CN110468060A - A kind of general bacteria strain and its application in biological control - Google Patents
A kind of general bacteria strain and its application in biological control Download PDFInfo
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Abstract
The present invention provides a kind of general bacteria strain and its applications in biological control.The culture presevation number of the bacterial strain is CGMCC NO.14361.The present invention also provides the strain preparation comprising the bacterial strain or the biological control agents including the strain culture.The present invention also provides the bacterial strain inhibit disease caused by the pathogens such as verticillium dahliae or caused by application in pollution.Biological control is carried out to such as smoke tree wilt disease as caused by verticillium dahliae using bacterial strain of the present invention, the disease index of smoke tree wilt disease can be effectively reduced, decline 20% or more compared with the control.
Description
Technical field
The invention belongs to Ornamental Diseases prevention and control field, it is related to a kind of bacterial strain for biological control and its application, especially
It is applied in the biological control of smoke tree wilt disease.
Background technique
Smoke tree (Cotinus coggygria) is used as a kind of main red-leaf tree species, has not in the succession of red autumnal leaves culture
Alternative effect.
By taking the Fragrant Hill Park of Beijing as an example.Since the eighties in last century, the smoke tree woods in Xiangshan park goes out because of wilt disease
The withered phenomenon of large area is showed, it is in the landscape ecology system Construction of Beijing city that annual withered smoke tree, which was once reaching thousands of strains,
One of letter problem to be solved (north the Fragrant Hill Park Song Lizhou, Du Wanguang, Li Weiwei smoke tree wilt disease Study of Prevention Technology [J]
Capital gardens, 2011,27 (2): 51-56；Ge Yuxuan, Zhou Xiaohong, Liu Yang, Wang Liangsheng smoke tree category germ plasm resource, Lycopersicum esculentum, change
It studies point, leaf color study on regulation progress gardening journal, 2014,41 (9): 1833-1845.).
The disease is that the systematicness as caused by verticillium dahliae (Verticillium dahlia) infects disease, cause of disease
Bacterium has extensive host range, widely distributed in soil and plant, can infect 660 kinds of 40 section or more plant, including agriculture
Make species, greengrocery, fruits, ornamental flower class, fiber and oil crops and various xylophytas etc., utilizes chemical bactericide
It is thoroughly removed and is difficult to realize, and the use of excess chemical pesticide does great damage to soil microenvironment,
Keep plant growing way weak, to more aggravate the severity of disease generation.
The following aspects is concentrated mainly on to the specific control measure of the disease at present: (1) emphasizing to put prevention first, strictly
Disease diffusion is controlled, strengthens quarantine supervision, nursery stock and soil enter no lesion to strict control in spite of illness.(2) in terms of physical control, often
Soil solarization method and the two kinds of measures of biological soil fumigating system are taken, but both physical control methods take effect slowly.(3) chemistry is anti-
It is often used bromomethane, trichloronitromethane, methylisothiocyanate ester etc. in controlling and carries out soil-fumigating, or uses plant growth regulator
Alleviate symptom, or is injected directly into limb prevention and treatment (Li Lunguang edaphic factor pair with benzimidazole, benomyl, thiophanate methyl etc.
Smoke tree withers Beijing Primary Study [D] of pathogenetic influence and disease control: Beijing Forestry University, 2008.).Pass through medicament
Screening test, Han Jing et al. confirm net 300 times of bacterium control efficiency most preferably (Korea Spro with 400 times of liquid of carbendazim to smoke tree wilt disease that withers
Jing, 2009).However with the serious consequences such as the environmental pollution of chemical pesticide bring, pesticide residue are used, application dynamics is gradually
It is controversial.(4) Synthetical prevention that biological control is smoke tree wilt disease is carried out using Antagonistic Fungi bring new hope (Ge Jin, face
Rong, Song Lizhou wait Comprehensive Control Strategy [J] Chinese Urban Forestry of smoke tree wilt disease, 2007,5 (3): 43-44.；Wang Jian
Beautiful smoke tree Pathogen of Fusarium Wilt and the Beijing pathogenesis [D]: Beijing Forestry University, 2009.), but current Antagonistic Fungi
In terms of screening is concentrated mainly on crops, and less (Wang Yan smoke tree and big is then reported for the screening of the Antagonistic Fungi of forest wilt disease
Beautiful Verticillium dahliae Interaction and the Beijing pathogen quantitative study [D]: Beijing Forestry University, 2012.).Therefore, acquisition is screened
There is the bacterial strain of inhibiting effect that will there is important application value in terms of Ornamental Diseases biological control smoke tree wilt.
Summary of the invention
(1) technical problems to be solved
The purpose of the present invention is find one kind effectively to carry out the method for biological control to smoke tree wilt disease to solve
Above-mentioned deficiency in existing method.Presently, there are technical problem underlying be to find effectively inhibit smoke tree wilt disease not yet
The bacterial strain that can be used in biological control of bacterium (i.e. verticillium dahliae), therefore it is anti-effective biology can not to be carried out to smoke tree wilt disease
It controls.
(2) technical solution
The present inventor screened from smoke tree limb obtain endogenetic bacteria bacterial strain CCBC3-3-1 (hereinafter be occasionally referred to as this hair
Bright bacterial strain), it has been surprisingly found that the bacterial strain has strong inhibiting effect to numerous strains including verticillium dahliae, then will
The bacterial strain carries out culture presevation and microbial inoculum is made using the culture that the strain culturing obtains to be used to by including big beautiful wheel
Disease such as smoke tree wilt disease caused by pathogen including branch bacterium carries out biological control, so as to complete the present invention.
Then, the present invention provides a kind of bacterial strain for biological control, the culture presevation of the bacterial strain in first aspect
Number for CGMCC NO.14361 (hereinafter be occasionally referred to as bacterial strain of the present invention, may be used interchangeably with bacterial strain CCBC3-3-1).
The present invention provides a kind of strain preparation in second aspect, and the strain preparation has great-hearted first party of the present invention
Bacterial strain described in face is bacterial strain of the present invention.
Third aspect present invention provides a kind of biological control microbial inoculum, and the microbial inoculum includes as described in first aspect present invention
The obtained culture of strain culturing.
The present invention provides bacterial strain described in first aspect present invention in fourth aspect and is inhibiting selected from by verticillium dahliae
(Verticillium dahlia), grape seat chamber bacterium (Botryosphaeria dothidea), Rhizoctonia solani Kuhn
(Rhizoctonia solani), willow rotten pathogenic bacteria (Valsa sordida) and Valsa sordida bacterium (Valsa sordida)
Application at least one of group of composition pathogen.
Fifth aspect present invention provides bacterial strain described in first aspect present invention in prevention and treatment by verticillium dahliae
(Verticillium dahlia), grape seat chamber bacterium (Botryosphaeria dothidea), Rhizoctonia solani Kuhn
(Rhizoctonia solani), willow rotten pathogenic bacteria (Valsa sordida) and Valsa sordida bacterium (Valsa sordida)
Application in disease caused by least one of group of composition pathogen.
Sixth aspect present invention provides a kind of method that the bacterial strain described to the first aspect of the present invention carries out separation identification.
(1) solid LB media preparation and making sheet: formula is as follows: including 10g/L sodium chloride, 10g/L tryptone, 5g/L
Yeast extract and 8g/L agar, and pH is adjusted to 7.2, heating sterilizes after dissolving, and is subsequently poured into culture dish and LB plate is made；
(2) strain isolation: smoke tree branch section is carried out disinfection using 70% ethyl alcohol, then uses aseptic water washing, then put into
It is sterilized in 5% liquor natrii hypochloritis, then reuses aseptic water washing, the resulting smoke tree branch section by disinfection is seeded in
On LB plate, cultivated under the conditions of 28 DEG C；After bacterial clump is grown, picking bacterium is transferred on other LB plate, grows list
After bacterium colony, picking single colonie continues to be trained pure bacterial strain.
(3) bacterial strain is identified
The DNA for extracting the pure bacterial strain is led to as shown in SEQ ID NO.1 then using gained DNA as template using sequence
With primer 2 7F and sequence, the universal primer 1495R as shown in SEQ ID NO.2 carries out PCR amplification, obtains amplified production and surveys
Sequence confirms that sequence similarity shown in the sequence and SEQID NO.3 reaches 90 or more, preferably reaches 95% or more, more preferably up to
99% or more, most preferably up to 100%, confirmation obtained strains are bacterial strain of the present invention.
(3) beneficial effect
The present inventor's bacterial strain of the present invention isolated from smoke tree limb is not only to verticillium dahliae (Verticillium
Dahlia) there is strong inhibiting effect, and the disease index of such as smoke tree wilt disease is effectively reduced, decline compared with the control
20% or more, so as to the biological control for smoke tree wilt disease.Moreover, bacterial strain of the present invention is also to such as grape seat chamber bacterium
(Botryosphaeria dothidea), Rhizoctonia solani Kuhn (Rhizoctonia solani), willow rotten pathogenic bacteria (Valsa
Sordida) and Valsa sordida bacterium (Valsa sordida) also has significant inhibitory effect, therefore can also be by the bacterial strain
Broad spectrum activity biological control microbial inoculum such as pulvis or suspending agent is made with the biological control for these germs in culture.
Detailed description of the invention
Fig. 1 is colonial morphology of the bacterium CCBC3-3-1 when cultivating 10d in PDA culture medium.
Fig. 2 is the thalli morphology of (10500X) bacterium CCBC3-3-1 under scanning electron microscope.
Fig. 3 is using bacterial universal primers to the band of the bacterium CCBC3-3-1 1510bp amplified.In figure, M: nucleic acid
Molecular weight D2000；1-5:CCBC3-3-1；6: negative control (distilled water).
Fig. 4 is the CCBC3-3-1 systematic evolution tree of the 16S rDNA sequence construct according to CCBC3-3-1.
Fig. 5 is smoke tree wilt (i.e. verticillium dahliae may be used interchangeably with verticillium dahliae herein) and thin
Colonial morphology when bacterium CCBC3-3-1 opposite culture 21d (left side is smoke tree wilt, and the right is bacterium CCBC3-3-1).
Fig. 6 is the colonial morphology that smoke tree wilt cultivates 21d on PDA culture plate.
Fig. 7 cultivates 21d's in PDA culture medium+bacterium CCBC3-3-1 for smoke tree wilt on without fermented liquid plate
Colonial morphology.
Fig. 8 be grape seat chamber bacterium and bacterium CCBC3-3-1 opposite culture 6d colonial morphology (left side is grape seat chamber bacterium,
The right is CCBC3-3-1).
Fig. 9 be Rhizoctonia solani Kuhn and bacterium CCBC3-3-1 opposite culture 6d colonial morphology (left side is Rhizoctonia solani Kuhn,
The right is bacterium CCBC3-3-1).
Figure 10 is that willow rotten pathogenic bacteria and the colonial morphology of bacterium CCBC3-3-1 opposite culture 6d (rot on the left side for willow
Germ, the right are bacterium CCBC3-3-1)
Figure 11 is that Valsa sordida bacterium and the colonial morphology of bacterium CCBC3-3-1 opposite culture 6d (rot on the left side for poplar
Germ, the right are bacterium CCBC3-3-1).
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill people
Member's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
As described above, the present invention provides a kind of bacterial strain for biological control, the strain of the bacterial strain in first aspect
Deposit number is CGMCC NO.14361.
The number of the bacterial strain is CCBC3-3-1, and Chinese microorganism strain preservation management committee is preserved on June 27th, 2017
Member can common micro-organisms center, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica,
Culture presevation number is CGMCCNO.14361, and classification naming is general bacterium (Pantoea SP.).
The Morphological Identification of bacterial strain: in PDA culture medium, (formula is as follows: removing the fresh potato 200g of skin, adds water
1000ml boils 20min, and glucose 15g, agar 8g is added after double gauze filtering, supplements suitable water to 1000ml, sufficiently
It is fitted into triangular flask after shaking up, 121 DEG C of sterilizing 30min are poured into and plate is made in sterilizes culture dish, what Examples below used
PDA culture medium is all made of the formula) on, 30 DEG C of culture 10d, colony diameter reaches 18mm, is faint yellow, surface elevation, moistens, light
Sliding (as shown in Figure 1), is detected as Gram-negative through 3%KOH solution.Through scanning electron microscopic observation, thallus is in direct rod shape, 0.5-
1.5 μm (as shown in Figure 2).
The Molecular Identification of bacterial strain: paramagnetic particle method bacterial genomes DNA extraction agent box (the limited public affairs of Shanghai bioengineering are used
Department) extract CCBC3-3-1 DNA, using 16S rDNA universal primer 27F (as shown in SEQ ID NO.1) and universal primer
1495R (as shown in SEQ ID NO.2) carries out PCR amplification, obtains the PCR product of 1510bp (referring to Fig. 3)；Recycle T-
Obtained PCR product is connected to carrier T by carrier PCR product Cloning Kit (Shanghai bioengineering Co., Ltd), then into
Heat-shock transformed, the picking positive colony of row, serves marine growth Engineering Co., Ltd and is sequenced, obtain the 1510bp's of the bacterial strain
16S rDNA sequence (as shown in SEQ ID NO.3), which is carried out in www.ncbi.nlm.nlh.gov database
BLAST is compared.As comparison result it is found that known to the 16S rDNA sequence of bacterium CCBC3-3-1 and general Pseudomonas three kinds it is agglomerating general
Bacterium (Pantoea agglomerans), the general bacterium of pineapple (Pantoea ananatic) and the similitude of Pantoea vagans are equal
It is 98%, so that it is determined that bacterium CCBC3-3-1 is a variation kind Pantoea sp. of general Pseudomonas (Pantoea).Utilize MEGA
Neighbor-joining Tree in software makees phylogenetic analysis, the results showed that bacterium CCBC3-3-1 and pantoea agglomerans
(Pantoea ananatic) has closer affiliation (as shown in Figure 4).
The biological control is to be suppressed or eliminated using the bacterial strain selected from by big in some preferred embodiments
Beautiful Verticillium dahliae (Verticillium dahlia), grape seat chamber bacterium (Botryosphaeria dothidea), Rhizoctonia solani Kuhn
(Rhizoctonia solani), willow rotten pathogenic bacteria (Valsa sordida) and Valsa sordida bacterium (Valsa sordida)
Disease caused by least one of group of composition pathogen or caused by pollute.
In some preferred embodiments, the biological control be inhibited using the bacterial strain verticillium dahliae to
Biological control is carried out to smoke tree wilt disease.
The present invention provides a kind of strain preparation in second aspect, and the strain microbial inoculum includes to have great-hearted bacterium of the present invention
Strain.The strain preparation can be used for for example producing the culture obtained comprising the strain culturing as described in first aspect present invention
Biological control microbial inoculum.
Then, third aspect present invention provides a kind of biological control microbial inoculum, and the microbial inoculum includes to be trained by bacterial strain of the present invention
Support obtained culture.
In some preferred embodiments, the microbial inoculum is suspending agent, and the culture is fermentation liquid；Preferably
It is that the fermentation liquid is cultivated in the following way: the strain inoculated is activated on LB plate, obtains activated strains,
Then by the strain inoculated of activation into LB liquid medium, 7 days after 30 DEG C of shaken cultivations, the fermentation liquid is obtained.
In some preferred embodiments, the biological control microbial inoculum also includes auxiliary agent and stabilizer.The present invention is to institute
It states auxiliary agent to be not particularly limited, it is preferred that the auxiliary agent is selected from by polyethylene glycol (PEG) 8000, polyvinyl alcohol
(PVA), lauryl sodium sulfate (SDS), the group of Tea Saponin and gum arabic composition.The present invention is to the stabilizer without spy
Other limitation, it is preferred that the stabilizer is selected from by calcium carbonate, potassium phosphate, dipotassium hydrogen phosphate and hydroxymethyl cellulose
The group of sodium composition.
In some preferred embodiments, the biological control microbial inoculum is pulvis, and the culture is solid training
Support object；Preferably, the solid culture obtains in the following way: by the strain inoculated in solid medium such as wheat
On bran, 30 DEG C obtain solid culture after dark culture 15 days, then by solid culture crushing and processing, obtain the pulvis.
The biological control microbial inoculum is injected in smoke tree limb or is spread in the transplanting of spring smoke tree in which can be convenient when in use
It imposes on around rhizosphere, so as to which the disease incidence of smoke tree wilt disease is effectively reduced.
The present invention provides bacterial strain described in first aspect present invention in fourth aspect and is inhibiting selected from by verticillium dahliae
(Verticillium dahlia), grape seat chamber bacterium (Botryosphaeria dothidea), Rhizoctonia solani Kuhn
(Rhizoctonia solani), willow rotten pathogenic bacteria (Valsa sordida) and Valsa sordida bacterium (Valsa sordida)
Application at least one of group of composition pathogen.
Fifth aspect present invention provides bacterial strain described in first aspect present invention in prevention and treatment by verticillium dahliae
(Verticillium dahlia), grape seat chamber bacterium (Botryosphaeria dothidea), Rhizoctonia solani Kuhn
(Rhizoctonia solani), willow rotten pathogenic bacteria (Valsa sordida) and Valsa sordida bacterium (Valsa sordida)
Application in disease caused by least one of group of composition pathogen.
The present invention provides a kind of method that the bacterial strain described to the first aspect of the present invention is separated at the 6th aspect.
(1) solid LB media preparation and making sheet: formula is as follows: 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast
Powder and 8g/L agar are soaked, and pH is adjusted to 7.2, heating sterilizes after dissolving, and is subsequently poured into culture dish and LB plate is made；
(2) strain isolation: smoke tree branch section is carried out disinfection using 70% ethyl alcohol, then uses aseptic water washing, then put into
It is sterilized in 5% liquor natrii hypochloritis, then reuses aseptic water washing, the resulting smoke tree branch section by disinfection is seeded in
On LB plate, cultivated under the conditions of 28 DEG C；After bacterial clump is grown, picking bacterium is transferred on other LB plate, grows list
After bacterium colony, picking single colonie continues to be trained pure bacterial strain.
(3) bacterial strain is identified
The DNA for extracting the pure bacterial strain is led to as shown in SEQ ID NO.1 then using gained DNA as template using sequence
With primer 2 7F and sequence, the universal primer 1495R as shown in SEQ ID NO.2 carries out PCR amplification, obtains amplified production and surveys
Sequence confirms that sequence similarity shown in the sequence and SEQID NO.3 reaches 90 or more, preferably reaches 95% or more, more preferably up to
99% or more, most preferably up to 100%, confirmation obtained strains are bacterial strain of the present invention.
Hereafter by invention is further explained by way of embodiment.But these embodiments are only for example
Illustration purpose.Protection scope of the present invention is not limited to these examples.
Embodiment
Embodiment 1: bacterium CCBC3-3-1's isolating and purifying and identifying
(1) preparation of solid LB media, formula are as follows: sodium chloride 10g/L, tryptose old 10g/L, yeast extract 5g/
(solid LB media used in other embodiments is trained by this preparation, LB liquid medium and solid LB by L, agar 8g/L
Base phase ratio is supported, agar is only added without), regulated value pH to 7.2, heating is fitted into triangular flask after dissolving fully, 121 DEG C of sterilizings
30min is poured into sterilizes culture dish and LB plate is made.
(2) CCBC3-3-1 is isolated and purified: segment by fresh smoke tree secateurs at 0.5cm, with 70% ethanol disinfection
5min aseptic water washing 1 time, then puts into 5% liquor natrii hypochloritis and sterilizes 30min, aseptic water washing 3 times, is seeded in LB
It on plate, is cultivated under the conditions of 28 DEG C, after bacterial clump is grown, a small amount of bacterium scribing line of picking is transferred on new LB plate, is grown
After single colonie, picking single colonie continues to be trained pure bacterial strain.
(2) identification of bacterium CCBC3-3-1: preparation scanning electron microscope sample carries out morphologic observation (result such as Fig. 2
It is shown).In addition, extracting the DNA of the pure bacterial strain of gained, using bacterial universal primers 27F (sequence is as shown in SEQ ID NO.1) and lead to
With primer 1495R (sequence is as shown in SEQ ID NO.2), PCR amplification is carried out, PCR product sequencing result shows to obtain
The 16s rDNA sequence (gained sequence is as shown in SEQ ID NO.3) of 1510bp, sequence alignment is carried out on NCBI, is then utilized
Neighbor-joining Tree in MEGA software makees phylogenetic analysis, the results showed that CCBC3-3-1 and pantoea agglomerans
(Pantoea ananatic) has closer affiliation.
The sequence of table 1. universal primer 27F and universal primer 1495R and amplified production AP.
Embodiment 2: the opposite culture of bacterium CCBC3-3-1 and smoke tree wilt
It is inoculated with the mycelia block of smoke tree wilt disease bacterial strain CCW2-4-2 on PDA plate, is placed in 25 DEG C of incubators and cultivates 7 days
Afterwards, bacterium CCBC3-3-1 is inoculated, the colony edge with smoke tree wilt disease bacterial strain CCW2-4-2 is at a distance of 3cm, then is placed in 25 DEG C of trainings
It supports and is cultivated in case, with the size of vertical cross method measurement inhibition zone, every 7d is measured 1 time, and continuous measurement 3 times records data.As a result
It was found that bacterium CCBC3-3-1 and smoke tree wilt form apparent inhibition zone in opposite culture in PDA culture medium, cultivate
Antibacterial circle diameter reaches 3.2cm (as shown in Figure 5) at 21 days.
Embodiment 4: bacterium CCBC3-3-1 and grape seat chamber bacterium (Botryosphaeria dothidea), Rhizoctonia solani Kuhn
(Rhizoctonia solani), willow rotten pathogenic bacteria (Valsa sordida) and Valsa sordida bacterium (Valsa sordida)
Opposite culture
It is inoculated with grape seat chamber bacterium (Botryosphaeria dothidea), Rhizoctonia solani Kuhn respectively on PDA plate
(Rhizoctonia solani), willow rotten pathogenic bacteria (Valsa sordida) and Valsa sordida bacterium (Valsa sordida)
Mycelia block, be placed in 25 DEG C of incubators after cultivating 7 days, inoculate general bacterium CCBC3-3-1, with first time inoculation colony edge
It at a distance of 3cm, then is placed in 25 DEG C of incubators and cultivates, with the size of vertical cross method measurement inhibition zone, record data.Opposite culture
Experiment shows bacterium CCBC3-3-1 to grape seat chamber bacterium (Botryosphaeria dothidea) and Rhizoctonia solani Kuhn
The inhibiting effect of (Rhizoctonia solani) is weaker, and antibacterial circle diameter is respectively 2.5cm and 2.8cm at opposite culture 6 days
(Fig. 8 and Fig. 9)；The inhibition of willow rotten pathogenic bacteria (Valsa sordida) and Valsa sordida bacterium (Valsa sordida) is made
With relatively strong, antibacterial circle diameter all reaches 3.2cm (Figure 10 and Figure 11) at opposite culture 6 days.
Embodiment 4: inhibiting effect of the bacterium CCBC3-3-1 without fermented liquid to smoke tree wilt
Bacterium CCBC3-3-1 is seeded in LB liquid medium, 28 DEG C 200rpm shaken cultivation 5 days, fermentation liquid warp
0.22 μm of bacterial filter filtering, obtains without fermented liquid, makes plate after mixing with PDA culture 1:1, it is withered to be then inoculated with smoke tree
Germ (Fig. 7) is subsequently placed in 25 DEG C of incubators and cultivates to add the PDA plate of isometric sterile water for control (Fig. 6), with hanging down
The size of straight cross method measurement colony diameter, every 7d are measured 1 time, and continuous measurement 3 times records data.The result shows that bacterium
CCBC3-3-1 without fermented liquid has significant inhibiting effect (referring to Fig. 6 and Fig. 7) to smoke tree wilt, compared with the control, training
Bacteriostasis rate is 63.37% when supporting 21d.
Embodiment 5: the preparation of bacterium CCBC3-3-1 biological control microbial inoculum (suspending agent)
Bacterium CCBC3-3-1 strain is inoculated on LB plate and is activated, by the strain inoculated after activation to LB liquid
In culture medium (relative to solid LB media, be different only in that and do not add agar), 7 days after 30 DEG C of shaken cultivations, every 100mL
Fermentation liquid adds 10g polyvinyl alcohol (PVA) as auxiliary agent and 2g sodium cellulose glycolate as stabilizer, is processed into aqua.
Embodiment 6: the preparation of bacterium CCBC3-3-1 biological control microbial inoculum (suspending agent)
Bacterium CCBC3-3-1 strain is seeded on sterilizing wheat bran, 30 DEG C after dark culture 15 days, solid culture is carried out
After crushing and processing (300-2500 μm of partial size), it is made biological control microbial inoculum (pulvis).
Embodiment 7: the use of bacterium CCBC3-3-1 biological control microbial inoculum suspending agent
CCBC3-3-1 biological control microbial inoculum suspending agent is taken, the medical fluid that concentration is 105/mL is diluted with water to, in Huang in March
Before the expansion of smoke tree young leaves, it is to inject clear water by the medical fluid injection smoke tree xylem after dilution using the method for trunk injection
Control.It investigates the disease incidence and severity of smoke tree wilt disease June, and calculates disease index.
(1) disease incidence: as unit of whole strain, the morbidity strain number of certain strain number is investigated, calculates disease incidence.
Disease incidence=morbidity strain number/investigation total strain number × 100%
(2) severity: as unit of whole strain, according to the form below grade scale investigates the grade that disease occurs.
The grade scale of 2. disease severity of table.
| Grade | Grade scale |
| 0 | Complete stool blade is without wilting symptom |
| I | 1 branch blade has wilting symptom |
| II | 2-3 branch blade has wilting symptom |
| III | More than half blade of plant wilts and has partial blade to fall off |
| IV | Plant all wiltings or withered |
(3) disease index:
Disease index=diseased plant number at different levels multiplies the sum of corresponding series/investigation total strain number × highest series × 100%
The result shows that compared with the control, after injecting CCBC3-3-1 biological control microbial inoculum suspending agent, the disease of smoke tree wilt disease
Feelings index can reduce by 30%.
Embodiment 8: the use of bacterium CCBC3-3-1 biological control microbial inoculum pulvis
CCBC3-3-1 biological control microbial inoculum pulvis is taken uniformly to spread fertilizer over the fields in rhizosphere, every plant pit dosage when Yu Sanyue smoke tree is transplanted
250g, then earthing.Not apply the processing of biocontrol agent as control, the disease incidence of smoke tree wilt disease is investigated second year June
And severity, and calculate disease index.
(1) disease incidence: as unit of whole strain, the morbidity strain number of certain strain number is investigated, calculates disease incidence.
Disease incidence=morbidity strain number/investigation total strain number × 100%
(2) severity: as unit of whole strain, by the grade of the investigation disease generation of grade scale shown in table 2.
(3) disease index:
Disease index=diseased plant number at different levels multiplies the sum of corresponding series/investigation total strain number × highest series × 100%
The result shows that compared with the control, rhizosphere application CCBC3-3-1 biological control microbial inoculum pulvis is after 1 year when plant, smoke tree
The disease index of wilt disease can reduce by 20%.
In conclusion
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Sequence table
<110>Ornamental Plants of Beijing District research institute
<120>a kind of general bacteria strain and its application in biological control
<130> GY17100351
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>artificial synthesized
<400> 1
agagtttgat cctggctca 19
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized
<400> 2
ctacggctac cttgttacga 20
<210> 3
<211> 1510
<212> DNA
<213>pantoea agglomerans (Pantoea ananatic)
<400> 3
agagtttgat cctggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgaac 60
ggtagcacag aggagcttgc tccttgggtg acgagtggcg gacgggtgag taatgtctgg 120
ggatctgcct gacagagggg gataactact ggaaacggta gctaataccg cataacctcg 180
caagagcaaa gagggggacc ttcgggcctc tcgctgtcag atgaacccag atgggattag 240
ctagtaggtg gggtaatggc tcacctaggc gacgatccct agctggtctg agaggatgac 300
cagccacact ggaactgaga cacggtccag actcctacgg gaggcagcag tggggaatat 360
tgcacaatgg gcgcaagcct gatgcagcca tgccgcgtgt atgaagaagg ccttcgggtt 420
gtaaagtact ttcagcgggg aggaaggtgt tgaggttaat aacctcagca attgacgtta 480
cccgcagaag aagcaccggc taactccgtg ccagcagccg cggtaatacg gagggtgcca 540
agcgttaatt cggaattact gggcgtaaag cgcacgcagg cggtctgtca agtcagatgt 600
gaaatccccg ggcttaacct gggaactgca tttgaaactg gcaggctaga gtcttgtaga 660
ggggggtaga attccaggtg tagcggtgaa atgcgtagag atctggagga ataccggtgg 720
cgaaggcggc cccctggaca aagactgacg ctcaggtgcg aaagcgtggg gagcaaacag 780
gattagatac cctggtagtc cacgccgtaa acgatgtcga cttggaggtt gttcccttga 840
ggagtggctt ccggagctaa cgcgttaagt cgaccgcctg gggagtacgg ccgcaaggtt 900
aaaactcaaa tgaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgat 960
gcaacgcgaa gaaccttacc tactcttgac atccacggaa tttggcagag atgccttagt 1020
gccttcggga accgtgagac aggtgctgca tggctgtcgt cagctcgtgt tgtgaaatgt 1080
tgggttaagt cccgcaacga gcgcaaccct tatcctttgt tgccagcacg taatggtggg 1140
aactcaaagg agactgccgg tgataaaccg gaggaaggtg gggatgacgt caagtcatca 1200
tggcccttac gagtagggct acacacgtgc tacaatggcg catacaaaga gaagcgacct 1260
cgcgagagca agcggacctc ataaagtgcg tcgtagtccg gatcggagtc tgcaactcga 1320
ctccgtgaag tcggaatcgc tagtaatcgt ggatcagaat gccacggtga atacgttccc 1380
gggccttgta cacaccgccc gtcacaccat gggagtgggt tgcaaaagaa gtaggtagct 1440
taaccttcgg gagggcgctt accactttgt gattcatgac tggggtgaag tcgtaacaag 1500
gtagccgtag 1510
Claims (10)
1. a kind of bacterial strain for biological control, which is characterized in that the culture presevation number of the bacterial strain is CGMCC NO.14361.
2. bacterial strain according to claim 1, which is characterized in that the biological control is to inhibit or disappear using the bacterial strain
Except selected from by verticillium dahliae (Verticillium dahlia), grape seat chamber bacterium (Botryosphaeria dothidea),
Rhizoctonia solani Kuhn (Rhizoctonia solani), willow rotten pathogenic bacteria (Valsa sordida) and Valsa sordida bacterium
Disease caused by least one of the group pathogen of (Valsa sordida) composition or caused by pollute；Preferably, institute
Stating biological control is to inhibit verticillium dahliae using the bacterial strain to carry out biological control to smoke tree wilt disease.
3. a kind of strain preparation, which is characterized in that the strain microbial inoculum includes to have great-hearted bacterial strain described in claim 1.
4. a kind of biological control microbial inoculum, which is characterized in that the biological control microbial inoculum includes the training obtained by the strain culturing
Support object.
5. biological control microbial inoculum according to claim 4, it is characterised in that:
The biological control microbial inoculum is suspending agent, and the culture is fermentation liquid；
Preferably, the fermentation liquid is cultivated in the following way: the strain inoculated being activated on LB plate, is obtained
Activated strains 7 days after 30 DEG C of shaken cultivations, obtain the fermentation then by the strain inoculated of activation into LB liquid medium
Liquid.
6. biological control microbial inoculum according to claim 5, it is characterised in that:
The biological control microbial inoculum also includes auxiliary agent and stabilizer；
It may further be preferable that the auxiliary agent is selected from by PEG 8000, polyvinyl alcohol, lauryl sodium sulfate, Tea Saponin
With the group of gum arabic composition；And/or the stabilizer is selected from by calcium carbonate, potassium phosphate, dipotassium hydrogen phosphate and methylol fibre
Tie up the group of plain sodium composition.
7. biological control microbial inoculum according to claim 4, it is characterised in that:
The biological control microbial inoculum is pulvis, and the culture is solid culture；
Preferably, the solid culture obtains in the following way: by the strain inoculated in solid medium such as wheat
On bran, 30 DEG C obtain solid culture after dark culture 15 days, then by solid culture crushing and processing, obtain the pulvis.
8. bacterial strain described in claim 1 is inhibiting selected from by verticillium dahliae (Verticillium dahlia), grape seat chamber
Bacterium (Botryosphaeria dothidea), Rhizoctonia solani Kuhn (Rhizoctonia solani), willow rotten pathogenic bacteria (Valsa
) and the application at least one of the group pathogen of Valsa sordida bacterium (Valsa sordida) composition sordida.
9. application according to claim 8, wherein bacterial strain described in claim 1 is for inhibiting verticillium dahliae with right
Smoke tree wilt disease carries out biological control.
10. the method that pair bacterial strain described in claim 1 is separated, which is characterized in that described method includes following steps:
(1) solid LB media preparation and making sheet: formula is as follows: including 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast
Powder and 8g/L agar are soaked, and pH is adjusted to 7.2, heating sterilizes after dissolving, and is subsequently poured into culture dish and LB plate is made；
(2) strain isolation: smoke tree branch section is carried out disinfection using 70% ethyl alcohol, then uses aseptic water washing, then put into 5% time
It is sterilized in sodium chlorate solution, then reuses aseptic water washing；The resulting smoke tree branch section by disinfection is seeded in LB to put down
On plate, cultivated under the conditions of 28 DEG C；After bacterial clump is grown, picking bacterium is transferred on other LB plate, grows single colonie
Afterwards, picking single colonie continues to be trained pure bacterial strain；
(3) bacterial strain is identified
The DNA for extracting the pure bacterial strain draws then using gained DNA as template using sequence is general as shown in SEQ ID NO.1
Object 27F and sequence the universal primer 1495R as shown in SEQ ID NO.2 carry out PCR amplification, obtain amplified production and are sequenced, really
Recognize AP sequence similarity shown in the sequence and SEQ ID NO.3 and reach 90% or more, preferably reaches 95% or more, more preferably up to
99% or more, most preferably up to 100%, confirmation obtained strains are bacterial strain of the present invention.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679881A (en) * | 2019-01-31 | 2019-04-26 | 湖北工程学院 | A kind of general bacterium of pineapple and its microbial inoculum and application |
| CN112760274A (en) * | 2021-04-07 | 2021-05-07 | 中国科学院上海高等研究院 | Organic solid waste high-temperature aerobic composting strain and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1965817A2 (en) * | 2005-12-20 | 2008-09-10 | Johnson and Johnson Consumer Companies, Inc. | Compositions containing cotinus coggygria extract and use thereof in treating wounds |
| US20100040707A1 (en) * | 2004-10-26 | 2010-02-18 | Violetta Iotsova Stone | Ingestible compositions containing extracts |
| CN102732443A (en) * | 2012-01-13 | 2012-10-17 | 广西壮族自治区农业科学院微生物研究所 | Sugarcane endogenous nitrogen-fixing Pantoea bacteria and application thereof |
| CN103981137A (en) * | 2014-05-23 | 2014-08-13 | 北京林业大学 | Bacterial strain antagonistic to pathogenic bacteria of jujube fruit shrink disease and application of bacterial strain |
| CN104017748A (en) * | 2014-03-21 | 2014-09-03 | 枣庄学院 | Endogeny biocontrol strain as well as preparation method and application of biocontrol bacterial agent prepared from endogeny biocontrol strain |
| CN104164394A (en) * | 2014-08-04 | 2014-11-26 | 北京林业大学 | Antagonistic phytopathogen strain and application thereof |
| CN104877926A (en) * | 2014-09-01 | 2015-09-02 | 北京林业大学 | Anti-Cotinus coggygria oxysporum strain and use thereof |
| CN106434490A (en) * | 2016-11-23 | 2017-02-22 | 吉林农业大学 | Ginseng bacterium TY15-2 with effects of disease prevention and growth promotion and application thereof |
-
2018
- 2018-05-15 CN CN201810463490.7A patent/CN110468060B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100040707A1 (en) * | 2004-10-26 | 2010-02-18 | Violetta Iotsova Stone | Ingestible compositions containing extracts |
| EP1965817A2 (en) * | 2005-12-20 | 2008-09-10 | Johnson and Johnson Consumer Companies, Inc. | Compositions containing cotinus coggygria extract and use thereof in treating wounds |
| CN102732443A (en) * | 2012-01-13 | 2012-10-17 | 广西壮族自治区农业科学院微生物研究所 | Sugarcane endogenous nitrogen-fixing Pantoea bacteria and application thereof |
| CN104017748A (en) * | 2014-03-21 | 2014-09-03 | 枣庄学院 | Endogeny biocontrol strain as well as preparation method and application of biocontrol bacterial agent prepared from endogeny biocontrol strain |
| CN103981137A (en) * | 2014-05-23 | 2014-08-13 | 北京林业大学 | Bacterial strain antagonistic to pathogenic bacteria of jujube fruit shrink disease and application of bacterial strain |
| CN104164394A (en) * | 2014-08-04 | 2014-11-26 | 北京林业大学 | Antagonistic phytopathogen strain and application thereof |
| CN104877926A (en) * | 2014-09-01 | 2015-09-02 | 北京林业大学 | Anti-Cotinus coggygria oxysporum strain and use thereof |
| CN106434490A (en) * | 2016-11-23 | 2017-02-22 | 吉林农业大学 | Ginseng bacterium TY15-2 with effects of disease prevention and growth promotion and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| C.NUNES ET AL.: "Post-harvest biological control by Pantoea agglomerans (CPA-2) on Golden Delicious apples", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| 周江鸿等: "16种园林植物粗提物对黄栌枯萎病菌的抑制作用", 《园林科技》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679881A (en) * | 2019-01-31 | 2019-04-26 | 湖北工程学院 | A kind of general bacterium of pineapple and its microbial inoculum and application |
| CN112760274A (en) * | 2021-04-07 | 2021-05-07 | 中国科学院上海高等研究院 | Organic solid waste high-temperature aerobic composting strain and application thereof |
| CN112760274B (en) * | 2021-04-07 | 2021-07-06 | 中国科学院上海高等研究院 | Organic solid waste high-temperature aerobic composting strain and application thereof |
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