CN103734196A - Application of Pseudomonas aeruginosa strain fermentation broth extract and validamycin compound - Google Patents
Application of Pseudomonas aeruginosa strain fermentation broth extract and validamycin compound Download PDFInfo
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- CN103734196A CN103734196A CN201410019061.2A CN201410019061A CN103734196A CN 103734196 A CN103734196 A CN 103734196A CN 201410019061 A CN201410019061 A CN 201410019061A CN 103734196 A CN103734196 A CN 103734196A
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Abstract
The invention belongs to the technical field of biopesticides, and in particular relates to an antagonistic bacteria-Pseudomonas aeruginosa SU8. The Pseudomonas aeruginosa SU8 is preserved in China Center for Type Culture Collection in May 7th, 2013, the preservation number is CCTCC NO:M2013178. Ethyl acetate extract of Pseudomonas aeruginosa SU8 fermentation broth is compounded with validamycin to achieve the synergism on preventing and controlling rice sheath blight disease, and the ethyl acetate extract has an inhibiting effect on Xanthomonas oryzae pv. Oryzicola and Xanthomonas axonopodis pv. Citri.
Description
The application is that application number is 201310250195.0, the applying date is on June 21st, 2013, and denomination of invention is dividing an application of a kind of pseudomonas aeruginosa strain.
Technical field
The invention belongs to biological pesticide technical field, particularly Antagonistic Fungi-pseudomonas aeruginosa (Pseudomonas aeruginosa) SU8 also relates to the application of this bacterial strain simultaneously.
Background technology
In agricultural production, often use the generation of chemical pesticide control crop pest.Although chemical pesticide has desirable control efficiency to crop pest, the negative issue being brought by chemical pesticide becomes increasingly conspicuous, for example: contaminated environment, destruction natural enemy, long-term use is also easily brought out pathogen and developed immunity to drugs.Secondly, the excessive chemical pesticide of using has not only increased drug cost, also can cause residue of pesticide.Rely on modern biological prevention, adopt Antagonistic Fungi to prevent and treat the very important effect of playing of crop pest, particularly utilize the active substance that Antagonistic Fungi produces to prevent and treat crop pest, there is safety, efficient, low toxicity, pollution-free, the cycle is short, be easy to research, be convenient to the advantages such as production, noresidue.
Early stage result of study shows: pseudomonad has larger potentiality to be exploited as a kind of Antagonistic Fungi.Pseudomonas aeruginosa (Pseudomonas aeruginosa) is again a kind of of pseudomonad, can produce phenazine-1-carboxylic acid, pyoluteorin, 2, the various active materials such as 4-diacetyl phloroglucinol, inhibited to various plants pathogens such as rice sheath blight disease, rice blast, black shank, sclerotinia rot of colza, Hybrid Bamboo top dries.Because this bacterium can produce multiple antibacterial substance, the antibacterial substance that different strains produces is different, same bacterial strain also can produce multiple antibacterial substance, and these antibacterial substances are inhibited to plant pathogenic fungi, but the compound of its extract and jinggangmeisu is to the rarely seen research of plant pathogenic fungi inhibitory action, and less to the research of plant pathogenetic bacteria, rarely seen its metabolite pyo is inhibited to bacterium, and other metabolites also have no report to the research of plant pathogenetic bacteria.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, provide a kind of pseudomonas aeruginosa SU8(Pseudomonas aeruginosa) SU8.
Pseudomonas aeruginosa SU8(Pseudomonas aeruginosa of the present invention) SU8, on May 7th, 2013 be deposited in Chinese Typical Representative culture collection center (address: Wuhan, China. Wuhan University), deposit number is CCTCC NO:M2013178.
The characteristic of bacterial strain SU8 of the present invention:
1. morphological feature
On beef extract-peptone agar medium, at temperature 28-30 ℃ of continuous culture 30h, by electric microscope scanning, bacterial strain SU8 is elongated rod shape, and thalline length is even, and general length is within the scope of 1-1.5 μ m, and paired or short catenation, is shown in Fig. 1 sometimes.Thalline after Gram’s staining takes on a red color, and indicating this bacterial strain is Gram-negative bacteria.
2. the feature of growth on various medium (temperature 28-30 ℃ cultivates 30h)
Potato glucose medium: bacterium colony is mountain range shape, moistening, rough surface the smooth of the edge, be faint yellow without metallic luster, long-time cultivate to produce under blackish green accumulation thing, uviol lamp have fluorescence.Soluble pigment has.
Beef-protein medium: bacterium colony is rounded, moistening, rough surface has little spot-like projections, be in early days yellow green, the later stage is under bronzing, uviol lamp without fluorescence.Soluble pigment has.
Gause I medium: bacterium colony is rounded, moistening, equal smooth, the water white transparency shape in surface and edge.Soluble pigment without.
KingShi medium: bacterium colony is mountain range shape, moistening, rough surface has little spot-like projections, the smooth of the edge, have verdigris color metallic luster, has fluorescence under uviol lamp.Soluble pigment has.
3. physiology feature
Can utilize D-wood sugar, D-Glucose, mannose, erythrose, arabitol, melezitose, glycerine, citrate, methyl red, can make gelatin liquefaction, lipolysis, nitrate reduction, oxidase positive, pyocyanin is positive, catalase is positive, hydrogen peroxide enzyme positive, ornithine decarboxylase is positive, but can not utilize lactose, Arabinose, L-rhamnose, inositol, galactose, D fructose, sucrose, raffinose, litmus milk, dextrin, maltose, sorbic acid sugar, salicin, aesculin, do not produce hydrogen sulphide, ammonia, indoles etc., belong to aerobic bacteria, can at 41 ℃, grow but can not be again growth at 4 ℃.
Reference literature < < common bacteria system identification handbook > >, cultivation proterties and physiological and biochemical property in conjunction with this bacterial strain SU8 on various medium, show that this bacterial strain meets the relevant identification mark of pseudomonas aeruginosa (P.aeruginosa).The pcr amplification product of bacterial strain SU816S rDNA is checked order, and sequence shows through BLAST comparison, reaches 99% with the autoploidy of pseudomonas aeruginosa strains P.aeruginosa SRDchr3 (EU714901) sequence.By systematic evolution tree, build, show that the false cell model bacterial strain of this bacterial strain SU8 and verdigris P.aeruginosa ATCC10145 gathers in same system evolutionary branching (see figure 2), autoploidy reaches 99%.In conjunction with this bacterial strain SU8 sequence analysis, morphological feature, cultural characteristic, physiology feature, judge that with < < common bacteria system identification handbook > > this bacterial strain belongs to pseudomonas aeruginosa and belongs to (P.aeruginosa), and called after pseudomonas aeruginosa (P.aeruginosa) SU8.
Bacterial strain SU8 of the present invention is pressed in 8-12% inoculum concentration access beef extract-peptone liquid nutrient medium, and magnetic agitation rotor speed is 100-120r/min, puts in 28-30 ℃ of shaken cultivation case and continuously ferments and cultivate 72-96h, can obtain zymotic fluid.Optimal conditions of fermentation is: inoculum concentration 10%, and rotating speed 115r/min, temperature is 29 ℃, time 85h.
The zymotic fluid of bacterial strain SU8 of the present invention is through ethyl acetate extraction and after concentrated, obtain ethyl acetate extract, after this extract and composite jinggangmycin, Rhizoctonia solani Kuhn is had to synergistic effect, and inhibited to plant pathogenetic bacterias such as xanthomonas oryzae pv. oryzicola and c itrus canker germs, for further developing novel pesticide, provide theoretical foundation, for controlling plant disease, provided fundamental basis.
Compared with prior art, the advantage that the present invention has is:
1, zymotechnique is simple, quick, method of operating is convenient.
2, the compound of the separated extract obtaining of the present invention and jinggangmeisu has synergistic effect to control Rhizoctonia solani Kuhn.
3, the separated acquisition of the present invention extract is inhibited to xanthomonas oryzae pv. oryzicola and c itrus canker germ, has expanded antimicrobial spectrum.
Accompanying drawing explanation
Fig. 1 is the electron-microscope scanning figure of bacterial strain SU8 of the present invention.
Fig. 2 is the phylogenetic tree of bacterial strain SU8 of the present invention.
Fig. 3 is that the ethyl acetate extract of bacterial strain of the present invention is to xanthomonas oryzae pv. oryzicola inhibitory action figure.Wherein: 1 represents ethyl acetate extract, and 2 represent ethyl acetate solvent.
Fig. 4 is that the ethyl acetate extract of bacterial strain fermentation liquor of the present invention is to c itrus canker germ inhibitory action figure.Wherein: 1 represents ethyl acetate extract, and 2 represent ethyl acetate solvent.
Embodiment
Below in conjunction with concrete test and embodiment, technical scheme of the present invention is described further, the percentage relating in test of the present invention and embodiment is mass percent.
The Rhizoctonia solani Kuhn of using in following examples (Rhizoctonia solani), xanthomonas oryzae pv. oryzicola (Xanthomonoasoryzaepv.oryzicola), c itrus canker germ (Xanthomonas Campestris pv.citri) are provided by Agricultural University Of Hunan's plant pathology laboratory.
The preparation of embodiment 1 bacterial strain of the present invention
Adopt isolation by dilution method to support altogether Tanaka from the rice duck of Liuyang City and screen Antagonistic Fungi.Accurately take 10g duck excrement and put into 90mL with the sterile water of bead, vibration 30min, gets suspension after standing 10min, by gradient concentration method, is diluted to 1 * 10
-7doubly, then dilution is coated on beef extract-peptone agar medium flat board and cultivates, obtain single bacterium colony.The single bacterium colony obtaining is made to suspension and made filter paper with sterile water, leave on the potato agar culture medium flat plate at central water Rhizoctonia solani Kuhn bacterium cake (5mm) bilateral symmetry 2cm place and cultivate, indicator bacteria bacterium colony covers with after flat board, picking has the bacterial clump of inhibition zone, purifying repeatedly, obtain single bacterium colony, be pseudomonas aeruginosa SU8 of the present invention.
Activation and the fermentation of example 2 bacterial strains of the present invention
Adopt oese by bacterial strain SU8 access beef extract-peptone slant medium (pH is 7.2-7.3 for beef extract 3-6g, peptone 8-10g, sodium chloride 4-5g, glucose 15-20g, water 1000mL) of the present invention, cultivate 24h at 28 ℃.By in 10% inoculum concentration access beef extract-peptone liquid nutrient medium, magnetic agitation rotor speed is 1000r/min, puts continuous culture 84h in 28-30 ℃ of shaken cultivation case, obtains zymotic fluid again.
The extraction of example 3 antibacterial substances
Get zymotic fluid and ethyl acetate and fully mix dipping extraction 48h with the ratio of 1:2, extract is inserted to Rotary Evaporators 40 ℃ of temperature, rotating speed 110r/min rotary evaporation, to paste, obtains antibacterial substance.
Example 4 antagonistic activities detect
The assay method of Antagonistic Fungus: c itrus canker germ is inoculated in to PDA solid culture medium central authorities, at 28 ℃, cultivate mycelia two side perforatings (φ=5mm) that newly growing up to after 24h, active substance to be measured (bacteriostatic extractive of mentioning in embodiment 3) is injected in a hole, in another side opening, inject corresponding solvent, every hole 50 μ L, and make blank with corresponding solvent, cultivate 24-48h for 28 ℃, observe antibacterial situation and inhibition zone size, each is processed 3 times and repeats.
The assay method of antagonistic bacterium: adopt plate punch method, by xanthomonas oryzae pv. oryzicola suspension (1 * 10
8cfu/mL) 60 μ L evenly coat NA flat board, then punching (φ=5mm), active substance to be measured (bacteriostatic extractive of mentioning in embodiment 3) is injected in a hole, in another side opening, inject corresponding solvent, every hole 50 μ L, and compare with corresponding solvent, 24-48h cultivated for 28 ℃, observe antibacterial situation and inhibition zone size, each is processed 3 times and repeats.
Result shows: this ethyl acetate extract has to xanthomonas oryzae pv. oryzicola and c itrus canker germ the activity of inhibition, and control solvent unrestraint is active, sees Fig. 3 and Fig. 4.
Example 5 ethyl acetate extracts and the toxicity test of composite jinggangmycin thing to Rhizoctonia solani Kuhn
Through prerun, determine each medicament valid density scope, each medicament is established respectively 5 dosage by active constituent content and is processed, and establishes corresponding solvent for contrast (water: ethyl acetate: acetone).With reference to < < farm-chemical indoor determination test rule bactericide > >, carry out, adopt mycelial growth rate method to measure the virulence of medicament to target fungus.Cultivate 72h and measure colony diameter by right-angled intersection method, calculate and respectively process mycelial growth inhibition rate, draw EC
50with the parameter such as virulence regression equation, according to Wadley method, calculate the different proportioning synergy ratios (SR) of two medicaments simultaneously, SR<0.5 is antagonism, and 0.5≤SR≤1.5 are synergy, and SR>1.5 is synergistic effect.
Result of the test shows: contrast is on Rhizoctonia solani Kuhn without impact, and compound is the EC of ethyl acetate extract and jinggangmeisu to the Toxicity Determination result of Rhizoctonia solani Kuhn
50be respectively 4120.17 μ g/mL and 404.81 μ g/mL, the virulence of ethyl acetate extract is only 0.1 times of jinggangmeisu, illustrates that the virulence of ethyl acetate extract is lower than jinggangmeisu; Ethyl acetate extract and jinggangmeisu are had to collaborative and synergistic effect after composite within the scope of 1-10 and 10-1, it is the most obvious that wherein ethyl acetate extract and jinggangmeisu are pressed 9:1 synergy, other proportionings 1:9,1:1,4:1 also have synergistic effect, 1:4 proportioning has synergy, sees the following form 1.
Table 1 ethyl acetate extract, jinggangmeisu and compound thereof the toxicity test to Rhizoctonia solani Kuhn
Claims (3)
1. the ethyl acetate extract of zymotic fluid of false unit cell (Pseudomonas aeruginosa) the SU8CCTCC NO.M2013178 of verdigris and the compound of the jinggangmeisu synergy application in control Rhizoctonia solani Kuhn.
2. application as claimed in claim 1, it is characterized in that, described preserving number is that the zymotic fluid of the pseudomonas aeruginosa strain SU8 of CCTCC NO.M2013178 is by 8-12% inoculum concentration access beef extract-peptone liquid nutrient medium by this bacterial strain, at 28-30 ℃, rotating speed, be that under 100-120r/min condition, fermented and cultured 72-96h makes.
3. application as claimed in claim 2, is characterized in that, described inoculum concentration is 10%, and rotating speed is 115r/min, and temperature is 29 ℃, and incubation time is 85h.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112522133A (en) * | 2020-11-09 | 2021-03-19 | 中国农业科学院农业资源与农业区划研究所 | Pseudomonas aeruginosa EZ-35, metabolite thereof and application thereof |
| CN112940979A (en) * | 2021-03-18 | 2021-06-11 | 深圳大学 | Bacterial strain, extract, nano pesticide and application and preparation method thereof |
| CN115039794A (en) * | 2022-06-30 | 2022-09-13 | 中国烟草总公司辽宁省公司 | Biocontrol microbial inoculum for preventing and treating tobacco target leaf spot disease as well as preparation method and application thereof |
-
2013
- 2013-06-21 CN CN201410019061.2A patent/CN103734196B/en not_active Expired - Fee Related
Non-Patent Citations (3)
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| 任小平等: "铜绿假单胞菌ZJ1999对水稻纹枯病的防治及其在水稻上的定殖", 《中国生物防治》 * |
| 苏品: "稻鸭种养生态系统抑制水稻纹枯病的发生流行规律及拮抗菌SU8生防潜力研究", 《中国博士学位论文全文数据库-农业科技辑》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112522133A (en) * | 2020-11-09 | 2021-03-19 | 中国农业科学院农业资源与农业区划研究所 | Pseudomonas aeruginosa EZ-35, metabolite thereof and application thereof |
| CN112522133B (en) * | 2020-11-09 | 2022-09-30 | 中国农业科学院农业资源与农业区划研究所 | Pseudomonas aeruginosa EZ-35, metabolite thereof and application thereof |
| CN112940979A (en) * | 2021-03-18 | 2021-06-11 | 深圳大学 | Bacterial strain, extract, nano pesticide and application and preparation method thereof |
| CN112940979B (en) * | 2021-03-18 | 2022-07-19 | 深圳大学 | Bacterial strain, extract, nano pesticide and application and preparation method thereof |
| CN115039794A (en) * | 2022-06-30 | 2022-09-13 | 中国烟草总公司辽宁省公司 | Biocontrol microbial inoculum for preventing and treating tobacco target leaf spot disease as well as preparation method and application thereof |
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