CN110079470A - One plant of pseudomonad with bacteriostatic activity - Google Patents
One plant of pseudomonad with bacteriostatic activity Download PDFInfo
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- CN110079470A CN110079470A CN201910160005.3A CN201910160005A CN110079470A CN 110079470 A CN110079470 A CN 110079470A CN 201910160005 A CN201910160005 A CN 201910160005A CN 110079470 A CN110079470 A CN 110079470A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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Abstract
The invention discloses one plant of pseudomonad with bacteriostatic activity, for the Pseudomonas in pseudomonadaceae (Pseudomonadaceae) Mo Shi pseudomonas (Pseudomonas moselii), deposit number is CCTCC NO:M2019081.The bacteriostatic activity of Mo Shi pseudomonad LWB10 of the invention is strong; its metabolite equally has bacteriostatic activity; it can be applied in prevention and treatment Agrobacterium tumefaciems, preparation Agrobacterium tumefaciems antibacterials; simultaneously can fermenting and producing obtain its metabolite, provide strong foundation for large-scale promotion application biocontrol microorganisms.
Description
Technical field
The present invention relates to a kind of pseudomonads, and in particular to the pseudomonad with bacteriostatic activity.
Background technique
Agrobacterium tumefaciems (Agrobacterium tumefaciens) is a kind of important plant pathogenetic bacteria, it is posted
Main range is very extensive, can infect more than 138 section 1193 such as peach, cherry, Lee, apricot, pears and apple plant.The bacterium is from plant
Wound intrusion after, form knob on rhizome, lateral root or limb, hinder transport of the root system to moisture and nutriment,
The development for influencing root, causes nutritional deficiency, tree vigo(u)r weak, or even dead tree occurs.Oriental cherry Breeding base in Ningbo area, root
Carninomatosis is the important disease of one kind on oriental cherry, causes very huge economic loss to the breeding of oriental cherry.
For many years, since the inspection and quarantine work of oriental cherry nursery stock root knot is not kept up with, root knot is in the big face in various regions
Product occurs.With the growth of planting time, root knot occurrence degree enhances year by year in same nursery；Due to various regions oriental cherry nursery stock
Frequently, root knot is propagated also with the circulation of nursery stock, more seriously for circulation.Investigation discovery early period, Ningbo City's oriental cherry
Main Cultivation area average attack rate retransmits block plant illness rate and is even as high as 92% (Wang Zhilong etc., 2014) up to 30%.It is raw
On producing before the onset of root knot, using lime sulfur root dipping or with the methods of streptomycin sulphate pouring root, have centainly to root knot
Prevention effect.But after the onset, this method preventive effect is poor.By soil-fumigating, Agrobacterium tumefaciems in disease ground can be reduced
Quantity, reduce the probability of Infected with Pathogenic Fungi.But since soil-fumigating not only kills pathogenic bacteria, while also killing soil
In some other antagonistic microbe, may cause crown gall palindromia (Yakabe et al., 2010).
In the prevention and treatment to fruit diseases, reported using antagonistic microbe prevention and treatment fruit diseases existing research.Liu Ting equal part
From to one plant of streptomyces lydicus strains A 102, which can synthesize a kind of activated product, when the concentration of activated product is greater than
When 30mg/L, there are stronger inhibitory activity, inhibiting rate to grape grey mould germ, jujube tree mould germ and Monilinia fructicola
It can achieve 100%.A kind of bacillus AiL3 being separated to out of mangrove can synthetic antimicrobial albumen, passing through influences thallus
It is metabolized and the growth of Yi Mango anthrax-bacilus.To in the biological control of fruit root knot disease, Australian scientist is isolated to
One plant of agrobacterium rhizogenes K84, the bacterial strain and its transformation bacterial strain K1026, WJK84-1 can be successfully used in the kernel approaches such as peach
Fruit root knot disease prevention and treatment (Kerr, 1980；Copping, 2001；Wang Guanlin etc., 2004).Flower bud of height et al. is isolated
The Alcaligenes faecalis 51-A and 51-B of two plants of peach rhizospheres, both bacterium have significant suppression to Agrobacterium tumefaciems ATCC 23308T
Production is used.Inoculation experiments show to infiltrate Antagonistic Fungi bacteria suspension on indicator plant tomato in advance, can be with to the preventive effect of root knot
Reach 86% or more.And above-mentioned Antagonistic Fungi and its metabolite with antibacterial action, large-scale application is also failed at present in life
It produces.Therefore, screening, which obtains new biocontrol microorganisms resource, has important practice significance to the prevention and control of Ningbo locality root knot.
Summary of the invention
In order to solve the above technical problems, the present invention provides a pseudomonas, with strong bacteriostatic activity, Neng Gouming
It is aobvious to inhibit Agrobacterium tumefaciems, for biological control and develops new antibacterials foundation is provided.
The technical solution of the present invention is to provide a kind of pseudomonads with bacteriostatic activity, and the Pseudomonas is in pseudomonadaceae
(Pseudomonadaceae) Mo Shi pseudomonas (Pseudomonas moselii), name are as follows: Pseudomonas
moselii LWB10.It is preserved in China typical culture collection center, preservation address is Wuhan University, Wuhan City, preservation date
For on January 24th, 2019, deposit number was CCTCC NO:M2019081, hereinafter referred to as Mo Shi pseudomonad LWB10.
Mo Shi pseudomonad LWB10 of the invention is gramnegative bacterium, and aerobic bacteria, cell size is (0.5-
0.8) μ m (1.5-1.8) μm has the raw clump flagellum in end, as shown in Figure 1.
The 16S rDNA sequence length of pseudomonad LWB10 of the present invention is 1407bp, and sequence is as shown in SEQ ID NO:1.
The advantages of the present invention: the bacteriostatic activity of Mo Shi pseudomonad LWB10 of the invention is strong, and metabolism produces
Object equally has bacteriostatic activity, can be applied in prevention and treatment Agrobacterium tumefaciems, preparation Agrobacterium tumefaciems antibacterials, together
When can fermenting and producing obtain its metabolite, provide strong foundation for large-scale promotion application biocontrol microorganisms.
Detailed description of the invention
Fig. 1 is the electron microscopic picture of pseudomonad LWB10 of the present invention.
Fig. 2 is inhibition zone picture of the pseudomonad LWB10 of the present invention to Agrobacterium tumefaciems C58.
Fig. 3 is the phylogenetic tree of pseudomonad LWB10 of the present invention.
Specific embodiment
The invention will be further described With reference to embodiment.
The present invention provides a pseudomonas LWB10, and it is false single to belong to pseudomonadaceae (Pseudomonadaceae) Mo Shi
Born of the same parents Pseudomonas (Pseudomonas moselii), is deposited in China typical culture collection center (CCTCC), and preservation address is military
Wuhan University, Chinese city, the deposit date is on January 24th, 2019, deposit number was CCTCC NO:M2019081, the present invention by its
It is named as Pseudomonas moselii LWB10.
Embodiment 1
Mo Shi pseudomonad LWB10 Gram-negative of the present invention, aerobic bacteria.Catalase test is positive, oxidation
Enzyme test is negative, and mobility test result is with motility.The bacterium (contains 5g albumen in YEB culture medium in every liter of culture medium
Peptone, 5g sucrose, 1g yeast extract, 5g beef extract, 10g agar powder, pH are adjusted to 7.0-7.2,121 DEG C of high pressure sterilizations
20min.After sterilizing, the 1M MgSO equally to sterilize is added into culture medium42ml；Also may be selected to be added to beef extract and
LB, KB culture medium of peptone) on well-grown, can grow on Mai Kangkai culture medium, be grown on blood plate normal, be formed
The flat wet bacterium colony of canescence, bacterium colony is smooth, neat in edge, no haemolysis.Optimum growth temperature is 28 DEG C, cannot be at 4 DEG C and 42
DEG C growth.Culture can produce a large amount of yellow pigments at 22-28 DEG C, and glucose oxidative fermentation tests (OF test) positive, hydrolysis
DNA is negative, and arginine dihydrolase is positive, nitrate reduction negative, and acetamide hydrolysis is negative, xylose, mannitol, maltose
It is positive to produce acid.
Embodiment 2
The separation process of Mo Shi pseudomonad LWB10 of the present invention is as follows: selecting the oriental cherry plant infected with Agrobacterium tumefaciems
(the present embodiment is locality to dig downwards in Ningbo City, Zhejiang Province Yinzhou District Ningbo City College of Vocational Technology campus along rhizosphere
Pick, takes the soil about 5g or so apart from surface soil 10-20cm, in sterile 50ml centrifuge tube, with 10ml sterile distilled water
It impregnates, is placed on turbine mixer and is uniformly mixed, the microorganism in soil is sufficiently discharged.It is then allowed to stand 10min, super-clean bench
It is middle to draw a small amount of upper liquid progress gradient dilution, setting 101-105Five different gradients, each gradient draw 50ul liquid
It is uniformly applied on 9cm YEB solid plate, 28 DEG C are incubated overnight.Second day, picking was without single bacterium on the culture dish of fungal contamination
It falls, is transferred in liquid YEB culture medium and is cultivated to get to different soil bacteria single colonies, be used for following antagonistic effect
Qualification process.
Soil bacteria identifies the antagonistic effect of Agrobacterium tumefaciems: by having seen whether inhibition zone generation, largely sieving
The antagonistic bacterium of Agrobacterium tumefaciems in soil is selected, inhibition zone test operation is as follows.15ml YEB solid medium is poured into sterile
In culture dish, drying is stand-by.It is 1.0 that 1ml OD600 value is added about in 60 DEG C of solid YEB culture medium to 10ml temperature
Agrobacterium tumefaciems bacterium solution, be uniformly mixed.The culture medium for being mixed with bacterium solution is poured into ready culture dish above rapidly, is opened
Lid drying.The aseptic filter paper disk that diameter is 0.6cm is carefully placed in media surface with tweezers, draws 10 μ l OD600 values
It is central in filter paper for 1.0 different soils bacterial solution drop, with sterile ddH2O is as blank control.To Liquid Penetrant culture medium
Afterwards, culture medium is carefully moved to 28 DEG C of overnight incubations, the generation of second day observation inhibition zone simultaneously photographs to record, after culture 2 days,
Select antibacterial circle diameter to be furtherd investigate greater than the bacterium solution of 2cm as candidate Antagonistic Fungi, the bacterial strain LWB10 screened and
Inhibition zone after Agrobacterium tumefaciems C58 is co-cultured 2 days is as shown in Figure 2.
Embodiment 3
It screens obtained bacterial strain LWB10 and carries out Molecular Identification, follow the steps below: extracting the base of bacterium with broken wall method
Because of a group DNA, international Bacteria Identification universal primer F8 (5 '-AGAGTTTGATCCTGGCTCAG-3 ')/R1492 (5 '-is utilized
ACGGCTACCTTGTTACGACTT-3 ') PCR amplification is carried out to genomic DNA.Then it is verified and is expanded by agarose gel electrophoresis
Increase primer size and be tapped and recovered, recovery product carries out TA clone, is connected on pMD-18-T carrier, and then conversion is big
Enterobacteria TG1 competent cell after being incubated overnight, selects positive colony, the positive gram by antibiotic-screening and PCR amplification
The grand commission Shanghai Sangon Biological Engineering Technology And Service Co., Ltd after expanding culture is sequenced.
The 16S rDNA sequence length of bacterial strain LWB10 is 1407bp, and sequence is as shown in SEQ ID NO:1, by sequencing result
Homologous comparison is carried out in GenBank, then uses software building phylogenetic tree such as Fig. 3, to determine the race relation of bacterial strain,
It is an independent branch in pseudomonas from LWB10 of the present invention known to phylogenetic tree.Homology analysis the result shows that,
The homology highest of the 16S rDNA sequence of the bacterial strain and Mo Shi pseudomonad, similitude 99% pass through mass spectroscopy, warp
Database compares, and is Mo Shi pseudomonad, reflects in addition combined with morphological features, growth conditions, Physiology and biochemistry in embodiment 1
It is fixed as a result, and Mass Spectrometric Identification interpretation of result, determine that bacterial strain LWB10 belongs to pseudomonadaceae (Pseudomonadaceae) and rubs
Family name pseudomonas (Pseudomonas moselii).
Embodiment 4
The preparation of Mo Shi pseudomonad LWB10 tunning: isolated Mo Shi pseudomonad LWB10 is inoculated in
In YEB fluid nutrient medium, cultivated 3 days in 250rpm shaking table at 28 DEG C.After 3 days, bacterium solution is centrifuged under 10,000rpm
10min collects supernatant, then carries out gradation extraction, then the sterile distilled water pair with 1/10 volume with isometric ethyl acetate
Organic phase is extracted again.Next the organic phase being collected into is placed on Rotary Evaporators and is concentrated, is concentrated by several times
Afterwards, remaining 2ml solution in drying up completely on nitrogen evaporator.200ul chromatographically pure grade dimethyl is finally added into the test tube of drying
Sulfoxide (DMSO), vortex oscillation, until being completely dissolved to get the purification tunning of pseudomonad LWB10 is arrived.
The verifying of tunning bacteriostatic activity: the method for tunning bacteriostatic activity verifying is reflected with antagonistic effect in embodiment 2
Fixed process, difference is to change the bacterium solution on filter paper into extract tunning, after cultivating for 24 hours at 28 DEG C, sees
Fermentation material is examined to the bacteriostatic activity of Agrobacterium tumefaciems different strains, and measures inhibition zone size, the results are shown in Table 1:
The pseudomonad LWB10 of the present invention of table 1 counts the fungistatic effect of Agrobacterium tumefaciems
| Test strain | C58 | GV3101 | LAB4404 | EHA105 | C58C1 | NT1RE |
| LWB10 | +++ | +++ | +++ | +++ | +++ | +++ |
In table 1+indicate antibacterial circle diameter < 10mm, ++ indicate antibacterial circle diameter 10mm-20mm, +++ indicate that inhibition zone is straight
Diameter > 30mm.Listed Agrobacterium tumefaciens strain has been recorded open in the literature in table.
Above-mentioned fungistatic effect illustrates that Mo Shi pseudomonad LWB10 of the present invention is strong to the bacteriostatic activity of Agrobacterium tumefaciems, generation
Thanking also has a large amount of Substances in product, the biocontrol microorganisms as Agrobacterium tumefaciems, which have, deeply develops application value.
The present embodiments relate to the material arrived, reagent and experimental facilities, are to meet plant protection unless otherwise instructed
The commercial product in field.
The above is merely a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, under the premise of not departing from core of the invention technology, improvements and modifications can also be made, these improvements and modifications
It should belong to scope of patent protection of the invention.With any change in the comparable meaning and scope of claims of the present invention,
It is all considered as being included within the scope of the claims.
Sequence table
<110>Ningbo City College of Vocational Technology
<120>one plants of pseudomonads with bacteriostatic activity
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1407
<212> DNA
<213>Mo Shi Pseudomonas alba (Pseudomonas moselii)
<400> 2
acatgcagtc gagcggatga cgggagcttg ctccttgatt cagcggcgga cgggtgagta 60
atgcctagga atctgcctgg tagtggggga caacgtttcg aaaggaacgc taataccgca 120
tacgtcctac gggagaaagc aggggacctt cgggccttgc gctatcagat gagcctaggt 180
cggattagct agtaggtgag gtaatggctc acctaggcga cgatccgtaa ctggtctgag 240
aggatgatca gtcacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg 300
gggaatattg gacaatgggc gaaagcctga tccagccatg ccgcgtgtgt gaagaaggtc 360
ttcggattgt aaagcacttt aagttgggag gaagggcagt aagttaatac cttgctgttt 420
tgacgttacc gacagaataa gcaccggcta actctgtgcc agcagccgcg gtaatacaga 480
gggtgcaagc gttaatcgga attactgggc gtaaagcgcg cgtaggtggt tcgttaagtt 540
ggatgtgaaa gccccgggct caacctggga actgcatcca aaactggcga gctagagtat 600
ggtagagggt ggtggaattt cctgtgtagc ggtgaaatgc gtagatatag gaaggaacac 660
cagtggcgaa ggcgaccacc tggactgata ctgacactga ggtgcgaaag cgtggggagc 720
aaacaggatt agataccctg gtagtccacg ccgtaaacga tgtcaactag ccgttggaat 780
ccttgagatt ttagtggcgc agctaacgca ttaagttgac cgcctgggga gtacggccgc 840
aaggttaaaa ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 900
ttcgaagcaa cgcgaagaac cttaccaggc cttgacatgc agagaacttt ccagagatgg 960
attggtgcct tcgggaactc tgacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg 1020
agatgttggg ttaagtcccg taacgagcgc aacccttgtc cttagttacc agcacgtcat 1080
ggtgggcact ctaaggagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaag 1140
tcatcatggc ccttacggcc tgggctacac acgtgctaca atggtcggta cagagggttg 1200
ccaagccgcg aggtggagct aatctcacaa aaccgatcgt agtccggatc gcagtctgca 1260
actcgactgc gtgaagtcgg aatcgctagt aatcgcaaat cagaatgttg cggtgaatac 1320
gttcccgggc cttgtacaca ccgcccgtca caccatggga gtgggttgca ccagaagtag 1380
ctagtctaac ctcggaggac ggtacca 1407
Claims (10)
1. the pseudomonad with bacteriostatic activity, which is characterized in that deposit number is CCTCC NO:M2019081, is belonged to false single
Born of the same parents Cordycepps (Pseudomonadaceae) Mo Shi pseudomonas (Pseudomonas moselii).
2. as described in claim 1 with the pseudomonad of bacteriostatic activity, which is characterized in that it is gramnegative bacterium, it is good
Oxygen bacterium, cell size are (0.5-0.8) μ m (1.5-1.8) μm, have the raw clump flagellum in end, have motility.
3. as described in claim 1 with the pseudomonad of bacteriostatic activity, which is characterized in that catalase test is positive, oxygen
Change enzyme test to be negative, bacterium colony is that canescence is flat wet, and bacterium colony is smooth, neat in edge, no haemolysis；Optimum growth temperature is 28
DEG C, it cannot be grown at 4 DEG C and 42 DEG C, culture can produce a large amount of yellow pigments at 22-28 DEG C；Glucose oxidative fermentation test sun
Property, hydrolysis DNA is negative, and arginine dihydrolase is positive, nitrate reduction negative, and acetamide hydrolysis is negative, xylose, mannitol,
It is positive that maltose produces acid.
4. as described in claim 1 with the pseudomonad of bacteriostatic activity, which is characterized in that grown on YEB culture medium good
It is good, it can grow, be grown on blood plate normal on Mai Kangkai culture medium.
5. as described in claim 1 with the pseudomonad of bacteriostatic activity, which is characterized in that its 16S rDNA sequence length is
1407bp, nucleotide sequence is as shown in SEQ ID NO:1.
6. the tunning preparation method of the described in any item pseudomonads of Claims 1 to 5, which is characterized in that step includes:
(1) pseudomonad is inoculated in YEB fluid nutrient medium, shaking table culture 3 days at 28 DEG C；
(2) supernatant then is collected by centrifugation in bacterium solution；
(3) the isometric organic solvent of supernatant is extracted；
(4) organic phase being obtained by extraction is concentrated, dried.
7. the tunning preparation method of pseudomonad as claimed in claim 6, which is characterized in that the step (4) obtains
Dry product redissolve through dimethyl sulfoxide further to purify tunning；Shaking speed is 200- in the step (1)
250rpm；Parameter of noncentricity is to be centrifuged 5-15min under 8000-12000rpm in the step (2).
8. the tunning preparation method of pseudomonad as claimed in claim 6, which is characterized in that have in the step (3)
Solvent is ethyl acetate and is divided into multiple extraction, and further includes the sterile distilled water pair with 1/10 volume after ethyl acetate extraction
The step of organic phase is extracted again.
9. the described in any item pseudomonads of Claims 1 to 5 are in prevention and treatment Agrobacterium tumefaciems, preparation Agrobacterium tumefaciems antibacterials
In application.
10. the resulting tunning of the described in any item preparation methods of claim 6~8 is preventing and treating Agrobacterium tumefaciems, is preparing root
Application in cancer Agrobacterium antibacterials.
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Cited By (5)
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| CN111718864A (en) * | 2020-01-21 | 2020-09-29 | 武汉工程大学 | Rare earth leaching site soil indigenous high-efficiency denitrification strain pseudomonas flaviviridis K3 and pseudomonas morganii K17 and application thereof |
| CN111718864B (en) * | 2020-01-21 | 2022-05-10 | 武汉工程大学 | Rare earth leaching site soil indigenous high-efficiency denitrification strain pseudomonas flaviviridis K3 and pseudomonas morganii K17 and application thereof |
| CN113930363A (en) * | 2020-07-01 | 2022-01-14 | 山东五福生生态工程有限公司 | Pseudomonas chlororaphis orange subspecies and preparation and application of microbial agent thereof |
| CN113930363B (en) * | 2020-07-01 | 2023-03-24 | 山东五福生生态工程有限公司 | Pseudomonas chlororaphis orange subspecies and preparation and application of microbial agent thereof |
| CN114907985A (en) * | 2022-04-29 | 2022-08-16 | 华南农业大学 | Microbial preparation for improving saponin content of plant and application thereof |
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