CN105087428A - Pseudomonas aeruginosa and application thereof - Google Patents
Pseudomonas aeruginosa and application thereof Download PDFInfo
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- CN105087428A CN105087428A CN201510400702.3A CN201510400702A CN105087428A CN 105087428 A CN105087428 A CN 105087428A CN 201510400702 A CN201510400702 A CN 201510400702A CN 105087428 A CN105087428 A CN 105087428A
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Abstract
The invention provides pseudomonas aeruginosa. A classification name of the pseudomonas aeruginosa is pseudomonas aeruginosa T1-3, the pseudomonas aeruginosa is preserved in the Common Microorganism Center of the China Committee for Culture Collection of Microorganisms, the address is the Institution of Microbiology of the Chinese Academy of Sciences in the No.3 yard of the No.1 Beichen West Road in the Chaoyang district of Beijing, the postal code is 100101, a preservation number is CGMCC No.10433, and a preservation date is January 23, 2015. The pseudomonas aeruginosa has the advantages that excellent prevention and treatment effects can be realized by the pseudomonas aeruginosa for diversified diseases of plants such as corn, cow peas, chili, cabbage and leek, and the pseudomonas aeruginosa has wide antibacterial spectra; the pseudomonas aeruginosa is high in reproduction speed and stress resistance and favorable for production application, and can be artificially cultivated, and cultivation conditions are simple; the pseudomonas aeruginosa is free of pathogenicity to people and livestock, is safe and effective and accordingly has an excellent development prospect.
Description
Technical field
The present invention relates to microorganism field, particularly a kind of Pseudomonas aeruginosa, also relate to the application of this Pseudomonas aeruginosa in controlling plant diseases.
Background technology
Fungi is the important pathogen causing Plant diseases, and after it infects host, root of the crop, stem, leaf, flower, fruit and seed can produce variable color, necrosis, rot, wilt or lopsided symptom.The corps diseases such as current corn, cowpea and capital green onion mainly still rely on chemical bactericide to prevent and treat, these medicaments have potential harm to the health of environment and people on the one hand, the resistance of pathogenic fungi can be caused to strengthen on the one hand, therefore, the meter that fungal disease is not permanent environmental protection prevented and treated by single dependence chemical bactericide.
Antagonistic bacterium can produce the growth of a series of meta-bolites suppression pathogenic bacteria by secretion, as bacteriocin, proteolytic enzyme, organic acid, chitinase and catalase etc.Pseudomonas, as a kind of biocontrol microorganisms, is extensively active in plant rhizosphere, is the important component part of plant growth-promoting rhizobacteria (PlantGrowth-PromotingRhizobacteria, PGPR).
Research shows, wherein Pseudomonas aeruginosa (Pseudomonasaeruginosa), pseudomonas putida (Pseudomonasputida) and Pseudomonas fluorescens (Pseudomonasfluorescens) etc. can produce active substance or meta-bolites etc. suppress or hinder pathogenic fungi grow, mainly contain antibiotics, relevant enzymes, addicted to iron element, lipopeptid class etc.The various active materials such as and Pseudomonas aeruginosa is as the one of pseudomonas, it can produce phenazine-1-carboxylic acid, gamboge pyocyanin rhzomorph, inhibited to various plants pathogenic bacterias such as rice sheath blight disease, sclerotinia rot of colza, Phytophthora capsici diseases.And in shift of crops obstacle or continuous cropping phenomenon, pathogenic fungi is an one major reason, and therefore antagonistic bacterium also has important DEVELOPMENT PROSPECT in shift of crops obstacle or continuous cropping.
Continuous enhancing along with people's environmental consciousness and the demand to non-polluted farm product increase, and the biological control of Plant diseases is extremely urgent, and antagonistic bacterium has widely distributed, One's name is legion, and the mode of action is various, safety non-pollution and receiving much concern.Therefore, have in this research and have the comparatively wide T1-3 bacterial strain of spectrum that suppresses to have wider application prospect in the diseases such as corn multiple diseases, asparagus bean rust and capital rust of onion to various plants pathogenic bacteria, this work has great importance
Summary of the invention
Goal of the invention: the first object of the present invention is to provide a kind of Pseudomonas aeruginosa leaf blight of corn, Helminthosporium carbonum, Causal Organism of Maize Basal Stalk, maize sheath blight, cabbage rust, asparagus bean rust, Folium Capsici corruption, capital rust of onion and capital green onion leaf spot fungi to antagonistic action.
The second object of the present invention is to provide the application of a kind of Pseudomonas aeruginosa in control of maize leaf blight, Helminthosporium carbonum, Causal Organism of Maize Basal Stalk, maize sheath blight, cabbage leaf rot, asparagus bean rust, Folium Capsici corruption, capital rust of onion and capital green onion leaf spot fungi Plant diseases.
Technical scheme: in order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of Pseudomonas aeruginosa, its Classification And Nomenclature is Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode is 100101, deposit number is: CGMCCNo.10433, and preservation date is on 01 23rd, 2015.
The 16SrDNA sequence of Pseudomonas aeruginosa is as shown in SEQIDNo.1.By surveyed 16SrDNA sequence by BLAST comparison, identify that it is Pseudomonas aeruginosa (Pseudomonasaeruginosa).
Described Pseudomonas aeruginosa is the application in control of maize leaf blight.
Described Pseudomonas aeruginosa is the application in control of maize Northern leaf spot.
Described Pseudomonas aeruginosa is the application in control of maize base rot disease.
Described Pseudomonas aeruginosa is the application in control of maize banded sclerotial blight.
Described Pseudomonas aeruginosa is the application in control cabbage leaf rot.
Described Pseudomonas aeruginosa is the application in control asparagus bean rust.
Described Pseudomonas aeruginosa is the application in control Folium Capsici maize ear rot.
Described Pseudomonas aeruginosa is the application in the rust of onion of control capital.
Described Pseudomonas aeruginosa is the application in the green onion leaf spot of control capital.
Beneficial effect: Pseudomonas aeruginosa provided by the invention has good prevention effect to plurality of plant diseases such as corn, cowpea, capsicum, cabbage and capital green onions, and antimicrobial spectrum is wide; This bacterium reproduction speed is fast, can artificial culture, and culture condition is simple, and strong stress resistance, is beneficial to production application; This bacterium is to people and animals' no pathogenicity, safe and effective, has good DEVELOPMENT PROSPECT.
Specifically, the present invention, relative to prior art, has following outstanding advantage:
(1) Pseudomonas aeruginosa provided by the invention (Pseudomonasaeruginosa) T1-3 microbial inoculum suppresses spectrum comparatively wide, has more extensively suppress spectrum to disease, pepper anthracnose, cabbage leaf rot, asparagus bean rust, capital rust of onion and Leaf blotch pathogeny such as maize sheath blight, leaf blight, Northern leaf spot, leaf are withered; Compensate for the deficiency that chemical agent preventive effect is not good enough, the integrated control for this type of fungal disease adds a kind of means;
(2) Pseudomonas aeruginosa of the present invention (Pseudomonasaeruginosa) T1-3 microbial inoculum is biotechnological formulation, and bacterial strain itself is separated the Rhizosphere Soil from corn, therefore its use can not bring a series of environmental problem, is conducive to the No-harmful apple orchard of seeding corn and other crops;
(3) in shift of crops obstacle or continuous cropping phenomenon, fungal pathogen becomes many, quantity change is greatly an one major reason, and therefore antagonistic bacterium also has important DEVELOPMENT PROSPECT in shift of crops obstacle or continuous cropping.
Accompanying drawing explanation
Fig. 1 is that Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3 is respectively to the antagonistic experiment result of Exserohilum turcicum disease (A), rotten bacterium disease (B) of Folium Capsici, corn brown foot rot bacterium disease (C), asparagus bean rust (D).
Fig. 2 is Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3 physiological and biochemical analysis result.
Fig. 3 is cultivation results in control group and treatment group liquid medium within.
Fig. 4 is cultivation results during control group and treatment group are cultivated on Ma Dingshi plate culture medium.
Embodiment
The separation screening of embodiment 1 pseudomonas aeruginosa strains
1. substratum:
Potato dextrose agar (PDA substratum): potato 200g, glucose 20g, agar 15-20g, natural PH, is settled to 1L.
Beef-protein medium (LB substratum): Tryptones 10g, yeast leaching powder 5g, sodium-chlor 10g, pH partial neutral, be settled to 1L.
2. the separation screening of pseudomonas aeruginosa strains
A) take leaf blight of corn morbidity compared with the rhizosphere soil 10g of the healthy milpa not having this disease to occur near critical regions, add in 90ml sterilized water, obtain 10
-1diluent, 130r/min in 30 DEG C of shaking tables, cultivates 2-3h, continues to add sterilized water dilution, obtains 10 respectively
-5, 10
-6, 10
-7, 10
-8diluent, get the above-mentioned diluent of 200ml respectively and be coated with on LB flat board, 30 DEG C cultivate 48h, picking list colony inoculation is preserved in LB culture medium slant;
B) get onesize bacterium dish be inoculated in the dull and stereotyped central authorities of PDA by activating to beat in the punch tool of the fungal disease diameter 5mm of PDA flat board, after being down to 24h, steps A by LB slant activation) inoculation that obtains is in the side of 90mm flat board, cultivate 3-5d for 28 DEG C, observe bacteriostasis, and record the width of antibacterial band; With the PDA flat board of aseptic inoculated fungi in contrast, preliminary screening goes out to have the bacterial strain of Antagonistic Fungus; Multiple sieve is carried out again by same procedure, obtain Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3, the significant Pseudomonas aeruginosa of the antagonistic effect screened (Pseudomonasaeruginosa) T1-3 carries out antagonistic effect (see Fig. 1) for other fungal pathogens again, carries out antagonistic effect assessment.As shown in Figure 1, in Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3 target pathogenic bacteria almost without growth, Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3 has significant restraining effect to the leaf blight of corn, Helminthosporium carbonum, Causal Organism of Maize Basal Stalk, maize sheath blight, cabbage leaf rot, asparagus bean rust, Folium Capsici maize ear rot, capital rust of onion, capital green onion Leaf blotch pathogeny.
Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3 all has restraining effect in various degree to Different Kinds of Pathogens fungi, and concrete outcome is as table 1.
Table 1T1-3 is to Different Kinds of Pathogens fungi restraining effect
The physiologically active feature of Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3 is as follows:
Its bacterium colony on LB substratum is rounded, and surface has little point-like protruding, surface wettability, cultivate early stage be yellow-green colour, the later stage is sorrel, generation soluble pigment.
Its bacterium colony on PDA substratum is that projection is uneven shape, surface wettability, surface irregularity and the smooth of the edge, faint yellow, and without metalluster, incubation time increases and produces blackish green, has soluble pigment to produce.
It can grow at 41 DEG C, and does not grow at 4 DEG C.
C) entrust Shanghai Sheng Gong biotechnology limited-liability company to carry out 16SrDNA order-checking Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3 bacterial strain, in NCBI network address, carry out BLST comparison.Result display reaches 99% with the 16SrDNA sequence similarity of the Pseudomonasaeruginosa of accession number GB_KJ819583, in conjunction with morphological specificity and the physiological and biochemical analysis of this bacterial strain, be Pseudomonas aeruginosa (Pseudomonasaeruginosa) by this identification of strains.
D) examination of Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3 Physiology and biochemistry is analyzed, and the results are shown in Table 2 and Fig. 2.It can produce urase, protein powder enzyme, amylase and addicted to iron element etc.
The physiological and biochemical test result of table 2T1-3 bacterial strain
Note :+be positive ,-be negative
Embodiment 2T1-3 bacterial strain affects the former bacteria growing of asparagus bean rust under liquid culture
Liquid nutrient medium preparation method: Semen Maydis powder 10g, analysis for soybean powder 5g, glucose 5g, peptone 2g, NaCl1g, K
2hPO
41g, MgS0
47H
2o0.2g, is settled to 1L, natural pH.
Ma Dingshi plate culture medium: peptone 5g, glucose 10g, K
2hPO
41g, MgS0
47H
2o0.5g, is settled to 1L, natural pH, adds 1% rose-bengal aqueous solution 3.3mL.During use, every 100mL substratum adds 1% streptomycin solution 0.3mL, makes plate culture medium.
Test method:
(1) cultivate in liquid nutrient medium: Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3 is inoculated in liquid nutrient medium, 30 DEG C, shaking speed 150r/min, 48h; Control group inoculates 3 asparagus bean rust bacterium dish, treatment group Simultaneous vaccination 3 asparagus bean rust bacterium dish and the liquid medium 100uL comprising Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3; The results are shown in Figure 3;
Liquid antagonistic effect shows: normal growth in asparagus bean rust bacterium liquid medium within, growth 2-3d hyphae length is constantly increased to thick, and after adding Antagonistic Fungi Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3 bacterium liquid, mycelia obviously reduces asparagus bean rust, and nutrient solution is still comparatively clarified.
(2) Ma Dingshi plate culture medium is cultivated: control group and treatment group are inoculated in different 200mL triangular flask liquid nutrient mediums respectively, 30 DEG C, 150r/min cultivates 48-72h, carries out gradient dilution respectively and coats 10
-2, 10
-3, 10
-428 DEG C of 48-72h on Ma Dingshi plate culture medium, observe fungal growth situation; The results are shown in Figure 4.
Ma Dingshi substratum is cultivated, do not add the asparagus bean rust pathogenic bacteria raised growth of Antagonistic Fungi, and after having added Antagonistic Fungi, Antagonistic Fungi amount reproduction, flat board does not have normal mycelia grow.Illustrate under this liquid culture condi, Antagonistic Fungi T1-3 can suppress the mycelial growth of asparagus bean rust pathogenic bacteria very well.
The preparation of embodiment 3T1-3 bacterial strain microbial inoculum
Green pseudomonas (Pseudomonasaeruginosa) T1-3 is inoculated in LB liquid nutrient medium, and at 30 DEG C, under 130r/min, 24h is cultivated in concussion, forms seed liquor.
Again seed liquor is inoculated into by the volume ratio of 1:400 in the culture material in fermentor tank and carries out fermentative production, the groundwork parameter of fermentor tank is: temperature: 30-35 DEG C, stirring velocity: 130r/min, pH:6.8-7.2, dissolved oxygen: 30%, tank pressure: 0.04MPa.Stop fermentation after 24 hours, the then centrifugal 10min of 6000r/min, microbial inoculum is made in the dilution of taking precipitate sterilized water.Detect in microbial inoculum the concentration of viable bacteria be 1 × 10
9-1 × 10
10cFU/mL.
The microbial inoculum made may be used for the biological control to diseases such as corn Common Diseases and asparagus bean rusts.
Claims (10)
1. a Pseudomonas aeruginosa, its Classification And Nomenclature is Pseudomonas aeruginosa (Pseudomonasaeruginosa) T1-3, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode is 100101, deposit number is: CGMCCNo.10433, and preservation date is on 01 23rd, 2015.
2. the application of Pseudomonas aeruginosa according to claim 1 in control of maize leaf blight.
3. the application of Pseudomonas aeruginosa according to claim 1 in control of maize Northern leaf spot.
4. the application of Pseudomonas aeruginosa according to claim 1 in control of maize base rot disease.
5. the application of Pseudomonas aeruginosa according to claim 1 in control of maize banded sclerotial blight.
6. the application of Pseudomonas aeruginosa according to claim 1 in control cabbage leaf rot.
7. the application of Pseudomonas aeruginosa according to claim 1 in control asparagus bean rust.
8. the application of Pseudomonas aeruginosa according to claim 1 in control Folium Capsici maize ear rot.
9. the application of Pseudomonas aeruginosa according to claim 1 in the rust of onion of control capital.
10. the application of Pseudomonas aeruginosa according to claim 1 in the green onion leaf spot of control capital.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106577058A (en) * | 2016-12-15 | 2017-04-26 | 隆回田源农业高科技发展有限公司 | Pesticide application method capable of reducing longya lilium successive cropping obstacle |
| CN113943678A (en) * | 2021-11-05 | 2022-01-18 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Pseudomonas aeruginosa, microbial seedling substrate prepared from pseudomonas aeruginosa and application of microbial seedling substrate |
| CN116555068A (en) * | 2022-12-16 | 2023-08-08 | 安徽农业大学 | Endophytic pseudomonas aeruginosa HB-10 and application thereof |
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| CN104498387A (en) * | 2014-11-25 | 2015-04-08 | 辽宁大学 | Pseudomonas aeruginosa and its application |
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| CN104498387A (en) * | 2014-11-25 | 2015-04-08 | 辽宁大学 | Pseudomonas aeruginosa and its application |
Non-Patent Citations (2)
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| SAITEKHA等: "Characterization of multiple plant growth promotion traits of Pseudomonas aeruginosa FP6,a potential stress tolerant biocontrol agent", 《ANNUALS OF BIOLOGICAL RESEARCH》 * |
| 周莲等: "高产申嗪霉素和吩嗪-1-酰胺的水稻根际铜绿假单胞菌PA1201分离、鉴定与应用潜力", 《微生物学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106577058A (en) * | 2016-12-15 | 2017-04-26 | 隆回田源农业高科技发展有限公司 | Pesticide application method capable of reducing longya lilium successive cropping obstacle |
| CN113943678A (en) * | 2021-11-05 | 2022-01-18 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Pseudomonas aeruginosa, microbial seedling substrate prepared from pseudomonas aeruginosa and application of microbial seedling substrate |
| CN113943678B (en) * | 2021-11-05 | 2023-03-21 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Pseudomonas aeruginosa, microorganism seedling culture substrate prepared from pseudomonas aeruginosa and application of microorganism seedling culture substrate |
| CN116555068A (en) * | 2022-12-16 | 2023-08-08 | 安徽农业大学 | Endophytic pseudomonas aeruginosa HB-10 and application thereof |
| CN116555068B (en) * | 2022-12-16 | 2024-01-16 | 安徽农业大学 | Endophytic pseudomonas aeruginosa HB-10 and application thereof |
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