kind by Ralstonia solanacearum (Ralstonia
Solanacearum the worldwide soil-borne disease for) causing, extensively betides the torrid zone, subtropical zone and temperate zone some areas,
In recent years due to the impact of greenhouse effects, occurs the trend spread to high latitude area.6 are included in Ralstonia solanacearum kind
Biochemical type (biovar I-VI) and 5 biological strains (race 1-5), diversity can contaminate more than 50 in complicated kind
Plant is planted in section, more than 200, is the most wide plant of host range from shrub to arbor from draft to woody from unifacial leaf to dicotyledonous
One of thing pathogenetic bacteria, it is annual to global diversified economy crop (such as tomato, potato, peanut, tobacco, strawberry, banana) and
Various forests (such as horse-tail, eucalyptus, mulberry, olive, teak, sinopimelodendron kwangsiense) bring massive losses, therefore are acknowledged as the world
Second largest most economic threat, while the also most pathogenic of researching value.
At present, the preventing and treating of bacterial wilt not yet forms effective means still in conceptual phase, and Biocontrol microorganism is wherein most
One of research direction of main flow, including the use of Antagonistic Fungi, endophyte and probio.Comprehensive literature looks into the knot of new and patent retrieval
Really, the microorganism for having biological and ecological methods to prevent plant disease, pests, and erosion active to Ralstonia solanacearum reported so far is mainly derived from the root soil of host plant
With endogenous cycle border, including bacillus (Bacillus), bacillus brevis (Brevibacillus), series bacillus
(Paenibacillus), ground bacillus (Geobacillus), lysine bacillus (Lysinibacillus), false unit cell
Bacterium (Pseudomonas), Stenotrophomonas (Stenotrophomonas), acinetobacter (Acinetobacter), enterobacteria
Category (Enterobacter), streptomycete (Streptomyces), arthrobacterium (Arthrobacter), frankia
(Frankia), aspergillus (Aspergillus), trichoderma (Trichoderma), mould (Penicillium) and bacteriophage etc.,
Ironically, some bacillus and streptomycete for being isolated from marine environment also show very high antagonistic activity.Gemma bar
Pseudomonas is the quasi-microorganism for having now been found that bacterial wilt biocontrol bacterium strain is most, have been reported that including bacillus amyloliquefaciens
(B.amyloliquefaciens), bacillus thuringiensis (B.thuringiensis), bacillus subtilis
(B.subtilis), Methylotrophic bacillus (B.methylotrophicus), Death Valley bacillus
(B.vallismortis), Bacillus cercus (B.cereus), bacillus megaterium (' B.magaterium ') etc..And this is specially
New strains B268 involved by profit is nearest with the affiliation of Siam bacillus (B.siamensis), although existing document from
Genome angle predicts the Biocontrol Potential of this kind of bacillus, but not yet has report in the study on prevention of any disease at present
Road.
On the other hand, horse-tail (Casuarina equisetifolia) is since introduce a fine variety to China, due to its salt tolerant
Alkali, humidity, check winds and fix drifting sand and the features such as fast-growing, it has also become the irreplaceable pioneer of the coastal trunk shelterbelt of China's Coastal Sandy Land
Seeds, so far afforestation area is more than 300,000 hectares.Bacterial wilt is one of Major Diseases of horse-tail, especially on southeastern coast ground
Area, because warmer climate is moist, is conducive to the growth and breeding of Ralstonia solanacearum, and summer typhoon is frequently, is infecting for germ
Create condition with propagation, therefore, from 1964 since Guangdong Province finds first horse-tail bacterial wilt, including Zhejiang, Fujian,
Almost each provinces and regions happens occasionally for Guangxi and Hainan etc..It is latent time length in Ralstonia solanacearum external environment out of office, hidden
Covering property is high, and can cause disease in seedling stage to the manhood of horse-tail, causes plant mortality, and the death rate is reachable when serious
More than 90%, while being also possible to intersect other high susceptible forests and crops for contaminating Infected regions, harmfulness is big, therefore,
In national forestry danger harmful organism list is listed horse-tail bacterial wilt in No. 4 bulletin of 2013 by the State Administration of Forestry.
So far, the study on prevention of horse-tail bacterial wilt is reported at most with breeding for disease resistance technology, but in view of Solanaceae Lei Er
The intraspecific variablity of Salmonella and toxicity are broken up, in combination with biological prevention, the integrated control theory of ' inside and outside minor '
Become a kind of trend.For example according to the salt tolerant moisture resistance properties of horse-tail, carry to be poured with seawater in Zhejiang, Fujian, Guangdong one and drench right
The local method that horse-tail bacterial wilt is prevented and treated, its general principle is exactly the micro-ecological environment that plant root is adjusted by seawater, all
Such as salinity, concentration of metal ions, biological community structure, make the existence of pathogen and contaminate environmental degradation, while being conducive to
The growth of specific Biocontrol microorganism, with preferable prevention effect.Salt tolerant bacillus involved by this patent
Siamensis B268 are isolated from the horse-tail root soil after Seawater immersion, the bacterial strain have concurrently pathogen antagonistic activity and
Promoting growth of plants activity, and it is wide to the tolerance range of sea salt, colonization ability is strong, has in the preventing and treating of horse-tail bacterial wilt certain
Application and development potentiality, meanwhile, under the increasingly severe overall background of China soil salination situation, the research of the bacterial strain is for each
Plant the control of plant disease under soil property and also there is certain reference.
The content of the invention
It is an object of the present invention to provide the horse-tail bacterial wilt biocontrol bacteria B268 of one plant of salt tolerant, is one plant of new bud
Spore bacillus.Bacterial strain B268 according to the present invention derives from susceptible horse-tail plant, gathers the root soil after Seawater immersion, passes through
Face-off plate screening active ingredients are obtained.Further analysis result shows that the bacterial strain belongs to Siam bacillus (Bacillus
Siamensis), and stronger biological and ecological methods to prevent plant disease, pests, and erosion activity is shown, including：(1) to being isolated from the Solanaceae thunder Er Shi in same horse-tail forest zone
The certainly Belgian Culture Collection (Belgian of bacterium pathogenic strain Ralstonia solanacearum YQ and purchase
Coordinated Collections of Microorganisms, BCCM) Ralstonia solanacearum type strain Ralstonia
solanacearum LMG 2299TIt is respectively provided with antagonistic activity；(2) promote to plant with phosphorus decomposing, the thermophilic iron element of generation and heteroauxin etc.
Thing growth activity；(3) tolerance range of sea salt is wide, and biological quilt can be formed under different culture mediums and multiple salinity
Film, shows stronger colonization ability.At present, Bacillus siamensis bacterial strains are in the research and application that bacterial wilt is prevented and treated
There is not been reported.Described bacterial strain B268 is by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation
(address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), classification life
Entitled Bacillus siamensis, preserving number is CGMCC No.13225, and preservation date is on November 1st, 2016.
It is a further object to provide purposes of the bacterial strain B268 in the biological control of horse-tail bacterial wilt.
Described bacterial strain Bacillus siamensis B268 can be used to prepare the biological control microbial inoculum or agricultural chemicals of horse-tail bacterial wilt,
Or for preparing the microbial-bacterial fertilizer for promoting Casuarina equisetifolia Growth and improving its disease resistance.Described bacterial strain Bacillus
Siamensis B268 are used to prepare the microbial-bacterial fertilizer for promoting Casuarina equisetifolia Growth and improving its disease resistance.Experiment proves described bacterium
Strain Bacillus siamensis B268, it is respectively provided with antagonistic activity for the pathogenic strain of horse-tail and bacterial wilt of tomato.
Described bacterial strain Bacillus siamensis B268, it has promoting growth of plants activity, including Decomposing phosphate activity, synthesizes and divide
Secrete the activity of thermophilic iron element, and synthesis auxin --- the activity of heteroauxin (IAA).Described bacterial strain Bacillus
Siamensis B268, it is isolated from the horse-tail root soil of Seawater immersion, with wide sea salt tolerance range (0-12%),
And (0-5%) generates the ability of stabilate film in the case where having salt and salt-free conditions.
At present, be related to bacterial wilt preventing and treating bacterial strain it is in the majority with tobacco bacterial wilt, other also include tomato, peanut, potato,
The crop bacterial wilt such as capsicum, ginger, eggplant, and horse-tail bacterial wilt then not yet has been reported that.
For achieving the above object, the technical scheme taken of the present invention is：
(1) separation of bacterial strain Bacillus siamensis B268 and antagonistic activity are screened.According to the literature, ocean ring
The microorganism much to Ralstonia solanacearum with high antagonistic activity is there is in border, meanwhile, seawater pours pouring method can effectively be pressed down
The horse-tail bacterial wilt of early stage processed, with reference to information above, specially contacted by Yueqing City of Zhejiang Province forest transition preventing and treating quarantine
Stand development cooperation, pouring seawater pouring method and carry out gathering root soil in the horse-tail forest zone of prevention effect checking at it carries out micro- life
The separation of thing.The xylem sample of grave illness horse-tail plant in forest zone is gathered simultaneously, carries out the separation of pathogenic strains.Finally by
The method screening of face-off plate obtains many plants of antagonistic microbes, wherein including bacterial strain B268.
(2) the antagonistic activity checking of bacterial strain Bacillus siamensis B268.Cause in acquired horse-tail bacterial wilt
On the basis of diseased plant Ralstonia solanacearum YQ, from Belgian Culture Collection (Belgian
Coordinated Collections of Microorganisms, BCCM) purchase bacterial wilt opportunistic pathogen type strain
Ralstonia solanacearum LMG 2299T, two plants of bacterium belong to different biochemistry types in the kind of Ralstonia solanacearum, after
Person is isolated from tomato (Lycopersicon esculentum) bacterial wilt diseased plant, while by both as target to bacterial strain B268
Antagonistic activity verified (Fig. 3).
(3) promoting growth of plants activity analysis.Research shows that plant in soil can be difficult to absorb not by phosphate solubilizing bacteria
Dissolubility phosphorus is converted into the absorbable titanium pigment for utilizing, meanwhile, some microorganisms can secrete the iron chelating of small molecule ---
Thermophilic iron element (Siderophore), by helping plant that this restricted battalion is enriched with from soil ferric specific binding
Foster element, these physiological properties are conducive to improving the nutrient environment of plant root, so as to promote plant growth indirectly.Additionally,
According to document report, many biocontrol microorganisms can synthesize the growth hormone of plant endo --- heteroauxin (indole-3-
Acetic acid,
IAA), the invasion and attack of pathogen are resisted by direct coordinate plant growth.Based on above- mentioned information, respectively to bacterial strain
The Decomposing phosphate activity of B268, heteroauxin are generated and thermophilic iron element is generated and detected (Fig. 4-6).
(4) the tolerance analysis of salt.In view of bacterial strain B268 is isolated from the soil of the root after Seawater immersion, metallic element analysis result
Show, the sodium magnesium ion concentration in soil sample is obviously higher than normal soil.Meanwhile, the bacterial strain nearest with B268 affiliations
Bacillus siamensis PD-A10TSalt marsh crab is isolated from, the NaCl concentration of 14% (w/v) is resistant to.Based on above-mentioned
Background, to the salt tolerance of bacterial strain B268 analysis verification (Fig. 7) has been carried out.
(5) the Forming ability analysis of biofilm.Biofilm (biofilm) is a kind of special life that bacterium adapts to environment
Long status, i.e., in the face of environment-stress when a kind of self regulating and control mode, it is close with the multifrequency nature such as the colonizing, contaminate of bacterium, virulence
Cut is closed, and colonization ability is to determine that can biocontrol bacterial strain play for a long time in woodland to prevent and treat effectiveness, and then determines whether it has
There is one of important indicator of actual application value, for these reasons, the biofilm Forming ability of bacterial strain B268 is carried out
Investigate (Fig. 8).
(6) the preventing and treating activity checking of horse-tail bacterial wilt.Potted plant horse-tail seedling with hot-house culture as host,
Ralstonia solanacearum YQ are cause of disease, and the biological and ecological methods to prevent plant disease, pests, and erosion activity of bacterial strain B268 is compared and verified (Fig. 9).
In sum, the bacterial strain Bacillus siamensis B268 that the present invention is provided enrich Biocontrol Bacillus
Species and its apply category, the bacterial strain shows strong energy for growth and stable activity in different culture media, have into
The value of one step research and development.
The present invention provides one plant of salt tolerant bacillus B268, the strain isolation from the horse-tail root soil of Seawater immersion,
Antagonistic activity is respectively provided with to the pathogenic strain of horse-tail and bacterial wilt of tomato, while there is phosphorus decomposing, synthesizing thermophilic iron element and plant
The promoting growth of plants such as growth hormone activity.Additionally, the bacterial strain is wide to the tolerance range of sea salt, can be formed under multiple salinity stable
Biomembrane, growth and colonization ability it is strong.In view of above-mentioned biological and ecological methods to prevent plant disease, pests, and erosion activity, the bacterial strain B268 that the present invention is provided can be in preparation horse-tail
Application in the biological control of bacterial wilt, based on antagonistic activities and strong biofilm formation energy of the bacterial strain B268 to pathogen
Power, can develop as disease control microbial inoculum or be directly produced as biological pesticide, based on phosphorus decomposing, secrete the activity of thermophilic iron element and IAA,
Bacterial strain B268 can be developed as bacterial manure.
Description of the drawings
The cellular morphology of Fig. 1 bacterial strain Bacillus siamensis B268.
The Neighbor-Joining systematic growths of Fig. 2 bacterial strains Bacillus siamensis B268 and its nearly edge bacterial strain
Tree.
Antagonistic activities of Fig. 3 bacterial strain Bacillus siamensis B268 to Ralstonia solanacearum, wherein A：Eutrophy people
Work sea water medium+pathogenic strains Ralstonia solanacearum YQ+B268 (screening active ingredients), B1：Eutrophy is manually extra large
Water culture medium+pathogenic strains Ralstonia solanacearum YQ+B268 (activity checking), B2：Eutrophy artificial seawater is trained
Foster base+pathogenic strains Ralstonia solanacearum LMG 2299T+ B268, B3：Modified YPG the free mediums+
Pathogenic strains Ralstonia solanacearum YQ+B268, B4：Modified YPG the free mediums+pathogenic strains
Ralstonia solanacearum LMG 2299T+B268。
The Decomposing phosphate activity of Fig. 4 bacterial strain Bacillus siamensis B268, wherein 4-1：Pikovskaya Agar flat boards,
4-2：NBRI-BPB flat boards.
The thermophilic iron element of Fig. 5 bacterial strain Bacillus siamensis B268 is generated.
The IAA of Fig. 6 bacterial strain Bacillus siamensis B268 is generated, wherein A：Modified YPG the free mediums
Testing result, B：2% sea salt+Modified YPG the free medium testing results；Control 1：Add tryptophan but be not inoculated with training
Support pipe, parallel 1-3：Add tryptophan and inoculated and cultured pipe, control 2：Tryptophan but inoculated and cultured pipe are not added.
The salt tolerance of Fig. 7 bacterial strain Bacillus siamensis B268.
The crystal violet wash-out that biofilms of Fig. 8 A bacterial strain Bacillus siamensis B268 under different salinity is formed
The color contrast of liquid.
The crystal violet wash-out that biofilms of Fig. 8 B bacterial strain Bacillus siamensis B268 under different salinity is formed
The OD of liquid570Testing result.
Prevention effects of Fig. 9 bacterial strain Bacillus siamensis B268 to horse-tail bacterial wilt, wherein 9-1：Different bacterium
The root outward appearance of the horse-tail seedling after liquid irrigating root process, 9-2：The basal part of stem of the horse-tail seedling after different bacterium solution root irrigations is crosscutting
Face；A in figure：Aseptic culture medium+pathogenic strains Ralstonia solanacearum YQ nutrient solutions process；B：Bacillus
Subtilis NH2-51-2 nutrient solutions+pathogenic strains Ralstonia solanacearum YQ nutrient solutions process；C：
Bacillus siamensis B268 nutrient solutions+pathogenic strains Ralstonia solanacearum YQ nutrient solutions process.
Specific embodiment
In conjunction with the accompanying drawings and embodiments the present invention is further illustrated.
Embodiment 1：The separation of bacterial strain Bacillus siamensis B268 and screening active ingredients
From Yueqing City of Zhejiang Province strand Casuarina equisetifolia Plantation, the forest zone once carried out seawater and poured pouring to horse-tail sample collection
The replication experiment of bacterial wilt prevention effect, in the forest zone that Seawater immersion is crossed, onset state visually chooses respectively nothing
Illness and there is an illness plant, gather rhizosphere 10cm soil below.
Before strain isolation, sample salinity is verified, i.e., using inductively coupled plasma emission spectrography
The gold of (Inductively Coupled Plasma Optical Emission Spectrometry, ICP-OES) to soil sample
Category element composition and content are analyzed, and as a result show, sodium magnesium ion concentration therein is respectively 0.5-0.7% and 0.3-
0.5%, hence it is evident that higher than the common soil sample as control.
10g soil samples are taken with the suspension of 20ml SPSSs, is extracted 2 days under the conditions of 28 DEG C, 140rpm.Suspension is with nothing
Oligotrophic artificial seawater flat board is coated with after bacterium physiological saline gradient dilution, is cultivated in 28 DEG C to forming obvious bacterium colony.According to form
Difference distinguishes picking single bacterium colony, with the purifying that same plane carries out bacterial strain, and with 20% (v/v) glycerine form of tubes preservation.
With the activation of eutrophy artificial seawater plate streaking, 28 DEG C are cultivated 24 hours isolated strains, pathogenic strains Ralstonia
Solanacearum YQ with eutrophy artificial seawater liquid shake pipe activation, 28 DEG C, 140rpm cultivate 48 hours.Take 50 μ l bacterial strains
Nutrient solution (the OD of YQ600=1.0) rule eutrophy artificial seawater flat board, then the single bacterium colony dibbling of 3 isolated strains of picking this put down
Plate, made by stand facing each other plate in 28 DEG C cultivate 3 days, day by day observe inhibition zone formational situation.The active ingredients result of bacterial strain B268
See Fig. 3 A.
Artificial seawater：NaCl 4.86g,MgCl2·6H2O 3.15g,MgSO4·7H2O 1.66g,CaCl2 0.45g,KCl
0.138g,NaHCO340mg, ironic citrate 2.5mg, SrCl2·6H2O 14.25mg,KBr 20mg,H3BO3 5.5mg,
Na2SiO3·9H2O 2.32mg,NaF 0.6mg,NH4NO3 0.6mg,Na2HPO42mg, deionized water 1L.
Oligotrophic artificial seawater culture medium：Yeast Extract(BD 212750)0.5g,Trypticase Peptone
(BD 211921) 0.5g, artificial seawater 1L, pH 7.0.
Eutrophy artificial seawater culture medium：Yeast Extract(BD 212750)1g,Trypticase Peptone(BD
211921) 5g, artificial seawater 1L, pH 7.0.
At present, bacterial strain B268 is in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation (address：
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is
Bacillus siamensis, preserving number is CGMCC No.13225, and preservation date is on November 1st, 2016.
Embodiment 2：The phylogenetic identification of bacterial strain Bacillus siamensis B268
The flat board single bacterium colony of picking bacterial strain B268, eutrophy artificial seawater culture medium of transferring, 28 DEG C, 140rpm cultures it is 24 little
When after collect cell, using bacterial genomes Rapid extraction kit (east victory biological) extracting genomic templates while microscopy.
Using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-
ACGGTTACCTTGTTACGACTT-3') 16S rRNA genes, the product Jing AxyPrep PCR cleaning agents boxes of acquisition are expanded
(Axygen) it is connected (TaKaRa) with pMD 19-T Vector after purification, and converts Trans1-T1 competent cells
(TransGen Biotech), picking positive recombinant is delivered sequencing company and is sequenced.
Bacterial strain B268 can be observed the formation (Fig. 1) of gemma into shaft-like under microscope.
16S rRNA full length gene 1513nt (referring to sequence table) of bacterial strain B268, sequence has been forwarded to GenBank data
Storehouse, sequence accession number is KY129717.Using EzBioCloud (http://www.ezbiocloud.net/) comparison instrument enters
Row similarity analysis two-by-two between sequence, as a result show, B268 and Bacillus siamensis KCTC 13613TSequence
Similitude highest, is 99.93%.Multiple Sequence Alignment and Phylogenetic Relationships analysis are carried out using Mega 6.0, is as a result shown, bacterium
Strain B268 on Neighbor-Joining phylogenetic trees with the standard species B.subtilis of bacillus, and classics
Bacterial wilt biocontrol bacterium B.amyloliquefaciens, B.methylotrophicus, at potential biocontrol microorganisms B.siamensis
In same evolutionary branching, and with Bacillus siamensis KCTC 13613TBetween evolutionary distance most short (Fig. 2).
The above analysis result, Bacillus siamensis kinds bacterial strain B268 being divided in bacillus.
Bacterial strain Bacillus siamensis B268 are preserved in the common micro- life of China Committee for Culture Collection of Microorganisms
Thing center, preserving number is CGMCC No.13225.
Embodiment 3：The antagonistic activity checking of bacterial strain Bacillus siamensis B268
Prepare face-off plate using eutrophy artificial seawater culture medium and Modified YPG the free mediums respectively, respectively with
This laboratory is isolated from the Ralstonia solanacearum YQ of horse-tail diseased plant and purchase is isolated from tomato from BCCM
The Ralstonia solanacearum LMG 2299 of diseased plantTFor the checking that target carries out antagonistic activity.Bacterial strain B268 is with flat board
Line activation, 28 DEG C are cultivated 24 hours, and two pathogen strain bacterial strains shake pipe activation with liquid, using eutrophy artificial seawater culture medium
When, cultivate 48 hours in 28 DEG C, 140rpm, during using Modified YPG the free mediums, in 28 DEG C, 140rpm cultures it is 24 little
When.The preparation method of face-off plate observes the formational situation of inhibition zone with embodiment 1 after cultivating 3 days in 28 DEG C.
As shown in Figure 3 B, pathogenic strains YQ and LMG 2299TGrow more preferably on the free medium, and bacterial strain B268 is two
Equal well-grown on culture medium is planted, and two plants are infected with the pathogen of different hosts and show obvious antagonistic activity.
Modified YPG the free mediums：Beef extract powder 3g, peptone 5g, glucose 10g, yeast extract 0.5g,
Distilled water 1L, pH 7.0；
Embodiment 4：The phosphorus decomposing test of bacterial strain Bacillus siamensis B268
Dibbling Pikovskaya after bacterial strain B268 line eutrophy artificial seawater flat board activation, 28 DEG C of cultures 24 hours
, to identify its Decomposing phosphate activity, 28 DEG C are cultivated to there is obvious phosphorus decomposing circle for Agar flat boards and NBRI-BPB flat boards.As a result such as Fig. 4-1
And shown in 4-2, transparent circle occur and occur yellow circle on NBRI-BPB flat boards on Pikovskaya Agar flat boards, show bacterial strain
B268 has Decomposing phosphate activity.
Pikovskaya Agar culture mediums：Yeast Extract (BD 212750) 0.5g, glucose 10g, Ca3(PO4)2
5g, (NH4)2SO40.5g, K2SO40.2g, MgSO4·7H2O 0.1g, MnSO42mg, FeSO4·7H2O 2mg, agar powder
15g, distilled water 1L, pH 7.0；
NBRI-BPB culture mediums：Glucose 10g, Ca3(PO4)25g, MgCl2·6H2O 5g, MgSO4·7H2O 0.25g,
KCl 0.2g, (NH4)2SO40.1g, bromophenol blue 25mg, agar powder 15g, distilled water 1L, pH 7.0；
Embodiment 5：The thermophilic iron element synthetic test of bacterial strain Bacillus siamensis B268
Dibbling LBA flat boards are identifying after bacterial strain B268 line eutrophy artificial seawater flat board activation, 28 DEG C of cultures 24 hours
Its thermophilic iron element synthesizing activity, 28 DEG C are cultivated to there is significant reaction circle.As a result as shown in figure 5, occurring yellow transparent circle on flat board,
Show that bacterial strain B268 can synthesize and secrete thermophilic iron element.
LBA culture mediums：60.5mg chrome azurol Ss are dissolved in 50ml deionized waters, with the FeCl that 10mM HCl prepare 1mM3·
6H2O solution, takes ferrous solution 10ml and adds mixing in the chrome azurol S aqueous solution, forms CAS solution；72.9mg CTAB are dissolved in
In 40ml deionized waters, above-mentioned CAS solution is slowly added to while stirring, sterilize after mixing standby；
Tryptone (BD 211705) 10g, Yeast Extract (BD 212750) 5g, NaCl 10g, PIPES
30.2g, deionized water 900mL is standby to sterilize after 50%NaOH solution tune pH 6.8, plus agar powder 15g；
Above two matrix mixing after fall LBA flat boards processed；
Embodiment 6：The IAA synthetic tests of bacterial strain Bacillus siamensis B268
, with the activation of Modified YPG the free mediums, supplement is final concentration of after switching same medium for bacterial strain B268
The tryptophan of 40mg/l, 28 DEG C, 140rpm cultures, to connect bacterium but not add tryptophan and add tryptophan but do not connect the flat of bacterium
Row culture tube is control.The Modified YPG the free mediums of 2% sea salt (Sigma S9883) are added in switching simultaneously, remaining
Operation is identical.
7 days nutrient solution 1ml are taken, after mixing with equal-volume detection reagent, room temperature avoid light place 24 hours, period observation color
Change.As a result as shown in Figure 6A, the culture tube of all inoculations, regardless of whether adding tryptophan, adds and show after detection reagent redness,
Show that bacterial strain B268 can synthesize IAA.And the result of B is close with A in Fig. 6, show that IAA of the salt for bacterial strain B268 synthesizes without bright
Development rings.
Detection reagent：12g FeCl3·6H2O, 7.9M sulfuric acid 1L；
Embodiment 7：The Salt Tolerance Analysis of bacterial strain Bacillus siamensis B268
Add 0-12% sea salt (Sigma S9883) on the basis of Modified YPG the free mediums, with salt manufacturing
Gradient media, difference inoculating strain B268 and pathogenic strains YQ and LMG 2299T, 28 DEG C, 140rpm cultivate 24 hours after
Detection OD600Value.As a result as shown in fig. 7, bacterial strain B268 is wider to the tolerance range of sea salt, when cultivating 24 hours, 1-7%'s
Equal well-grown (OD under sea salt concentration600>1), and during the Extending culture time, the equal well-grown under the sea salt concentration of 0-12%
(OD600>1).It is contrary, pathogenic strains YQ and LMG 2299TEqual not salt tolerants, hence it is evident that like salt-free or low-salt environment (<1%), when
Sea salt concentration can not grow when being more than 3%.And according to document report, the salt tolerance range of horse-tail is 0.3-1.5%, with disease
Opportunistic pathogen is suitable.Can consider based on above-mentioned data：The salt tolerance of one, bacterial strain B268 is separated to be had from the soil of Seawater immersion
Close；Two, wide sea salt tolerance range causes bacterial strain B268 to be easy to the horse-tail in the different salt content of inland, strand even beach etc.
Survive in growth belt soil；Three, the difference of salt tolerance may cooperate with the common antagonism pathogen of saline environment predictive of bacterial strain B268,
And this to be probably seawater pour that pouring method suppresses one of mechanism of horse-tail bacterial wilt.
Embodiment 8：The biofilm formation test of bacterial strain Bacillus siamensis B268
Add the sea salt (Sigma S9883) of variable concentrations, system on the basis of Modified YPG the free mediums
Standby salt gradient culture medium, 96 well culture plates are separately added into the dosage in 195 μ l/ holes by above-mentioned sea salt gradient media, each ladder
6 parallel holes of degree.Pathogenic strains YQ, LMG 2299 are prepared with Modified YPG the free mediumsTAnd the kind of bacterial strain B268
Sub- liquid, adjusts OD600After=0.3, it is inoculated with by the dosage in 5 μ l/ holes, while to add the hole of 5 μ l aseptic culture mediums right as feminine gender
According to.As shown in table 1,28 DEG C of quiescent cultures carry out biomembranous detection to the concrete layout of culture plate after 20 hours, concrete grammar is such as
Under：
The mycoderm that bacterium solution and liquid surface in every hole are formed carefully is siphoned away with micropipettor, is softly washed with sterilized water
Wash every hole twice；Blot and add 1% (w/v) crystal violet aqueous solution by the dosage in 300 μ l/ holes after residual liquid, stand under room temperature
Dyeing 30 minutes；Dye liquor is carefully siphoned away, orifice plate is softly rinsed to without obvious loose colour with sterilized water in suitable container；Inhale
33% (v/v) acetic acid solution is added by the dosage in 300 μ l/ holes after dry residual liquid, is stood under room temperature, its objective is that wash-out is given birth to
OD is detected after the crystal violet that thing film is fixed, fully wash-out570Value.
Table 1
As shown in Fig. 8 A and Fig. 8 B, bacterial strain B268 can form stable biomembrane under the sea salt concentration of 0-5%, especially with
The sea salt concentration of 1-3% for it is most suitable (wash-out crystal violet color is most deep or OD570Value highest), this trend and its salt tolerance base
This is consistent, and illustrating the sea salt of debita spissitudo can promote colonizing for bacterial strain B268.And pathogenic strains YQ are only in 0-1% sea salt concentration
It is lower to form faint biomembrane, LMG 2299TOnly biomembrane is formed under 0.5% sea salt concentration, shown in identical nutrition bar
Under part, bacterial strain B268 has more growth vigor, if in the field environment, means preferentially occupying for ecological niche, so as to reach
Suppress the purpose of pathogen propagation.
Embodiment 9：Controlling experiments of the bacterial strain Bacillus siamensis B268 to horse-tail bacterial wilt
Bacterial strain B268, Bacillus subtilis NH2-51-2 and pathogenic strains YQ be inoculated with respectively Modified YPG without
Salt culture medium, 28 DEG C, 140rpm cultivate 24 hours.Bacterium solution adjusts OD600After=1, respectively by NH2-51-2 and YQ, B268 and YQ, nothing
Bacterium culture medium mixes with YQ equal-volumes.Wherein, bacterial strain NH2-51-2 is isolated from South Sea surface seawater, and face-off plate the selection result shows
It has faint antagonistic activity to pathogenic strains YQ, in this experiment as positive contrast's bacterial strain.Add the YQ of aseptic culture medium
Bacterium solution is used as negative control.Above-mentioned 3 kinds of mixed bacteria liquids are inoculated with by potted plant horse-tail seedling, greenhouse training with 5ml/ strains with root-pouring method respectively
Root is taken after supporting 1 month infect situation to stem foot Partial Characterization.
As shown in figure 9, under identical incubation time, be only inoculated with the plant of pathogenic strains YQ, root table in black (Fig. 9-
1A), stem foot cross section is withered and vascular tissue has obvious putrefactive phenomenon (Fig. 9-2A), occurs in that the symptom that bacterial wilt infects；
The plant of inoculating strain B268 and YQ mixed liquor, fulvescent of the root table in health, and glossy (Fig. 9-1C), stem foot cross section is new
, there is not the symptom that bacterial wilt infects in fresh moistening (Fig. 9-2C)；And the plant of inoculating strain NH2-51-2 and YQ mixed liquor, root table
In crineous (Fig. 9-1B), stem foot cross section shows slightly withered, and vascular tissue has slightly rotten vestige (Fig. 9-2B).The above results table
Bright, bacterial strain NH2-51-2 antagonistic activities are weaker, can delay but can not suppress the generation of disease, and the presence of bacterial strain B268 can be effective
Suppress intrusion of the pathogen to horse-tail root, the effect with disease control.
<110>Zhejiang A ＆ F University
<120>The horse-tail bacterial wilt biocontrol bacteria B268 of one plant of salt tolerant and its application
<160> 3
<210> 1
<211> 1513
<212> DNA
<213> Bacillus siamensis
<400> 1
1 AGAGTTTGAT CCTGGCTCAG GACGAACGCT GGCGGCGTGC CTAATACATG CAAGTCGAGC
61 GGACAGATGG GAGCTTGCTC CCTGATGTTA GCGGCGGACG GGTGAGTAAC ACGTGGGTAA
121 CCTGCCTGTA AGACTGGGAT AACTCCGGGA AACCGGGGCT AATACCGGAT GGTTGTTTGA
181 ACCGCATGGT TCAGACATAA AAGGTGGCTT CGGCTACCAC TTACAGATGG ACCCGCGGCG
241 CATTAGCTAG TTGGTGAGGT AACGGCTCAC CAAGGCGACG ATGCGTAGCC GACCTGAGAG
301 GGTGATCGGC CACACTGGGA CTGAGACACG GCCCAGACTC CTACGGGAGG CAGCAGTAGG
361 GAATCTTCCG CAATGGACGA AAGTCTGACG GAGCAACGCC GCGTGAGTGA TGAAGGTTTT
421 CGGATCGTAA AGCTCTGTTG TTAGGGAAGA ACAAGTGCCG TTCAAATAGG GCGGCACCTT
481 GACGGTACCT AACCAGAAAG CCACGGCTAA CTACGTGCCA GCAGCCGCGG TAATACGTAG
541 GTGGCAAGCG TTGTCCGGAA TTATTGGGCG TAAAGGGCTC GCAGGCGGTT TCTTAAGTCT
601 GATGTGAAAG CCCCCGGCTC AACCGGGGAG GGTCATTGGA AACTGGGGAA CTTGAGTGCA
661 GAAGAGGAGA GTGGAATTCC ACGTGTAGCG GTGAAATGCG TAGAGATGTG GAGGAACACC
721 AGTGGCGAAG GCGACTCTCT GGTCTGTAAC TGACGCTGAG GAGCGAAAGC GTGGGGAGCG
781 AACAGGATTA GATACCCTGG TAGTCCACGC CGTAAACGAT GAGTGCTAAG TGTTAGGGGG
841 TTTCCGCCCC TTAGTGCTGC AGCTAACGCA TTAAGCACTC CGCCTGGGGA GTACGGTCGC
901 AAGACTGAAA CTCAAAGGAA TTGACGGGGG CCCGCACAAG CGGTGGAGCA TGTGGTTTAA
961 TTCGAAGCAA CGCGAAGAAC CTTACCAGGT CTTGACATCC TCTGACAATC CTAGAGATAG
1021 GACGTCCCCT TCGGGGGCAG AGTGACAGGT GGTGCATGGT TGTCGTCAGC TCGTGTCGTG
1081 AGATGTTGGG TTAAGTCCCG CAACGAGCGC AACCCTTGAT CTTAGTTGCC AGCATTCAGT
1141 TGGGCACTCT AAGGTGACTG CCGGTGACAA ACCGGAGGAA GGTGGGGATG ACGTCAAATC
1201 ATCATGCCCC TTATGACCTG GGCTACACAC GTGCTACAAT GGACAGAACA AAGGGCAGCG
1261 AAACCGCGAG GTTAAGCCAA TCCCACAAAT CTGTTCTCAG TTCGGATCGC AGTCTGCAAC
1321 TCGACTGCGT GAAGCTGGAA TCGCTAGTAA TCGCGGATCA GCATGCCGCG GTGAATACGT
1381 TCCCGGGCCT TGTACACACC GCCCGTCACA CCACGAGAGT TTGTAACACC CGAAGTCGGT
1441 GAGGTAACCT TTATGGAGCC AGCCGCCGAA GGTGGGACAG ATGATTGGGG TGAAGTCGTA
1501 ACAAGGTAGC CGT
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<223>16S rRNA upstream region of gene primers
<400> 2
AGAGTTTGAT CCTGGCTCAG
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<223>16S rRNA downstream of gene primers
<400> 3
ACGGTTACCT TGTTACGACT T
Claims (3)
1. the horse-tail bacterial wilt biocontrol bacteria B268 of one plant of salt tolerant, it is characterised in that the bacterial strain B268 is by Chinese micro- life
The common micro-organisms center preservation of thing culture presevation administration committee, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica, postcode 100101, Classification And Nomenclature is Bacillus siamensis, and preserving number is CGMCC
No.13225, preservation date is on November 1st, 2016.
2. lifes of the horse-tail bacterial wilt biocontrol bacteria B268 of one plant of salt tolerant according to claim 1 in horse-tail bacterial wilt
Application in thing preventing and treating, it is characterised in that the application in the biological control microbial inoculum or agricultural chemicals for preparing horse-tail bacterial wilt.
3. lifes of the horse-tail bacterial wilt biocontrol bacteria B268 of one plant of salt tolerant according to claim 1 in horse-tail bacterial wilt
Application in thing preventing and treating, it is characterised in that answering in the microbial-bacterial fertilizer for promoting Casuarina equisetifolia Growth and improving its disease resistance is prepared
With.
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