CN110387337A - Bei Laisi bacillus 118 and its bacteria agent and application - Google Patents

Bei Laisi bacillus 118 and its bacteria agent and application Download PDF

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CN110387337A
CN110387337A CN201910391310.3A CN201910391310A CN110387337A CN 110387337 A CN110387337 A CN 110387337A CN 201910391310 A CN201910391310 A CN 201910391310A CN 110387337 A CN110387337 A CN 110387337A
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bei laisi
laisi bacillus
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蔡燕飞
王玉琪
章家恩
刘晓玉
周晗
裴秦汉
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South China Agricultural University
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Abstract

The invention discloses a kind of Bei Laisi bacillus (Bacillus velezensis) 118 and its applications, the bacterial strain deposit number is GDMCC No.60625, is preserved in Guangdong Province's Culture Collection (GDMCC) on 04 04th, 2019；The bacterial strain prevention and control bacterial wilt of tomato and banana blight promote plant strain growth, high yield biomembrane and effective the advantages that colonizing rhizosphere；The bacterial strain shares 5 extracellular function delta antigens, including δM,δW,δX,δVAnd ylaC.Wherein δXGene has the function of that biomembrane gauffer and petal form, improve biomembrane yield, quickening wriggling and travelling speed and improve rhizosphere colonization rate；YlaC gene has the function of biomembrane form；Bacteria agent is prepared using the bacterial strain, can not only prevent and treat soil-borne disease but also plant growth can be promoted, is had a extensive future；Belong to agricultural biological technical field.

Description

Bei Laisi bacillus 118 and its bacteria agent and application
Technical field
It is a kind of microorganism and its application, and in particular to a kind of the present invention relates to plant soil-borne diseases field of biological control Bei Laisi bacillus 118 (Bacillus velezensis) and application.
Background technique
Soil-borne disease of crop be in agricultural production restriction one of yield and the main problem of quality, as wilt disease, bacterial wilt, Epidemic disease, root rot and banded sclerotial blight etc. generally occur in South China, gently the then underproduction, heavy then crop is caused to have no harvest.Banana blight It is the fungoid soil-borne disease as caused by Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense) Evil, bacterial wilt of tomato is the bacillary soil-borne disease as caused by Ralstonia solanacearum (Ralstonia solanacearum).Make Object soil-borne disease is mainly prevented and treated by improving the modes such as cultivation step, physical disinfection, chemical pesticide, but chemical pesticide is a large amount of It uses, not only causes crops pesticide residue, also cause environmental pollution, destroy ecological environment.Microbial pesticide has safe nothing It is harmful, double to subtract synergy, be not likely to produce the advantages that drug resistance, it is important in the Sino-Japan aobvious benefit of national green agricultural.For this purpose, excavating new and effective Biocontrol microorganism resource and its high efficiency gene resource, biological control and promotion plant strain growth to soil-borne disease of crop all have weight The meaning wanted.
Bei Laisi bacillus (Bacillus velezensis) is novel plant disease-proof growth-promoting microorganism, in order to Bacillus amyloliquefaciens DSM9 of industrial fermentation etc. is distinguished, and Fan is equal to the 2017 formal newnames that enable and is known as Bei Laisi gemma Bacillus (Bacillus velezensis).Bei Laisi bacillus has 5 extracellular function (extracytoplasmic Functional sigma factor, ECF) sigma factor (δM、δX、δW、δVAnd ylaC), extracellular function (ECF) sigma factor is response Various stressful environmentals transcription regulaton factor (Luo et al., 2010；Onozawa et al., 2015), there is regulating cell The function of correlation function gene expression, to the various existence energy such as improve bacterial strain competition in the environment, antagonism, move and colonize Power.For this purpose, probe into ECF sigma factor formed in bacterial biof iotalm, travelling and wriggle, in terms of regulating and controlling effect, to mentioning The field of high biocontrol agent, which is colonized, has important practical significance with Biocontrol Potential.
By moving about and wriggling, generates biomembrane and adhere to plant root and colonize, be biocontrol agent field efficacy and fertilizer The premise played is imitated, in order to probe into effect of the sigma factor in rhizosphere colonization, using long-armed homologous polymerase chain reaction (long Flanking homology polymerase chain reaction, LFH-PCR) technology constructs 5 sigma factors respectively (δM、δX、δW、δV, ylaC) gene-deleted strain.Compare the biomembrane form and yield, travelling between wild strain and mutant strain With wriggle, the adherency differences such as root system, screen corresponding high efficiency gene, it is expected to solve current biocontrol agent field efficacy unstability Technical bottleneck.
Summary of the invention
The first object of the present invention is that the novel diseases prevention that has both of one kind is provided according to the above problem in the prior art to be promoted Raw, high yield biomembrane and efficient root colonization function Bei Laisi bacillus 118 (Bacillus velezensis).
Second object of the present invention is to provide bacteria agent prepared by above-mentioned Bei Laisi bacillus 118.
Third object of the present invention is to be to provide the application of Bei Laisi bacillus 118 and its bacteria agent.
For this purpose, the first purpose of this invention is achieved through the following technical solutions:
A kind of Bei Laisi bacillus 118 (Bacillus velezensis), deposit number GDMCC No.60625, in It is preserved in Guangdong Province's Culture Collection (GDMCC), preservation address: Xianlie Middle Road, Guangzhou City on 04 04th, 2019 No. 100 5 building, the buildings of compound the 59th, Guangdong Microbes Inst.
118 bacterium is to isolate and purify gained in Guangzhou Fanyu banana planting Tanaka, belongs to gram-positive bacteria, there is gemma, bar Shape, it is aerobic, there is motility；It is round for white that the bacterium colony formed afterwards for 24 hours is cultivated on LB solid medium, surface wettability.By Physiology and biochemistry and Molecular Identification are Bei Laisi bacillus 118 (Bacillus velezensis), the 16S rRNA gene of bacterial strain Sequencing results are as shown in SEQ ID NO:1.
It is measured using plate spot lawn method (spot-on-lawn assay), the bacterial strain is to Pseudomonas solanacearum and reaping hook Bacterium all has significant antagonistic effect.Using potted plant efficiency test, which has banana blight and bacterial wilt of tomato Significant control efficiency and the effect for promoting banana plant growth.Method is formed using the thin skin of LBGM Liquid static culture (pellicle formation assay), bacterial strain 118 generate the thin skin (pellicle) of yellow gauffer, thin skin yield 17.5 (mg/well).Using the biofilm formation method (biofilm formation assay) of LBGM plate culture, in fermentation liquid table Face, bacterial strain 118 formed yellow, gauffer, concentric circles, three layers of petal biomembrane of different shapes.Using arabidopsis root colonization Test, confirmation bacterial strain 118 adhere in arabidopsis root system and form biomembrane and successfully colonize.Thus illustrate that 118 bacterium have to pass through The advantages of biomembrane adheres to root system and efficiently colonizes.
Preferential:
Extracellular function delta antigen regulating cell related gene expression in response to the external environment factor improves bacterium in the environment Survival ability.To verify the high efficiency regulatory gene that 118 bacterium efficiently colonize, using long flanking homologous PCR (long flanking Homology PCR, LFH-PCR) method, construct the extracellular function factor δ of bacterial strain 118M、δX、δW、δVWith ylaC gene delection Strain further carries out thin skin using LBGM culture medium and forms method experiment and biofilm formation experiment, more each gene-deleted strain Biomembrane and thin skin phenotype, the results showed that, under LBGM Liquid static culture, δXControlling gene has gauffer shape in regulation thin skin At function, to 118 thin skin of bacterial strain formed contribution rate be about 50%.Under LBGM solid plate culture, δXControlling gene tool There is the function that gauffer and petal are formed in regulation biomembrane.
The travelling of bacterial strain and wriggling, using the LBGM plate of 0.3% and 0.7% agar, by culture to OD600~0.4 bacterium 3 μ L of liquid is added to plate middle, is placed in 37 DEG C of cultures, respectively in the diameter of 3h observation swimming, in the diameter that 4h observation is wriggled. It takes pictures and records diameter.Experiment is in triplicate.The result shows that δXGene-deleted strain/wild strain 118 swim in 3h diameter difference For 59/49mm.In 4h, wriggling diameter is respectively 22/14mm；Show that δ X gene has the function of promoting bacterial strain swimming and wriggle.
Using SYTO13 green fluorescence nucleic acid dye method and laser confocal scanning microscope method, have detected 118 and its Arabidopsis root colonizes situation.Experimental procedure is as follows, and the 5 days Arabidopsis thaliana Seedlings that will germinate are placed in 96 hole microplates, and adds Enter 300 μ L LBGM culture mediums, then takes 118 bacterial strains, δX3 μ L (OD of gene-deleted strain and 3610 fermentation liquid of positive control strain600~ 0.4) it is added in every hole, at 23 DEG C, 100rpm shaken cultivation.Respectively in 0h, 12h, for 24 hours, 48h when sample, washed with PBS buffer solution Root is washed, and after dyeing 2 minutes, is washed with PBS buffer solution, In with SYTO13 solution (diluting 1500 times with PBS phosphate buffer) Leica SP5 CONFOCAL SCANNING LASER MICROSCOPE (CSLM) 488nm excitation laser and 509nm transmitting are imaged.The result shows that δXGene-deleted strain only forms small part membrane structure on arabidopsis root system surface, shows δXGene, which has, promotes biofilm formation Gene expression and reach and efficiently colonize the effect of root system.
Using Illumina high-flux sequence and CLC gene-splicing method, the whole genome sequence of bacterial strain 118 is obtained, is adopted Gene order is compared with NCBI, obtains the controlling gene δ of bacterial strain 118XSequence, as shown in SEQ ID NO.2.The sequence and mould The similarity of formula Strains B. subtilis 3610 is 87%.
It is preferred:
Using long flanking homologous PCR (long flanking homology PCR, LFH-PCR) method, bacterial strain is constructed 118 extracellular function factor δM、δX、δW、δVWith ylaC gene-deleted strain, thin skin is further carried out using LBGM culture medium and is formed Experiment and biofilm formation experiment, under LBGM Liquid static culture, the biofilm phenotype of ylaC gene-deleted strain and 118 bacterial strains are wild Strain is not different, but the specific life for having bacterium dizzy around the display of LBGM solid plate culture 1d, ylaC controlling gene gene-deleted strain Object film form, discovery ylaC controlling gene participate in petal in biomembrane and are formed.
Using Illumina high-flux sequence and CLC gene-splicing method, the whole genome sequence of bacterial strain 118 is obtained, is adopted Gene order is compared with NCBI, the controlling gene ylaC sequence of bacterial strain 118 is obtained, as shown in SEQ ID NO.3.The sequence and 3610 difference of type strain bacillus subtilis is very big, it is shown that unique.
Second object of the present invention is achieved through the following technical solutions:
A kind of bacteria agent containing above-mentioned 118 fermentation liquid of Bei Laisi bacillus, the Bei Laisi bacillus 118 Quantity is 1.0 × 108~9.0 × 109cfu/ml。
The bacterial preparation process are as follows: -80 DEG C of glycerol freeze-drying 118 bacterial strain of pipe scribing line of picking are on LB culture medium, and 37 DEG C overnight Culture.Picking single bacterium drops down onto LB liquid medium, and 150rpm, 37 DEG C of shaking table culture 14h, 3 obtain seed liquor；By 1% inoculum concentration, By in the seed liquor access fermentation medium of acquisition, 37 DEG C of shaking table culture 10-14h, revolving speed 150r/min obtain bacterial strain 118 Fermentation liquid.
Third object of the present invention is achieved through the following technical solutions
Application of 118 microbial inoculums in prevention and treatment banana blight, sharp born of the same parents sickle-like bacteria (F.oxysporum, Foc) sensitive varieties Brazil Any of several broadleaf plants 4-5 piece leaf seedling, is arranged four processing, and as follows respectively: (Foc is not added) in CK1, CK2 (Foc), Foc+118 wild strain with Foc+δXGene-deleted strain.30 banana seedlings of every processing.By CK2 (Foc), Foc+118 and Foc+ δXThe root of three processing group Banana Seedlings Foc (~10 is immersed in portion6Spores/ml 30 minutes in), two days later, Foc+118 processing group and Foc+ δXProcessing group respectively will preparation Good 50mL 118 and δXBacteria suspension be poured onto plant root (106Cfu/g soil.The banana plant of monitoring in every 3 days after transplanting Incidence, disease index record is as shown in table 5 (Ploetz et al., 1999)
118 microbial inoculums control of plant bacterial wilt application, using Pseudomonas solanacearum (Ralstonia Solanacearum, Rs) the prosperous carbuncle 5-6 piece leaf seedling replanting of susceptible kind tomato China is into potting soil, and processing is as follows: CK1 (No Rs), CK2 (Rs), Rs+118 wild strain and Rs+ δXGene-deleted strain processing.24 plants of tomato seedlings of every processing, 2 plants of every basin, each Handle 12 basins.7 days after transplanting, Rs+118 or Rs+ δXProcessing soil is inoculated with 118 or δ respectivelyXDeletion mutation strain suspension group (~ 106cfu·g-1Soil).CK1 and CK2 processing group is compared using 50mL sterile water respectively.It is inoculated with Antagonistic Fungi two days later, removes CK1 In addition, other three processing are all inoculated with Rs bacteria suspension (~107cfu·g-1Soil).It is placed in artificial climate room (PQX-450R- In 22HM), temperature is maintained at 25-32 DEG C, and relative humidity is 65% to 80%.Start to calculate morbidity after inoculating tomato bacterial wilt Rate.Disease index records (Chen et al., 2013) according to 6 grade scale of table.
Bacterial wilt of tomato and banana blight disease index and biological and ecological methods to prevent plant disease, pests, and erosion efficiency calculation are as follows:
Disease index (DI)=[∑ (plant number × number of levels of each disease grade)/(investigate total plant number × highest level Number)] × 100%
Biological and ecological methods to prevent plant disease, pests, and erosion efficiency (BE)=[(disease index-test process disease index of CK)/CK disease index] × 100%
The result shows that bacterial strain 118 is 40% to the biological and ecological methods to prevent plant disease, pests, and erosion efficiency of banana blight.Compare 118 wild strains and its δXBase Because of the difference of gene-deleted strain biological and ecological methods to prevent plant disease, pests, and erosion efficiency, show controlling gene δXIt is viscous by regulation 118 biomembrane of bacterial strain, travelling and wriggling, root system It is attached and the functions such as colonize, to promote prevention and treatment of the bacterial strain 118 to banana blight and bacterial wilt of tomato, wherein δXIn biological and ecological methods to prevent plant disease, pests, and erosion efficiency In contribution rate be 8%.
Compared with prior art, the invention has the following advantages:
The Bei Laisi bacillus 118 of offer of the invention, there is the soil-borne diseases such as bacterial wilt of tomato and banana blight Significant biocontrol effect.In addition, the present invention confirms the extracellular δ in 118XThe factor participates in regulation bacterial biof iotalm gauffer and petal Formation promotes functions, the extracellular ylaC factors such as bacterium moves about and wriggling, raising root system are adhered to and colonized to participate in biomembrane and spend Lobed is to achieve the effect that biological and ecological methods to prevent plant disease, pests, and erosion.Specific manifestation is as follows:
(1) Bei Laisi bacillus 118 provided by the invention has high-performance bio prevention and control capability to banana blight, right The biological control efficiency of banana blight is up to 40%.
(2) bacterial strain report associated with the prior art compares, and 118 abilities with high yield biomembrane form unique life Object film form: yellow, gauffer, concentric circles, three layers of petal of different shapes.
(3) 118 δ that the invention providesXFunction factor has regulation bacterial strain biomembrane yield and form (gauffer and flower Valve), travelling and wriggle, root system adheres to the function that colonizes.
(4) the ylaC function factor of the invention 118 participates in the function of regulation biomembrane form.
(5) present invention provides a kind of Bei Laisi bacillus money of new high-efficiency for the prevention and treatment of soil-borne disease of crop Tiny ecosystem Source.
Detailed description of the invention
Fig. 1 is antagonistic effect (1A plate spot lawn method, 118+Foc of 118 bacterium to pathogen；1B plate spot lawn Method, 118+Rs；1C plate face-off bacteriostatic method, 118+Foc；The microphoto of 118 bacterium antagonism Foc bacterium of 1D).
Fig. 2 is 118 bacterium systematic growth tree graphs.
Fig. 3 is 118 bacterium prevention and treatment banana blight and growth-promoting plant strain growth (3A disease index, the 3B number of blade, 3C plant height, 3D Plant weights, 3E potted plant photo).
Fig. 4 is extracellular function delta antigen δM/VInfluence (4A thin skin form with ylaC to 118 bacterium biofilm formations；4B biology Film form).
Fig. 5 is δXTo regulating and controlling effect (5A thin skin form, 5B biomembrane form, the 5C of 118 bacterium biomembrane forms and yield Thin skin yield).
Fig. 6 is δXThe effect for promoting 118 bacterium to move about and wriggle (6A travelling, 6B are wriggled, 6C wriggling photo).
Fig. 7 is δXParticipate in effect (the 7A banana blight disease index of the anti-hole banana blight of 118 bacterium and bacterial wilt of tomato Curve graph, 7B banana blight prevent and treat potting photo, 7C bacterial wilt of tomato disease index curve graph, and 7D bacterial wilt of tomato prevents and treats basin Plant photo).
Fig. 8 is the effect picture that δ X promotes 118 bacterium in arabidopsis root colonization.
Specific embodiment
With reference to embodiment, claim of the invention is described in further detail, but do not constituted pair Any restrictions of the invention, the modification of anyone limited times made within the scope of claim of the present invention, still of the invention Within claims.
It in following embodiment unless otherwise indicated, is routine experiment method and operating procedure in the art.
Screening, the purifying of 1 118 bacterial strain of embodiment
(1) primary dcreening operation
Take Pseudomonas solanacearum (Ralstonia solanacearum, Rs) CPG (0.1% peptone, 0.01% network albumen Amino acid, 0.05% glucose) culture solution (OD600~0.4) 200 μ L bacterium solutions are respectively put into aseptic flat board, Xiang Shengyou bacterium solution Plate in pour into sterilizing after be cooled to 45 DEG C or so of 20mL/CPG culture medium, set the rapid turn plate in horizontal position, make to train It supports base to be uniformly mixed with bacterium solution, after culture medium solidification, it is spare that the CPG plate containing Pseudomonas solanacearum is made in naturally dry.
It is screening soil sample with the plant rhizosphere soil such as Guangzhou banana, tomato, weighs 10g and be put into equipped with 90mL sterile water (add bead) in 250mL triangular flask, shaking table vibrate 30min, keep bacterium fully dispersed, stand 20~30s, take supernatant into Row 10 successively decreases dilution again, using 103~105Dilution pipettes dilution 0.1mL with liquid-transfering gun respectively, is coated on plate containing bacterium On, above-mentioned inoculated and cultured is based on cultivating in 30 DEG C of constant incubators, and after 48h, whether there is or not inhibition zones for observation, will there is the bacterium of antagonistic effect Strain inoculation test tube slant, 4 DEG C save for use.
(2) it purifies
The Antagonistic Fungi that preliminary screening goes out is crossed after purification on LB culture medium, single bacterium is chosen and falls within mistake in LB liquid medium Night culture, bacterium solution are stored in -80 DEG C of 15% glycerol tube.
(3) secondary screening
Plate spot lawn method (spot-on-lawn assay): the CPG culture of 1.5%, 0.7% agar is respectively configured Base.The culture medium of 1.5% agar of 15mL is first added in culture dish, it is spare as lower layer's plate after solidification.Then OD is taken600~ 0.4 Rs bacterium 200ul is added in 50 DEG C of 4mL of 0.7%CPG culture medium, and plate is poured into after mixing, dries up 30min.Use 5mm Diameter punch is punched in plate center, adds 50ul OD600~0.4 primary dcreening operation bacterial strain fermentation liquor after drying, is placed in 30 DEG C of cultures For 24 hours, antibacterial circle diameter is observed and recorded.For sporogenic banana blight opportunistic pathogen Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense, Foc) measures bacteriostatic diameter with PDA culture medium same method.It records short of money Anti- loop diameter (D).The bacterium bacterial strain of antagonism circle is filtered out, wherein 118 antagonistic effect is significant, the antibacterial circle diameter to Rs is 10.00mm (Figure 1A), the antibacterial circle diameter to Foc are 8.75mm (Figure 1B).
2 118 dientification of bacteria of embodiment is Bacillus velezensis
118 Pseudomonas gram-positive bacterias, there is gemma, rod-shaped, aerobic, there is motility；It is cultivated for 24 hours on LB solid medium The bacterium colony formed afterwards is that white is round, surface wettability.Using Illumina high-flux sequence and CLC gene-splicing method, obtain The whole genome sequence of bacterial strain 118 compares 16SrRNA gene order using NCBI, obtains the 16S rRNA sequence of 118 bacterium.It adopts The phylogenetic tree (Fig. 2) of 118 bacterium is constructed with neighbor-joining method.The 16S rRNA sequence of bacterial strain of the present invention Type strain Bacillus velezensis FZB42 homology with higher in (SEQ ID NO:1) and GenBank is arranged, Its similarity is 100%.In conjunction with flat-plate bacterial colony feature and 16S rRNA sequence build phylogenetic tree as a result, the Preliminary Identification bacterium Strain is Bei Laisi bacillus spore bacillus (Bacillus velezensis), is named as Bacillus velezensis 118。
The gene order of 3 118 bacterium δ X and the ylaC factor of embodiment
Using Illumina high-flux sequence and CLC gene-splicing method, the whole genome sequence of bacterial strain 118 is obtained, is adopted Gene order is compared with NCBI, obtains the 16SrRNA sequence and controlling gene δ of bacterial strain 118XSequence, such as SEQ IDNO.1, Shown in NO.2.The sequence and the similarity of type strain bacillus subtilis 3610 are 87%.Obtain the controlling gene of bacterial strain 118 YlaC sequence, as shown in SEQ ID NO.3.The sequence and 3610 difference of type strain bacillus subtilis are very big, it is shown that solely Characteristic.
4 δ of embodimentXHave the function of 118 bacterium biofilm formations with the ylaC factor
Regulating and controlling the effect in 118 bacterium biofilm formations to assess extracellular function delta antigen.This experiment is same using long flank Source PCR (long flanking homology PCR, LFH-PCR) method, constructs the extracellular function factor δ of bacterial strain 118M、 δX、δW、δVWith ylaC gene-deleted strain, thin skin formation method experiment is further carried out using LBGM culture medium and biofilm formation is real It tests, the biomembrane and thin skin phenotype of more each gene-deleted strain, the results showed that, under LBGM Liquid static culture, δXRegulate and control base Because having the function of that gauffer forms (Fig. 4 A and Fig. 5 A) in thin skin.The contribution rate formed to 118 thin skin of bacterial strain is about 50% (Fig. 5 C).Under LBGM solid plate culture, δXControlling gene has the function of gauffer and petal formation in biomembrane (Fig. 4 B and Fig. 5 B).
Under LBGM Liquid static culture, the biofilm phenotype of ylaC gene-deleted strain and 118 bacterial strain wild strains are not different, but The specific biomembrane form for having bacterium dizzy around the display of LBGM solid plate culture 1d, ylaC controlling gene gene-deleted strain, discovery YlaC controlling gene participates in petal in biomembrane and forms (Fig. 4 B).
5 δ of embodimentXThe factor has the function of the travelling of 118 bacterium and wriggles
Using the LBGM plate of 0.3% and 0.7% agar, by culture to OD600~0.4 3 μ L of bacterium solution is added to plate center Between, 37 DEG C of cultures are placed in, respectively in the diameter of 3h observation swimming, in the diameter that 4h observation is wriggled.It takes pictures and records diameter. Experiment is in triplicate.The result shows that δXThe diameter of swimming in 3h of gene-deleted strain/wild strain 118 is respectively 59/49mm (Fig. 6 A). In 4h, wriggling diameter is respectively 22/14mm (Fig. 6 B and 6C)；Show δXWhat controlling gene swam and wriggled with promotion bacterial strain Function.
6 δ of embodimentXThe factor has the function of promoting 118 bacterium adherency root system and colonizing
Using the dyeing of SYTO13 solution and laser confocal scanning microscope method, 118 are had detected and its in arabidopsis root Colonize situation.Experimental procedure is as follows, and the 5 days Arabidopsis thaliana Seedlings that will germinate are placed in 96 hole microplates, and 300 μ L are added Then LBGM culture medium takes 118 bacterial strains, δX3 μ L (OD of gene-deleted strain and 3610 fermentation liquid of positive control strain600~0.4) it is added In every hole, at 23 DEG C, 100rpm shaken cultivation.Respectively in 0h, 12h, for 24 hours, 48h when sample, wash root with PBS buffer solution, and It after dyeing 2 minutes, is washed with PBS buffer solution, in Leica with SYTO13 solution (diluting 1500 times with PBS phosphate buffer) SP5 CONFOCAL SCANNING LASER MICROSCOPE (CSLM) 488nm excitation laser and 509nm transmitting are imaged.The result shows that (Fig. 8), δ X gene gene-deleted strain only forms small part membrane structure on arabidopsis root system surface, shows that the missing of δ X causes bacillus to be formed Biomembrane is slack-off, the decline of root colonization ability.
7 δ of embodimentXThe factor participates in 118 bacterium biological control banana blights and bacterial wilt of tomato
Sharp born of the same parents sickle-like bacteria (F.oxysporum, Foc) sensitive varieties Brazil any of several broadleaf plants 4-5 piece leaf seedling, is arranged four processing, point Not as follows: (Foc is not added) in CK1, CK2 (Foc), Foc+118 and Foc+ δX.30 banana seedlings of every processing.By CK2 (Foc), Foc+ 118 and Foc+ δXFoc (~10 is immersed in the root of three processing group Banana Seedlings6Spores/ml 30 minutes in), two days later, Foc + 118 processing groups and Foc+ δXThe 50mL 118 and δ that processing group will prepare respectivelyXBacteria suspension be poured onto plant root (106 Cfu/g soil.The incidence of the banana plant of monitoring in every 3 days, disease index record (Ploetz as shown in table 5 after transplanting Et al., 1999).
Using the susceptible prosperous carbuncle 5-6 piece of kind tomato China of Pseudomonas solanacearum (Ralstonia solanacearum, Rs) Leaf seedling replanting is handled as follows: CK1 (No Rs), CK2 (Rs), Rs+118 and Rs+ δ into potting soilXProcessing.Every processing 24 Strain tomato seedling, 2 plants of every basin, 12 basins of each processing.7 days after transplanting, Rs+118 or Rs+ δXProcessing soil is inoculated with 118 or δ respectivelyXIt lacks Lose mutant strain suspension group (~106cfu·g-1Soil).CK1 and CK2 processing group is compared using 50mL sterile water respectively.It connects Two days later, in addition to CK1, other three processing are all inoculated with Rs bacteria suspension (~10 to kind Antagonistic Fungi7cfu·g-1Soil).It is placed in In artificial climate room (PQX-450R-22HM), temperature is maintained at 25-32 DEG C, and relative humidity is 65% to 80%.Inoculating tomato is green Start to calculate disease incidence after blight.Disease index records (Chen et al., 2013) according to 6 grade scale of table.
Bacterial wilt of tomato and banana blight disease index and biological and ecological methods to prevent plant disease, pests, and erosion efficiency calculation are as follows:
Disease index (DI)=[∑ (plant number × number of levels of each disease grade)/(investigate total plant number × highest level Number)] × 100%
Biological and ecological methods to prevent plant disease, pests, and erosion efficiency (BE)=[(disease index-test process disease index of CK)/CK disease index] × 100%
The result shows that (Fig. 7 A and 7B), bacterial strain 118 is 42% to the biological and ecological methods to prevent plant disease, pests, and erosion efficiency of banana blight.It is wild to compare 118 Strain and its δXThe difference of gene-deleted strain shows controlling gene δXIt is viscous by regulation 118 biomembrane of bacterial strain, travelling and wriggling, root system It is attached and the functions such as colonize, thus regulate and control bacterial strain 118 to the prevention and treatment of banana blight and bacterial wilt of tomato (Fig. 7 A, 7B, 7C and 7D), wherein δXContribution rate in biological and ecological methods to prevent plant disease, pests, and erosion efficiency is 8%.
The significant antagonizing pathogenic fungi point born of the same parents sickle-like bacteria of 8 118 bacterium of embodiment
With sharp born of the same parents sickle-like bacteria (Foc)) it is indicator bacteria, the sickle-like bacteria mycelia block of diameter about 5mm is accessed at PDA plate center, The 118 bacterium points inoculation of picking activation sets 28 DEG C of culture 5d with sickle-like bacteria mycelia block at a distance of 2.5cm or so, observes 118 bacterium to sickle The inhibitory effect of knife bacterium.Meanwhile the antibacterial belt edge mycelia of picking, hypha form situation of change is observed under the microscope.Not connect Endogenetic bacteria isolate plate is as control, if 3 repetitions.118 bacterium see Fig. 1 C to the inhibitory effect of Foc.In plate culture 5d Afterwards, 118 bacterium all have apparent inhibitory effect to sickle-like bacteria.The sickle-like bacteria mycelia for taking antibacterial belt edge suppressed is under the microscope Observation, the results showed that Fig. 1 D, the hyphal surface for compareing colony edge is smooth mellow and full, the bacterium colony of 118 bacterium and sickle-like bacteria opposite culture Mycelia is sparse at edge, and plasm in the antibacterial sickle-like bacteria mycelia taken at certain positions can be observed and be aggregated, and mycelia is expanded, Bulging deformation.
9 118 bacterium of embodiment significantly prevents and treats banana blight
118 bacterium are assessed using potting banana plant controlling experiment in the biocontrol effect of banana blight.Take susceptible sharp born of the same parents Sickle-like bacteria biological strain No. 4 (Foc) banana cavendish kind seedling are test material.Potting soil is big by Agriculture In South China It learns trees garden mould and is mixed with the northern proving ground vegetable garden soil of leap by 2: 1, basic physicochemical character: organic matter 23.1gkg-1, full nitrogen 1.0g·kg-1, full phosphorus 5.8gkg-1, rapid available phosphorus 15.7mgkg-1, alkali-hydrolyzable nitrogen 124.8mgkg-1, pH value 5.4.Every basin soil 1.5kg.Test sets CK1 (No Foc, water is added to make blank control), CK2 (Foc, sharp born of the same parents sickle-like bacteria), 118+Foc (sharp born of the same parents' reaping hook Bacterium and 118 bacterium) three processing groups.CK2 and 118+Foc processing is by banana seedlings in Foc physiological saline bacteria suspension (~106spore/ Ml 30min is impregnated in), is transplanted into potting soil.Two days later, 118+Foc processing inoculation 50mL (OD600~1.0) 118 bacterium is outstanding Liquid is into potting soil (~106Cfu/g soil), it is every to handle totally 30 seedlings.The morbidity feelings of banana plant of every 3 days records Condition, the determination method (Ploetz et al., 1999) of the disease index and grade scale of banana with reference to Ploetz.Calculation formula It is as follows:
Disease index (DI)=[∑ (plant number × number of levels of each disease grade)/(investigate total plant number × highest level Number)] × 100%
Biological and ecological methods to prevent plant disease, pests, and erosion efficiency (the BE)=[(disease of CKFeelingsIndex-test process disease index)/CK disease index] × 100%
As a result as shown in Fig. 3 A and 3E, in inoculation the 6th day, processing Foc started to fall ill, and handles 118+Foc and do not find to appoint What symptom, illustrates 118 generations for delaying banana blight at this time.After inoculation 30 days, the biological and ecological methods to prevent plant disease, pests, and erosion effect of 118 pairs of banana blights Rate reaches 42.31%.
10 118 bacterium of embodiment remarkably promotes banana plant growth
The variation dynamic of the banana plant number of blade of different disposal is shown in Fig. 3 B.After inoculation in the same time, different disposal The number of blade of plant are as follows: 118+Foc and CK2 (No Foc) healthy plant are not significantly different, and the two is all remarkably higher than CK1 (Foc).The variation dynamic of the banana plant plant height of different disposal is shown in Fig. 3 C.After inoculation in the same time, the plant height of different disposal Are as follows: 118+Foc and CK2 (No Foc) > CK1 (Foc), as the generation of banana blight handles the banana seedlings of CK1 (Foc) not There is the symptom here to lodge of withering in regrowth.This illustrates that wilt can significantly inhibit the growth of banana plant, and 118 bacterium can show Write the influence for reducing wilt to banana seedling leaf and plant height.After inoculation 30 days, the biology of banana plant different disposal Amount is as shown in Figure 3D.CK1 (Foc) the either fresh weight of aerial part or under ground portion and dry weight are significantly less than at other 2 Reason, and reach significant difference (p < 0.05) with other three processing.Handle the total fresh weight of 118+Foc plant, gross dry weight ratio CK1 (Foc) has increased separately 110% and 101%.
Sequence table
<110>Agricultural University Of South China
<120>Bei Laisi bacillus 118 and its bacteria agent and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 990
<212> DNA
<213>Bei Laisi bacillus (Bacillus velezensis)
<400> 1
tgctcctgat gttagcggcg gacgggtgag taacacgtgg gtaacctgcc tgtaagactg 60
ggataactcc gggaaaccgg ggctaatacc ggatggttgt ctgaaccgca tggttcagac 120
ataaaaggtg gcttcggcta ccacttacag atggacccgc ggcgcatagc tagttggtga 180
ggtaacggct caccaaggcg acgatgcgta gccgacctga gagggtgatc ggccacactg 240
ggactgagac acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga 300
cgaaagtctg acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg 360
ttgttaggga agaacaagtg ccgttcaaat agggcggcac cttgacggta cctaaccaga 420
aagccacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg 480
gaattattgg gcgtaaaggg ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg 540
ctcaaccggg gagggtcatt ggaaactggg gaacttgagt gcagaagagg agagtggaat 600
tccacgtgta gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcgactc 660
tctggtctgt aactgacgct gaggagcgaa agcgtgggga gcgaacagga ttagataccc 720
tggtagtcca cgccgtaaac gatgagtgct aagtgttagg gggtttccgc cccttagtgc 780
tgcagctaac gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag 840
gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag 900
aaccttacca ggtcttgaca tcctctgaca atcctagaga taggacgtcc ccttcggggg 960
cagagtgaca ggtggtgcat ggtgtcgtca 990
<210> 2
<211> 584
<212> DNA
<213>Bei Laisi bacillus (Bacillus velezensis)
<400> 2
taaccgccgg aagttgtctt aacaactcct ctactctttc ttcgttccaa tatgacttca 60
tcatccatta gctcctccct caaaagctcc atctgtttcc gaagtatttt gagccctctg 120
tgttgggtcg ttttgacttt gctttccgag aagcggagag cggcggctgt ttcctgaatg 180
gaatatccct gaataaatct caagataata acagctcttt gatcaatggt gcaatgatca 240
agcgcggtat aaatttcccg gacattttca tgctgtacgg cgagttcgtc cggaagcaaa 300
tgctgatctc tgacatcttg tgtatcccaa tcaaacgtac ccataatacg ctgtctgatc 360
gtctgctgct tgcggaacca gtcaatcgcc acatggcgtg cgatggacaa cagccatgtt 420
ttttcactgc ttcttccttc aaatgtatga taggaattca agacgcggat atacacttcc 480
tgcgtcaaat cttcagcctg cgacttatct tttaccatat aaaataaaaa ttgatataaa 540
tcttgatggt atgcatcgta taatatctga aaggtttctt ccat 584
<210> 3
<211> 516
<212> DNA
<213>Bei Laisi bacillus (Bacillus velezensis)
<400> 3
aaaaacaaga tgtcaattga acagctgtat cgtgaacatt atcaagatat tatgtattac 60
ctttacagac gcacacatca tgctgagacc gctaaagacc tcgctcagga tacatttatg 120
aaagcattta acgggcttga ttcgtttaag ggccattcgt ctatcagaac gtggctctat 180
gcaattgccc atcatacctt catcaattgg tacagacgtg atagaaaata tcaattttca 240
gatatcaccc tgaatgaagg gcttcagcaa acaacatatg aactgccgga gcattacgtc 300
gcccggacag acactcagaa tgcgctgacc gggcagattc agcgtctgaa aagcgatcag 360
cagaccgttt taattttaag ggaatttcaa gagctgtctt acgaagagat tgctgatgtt 420
ttggactgga gtctttcgaa ggtgaagagc accctgtaca gggcgagaat gcagctgaaa 480
cagcacttgg cggaaaaaag gaaggacgga cagata 516

Claims (9)

1. one plant of Bei Laisi bacillus (Bacillus velezensis) 118, deposit number GDMCC No.60625, in It is preserved in Guangdong Province's Culture Collection (GDMCC) on 04 04th, 2019.
2. Bei Laisi bacillus 118 according to claim 1, which is characterized in that its 16S rRNA nucleotide sequence is such as Shown in SEQ ID NO.1, extracellular function delta antigen δXNucleotide sequence is as shown in SEQ ID NO.2；Extracellular function delta antigen ylaC Nucleotide sequence is as shown in SEQ ID NO.3.
3. Bei Laisi bacillus 118 according to claim 1, which is characterized in that the Bei Laisi bacillus 118 Has the function of high yield biomembrane.
4. according to Bei Laisi bacillus 118 described in right 3, which is characterized in that in the Bei Laisi bacillus 118 Extracellular function delta antigen δXHave the function of promoting biomembrane yield.
5. Bei Laisi bacillus 118 according to claim 1, which is characterized in that the Bei Laisi bacillus 118 has There is the function of regulation bacterial strain biomembrane form, or the function of promoting bacterium travelling and wriggle；Or it improves root system adherency and colonizes Function.
6. Bei Laisi bacillus 118 according to claim 5, which is characterized in that in the Bei Laisi bacillus 118 Extracellular function factor δXHave the function of gauffer and petal formation in bacterial strain biomembrane form, or improves bacterium travelling With the function of peristaltic velocity；Or it improves root system and adheres to the function of colonizing；Extracellular function in the Bei Laisi bacillus 118 Factor ylaC also has the function of biomembrane form.
7. a kind of bacteria agent, which is characterized in that the bacteria agent be 1 is wanted as right described in Bei Laisi bacillus 118 Obtained, 118 quantity of Bei Laisi bacillus is 1.0 × 108~9.0 × 109cfu/ml。
8. 118 bacteria agent of Bei Laisi bacillus described in claim 1 in terms of control of plant bacterial wilt application or Prevent and treat the application in terms of banana blight.
9. application of 118 bacteria agent of Bei Laisi bacillus described in claim 1 in terms of promoting banana plant growth.
CN201910391310.3A 2019-05-12 2019-05-12 Bei Laisi bacillus 118 and its bacteria agent and application Pending CN110387337A (en)

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Application publication date: 20191029