CN103704274B - Application of pseudomonas aeruginosa strain - Google Patents
Application of pseudomonas aeruginosa strain Download PDFInfo
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- CN103704274B CN103704274B CN201410019910.4A CN201410019910A CN103704274B CN 103704274 B CN103704274 B CN 103704274B CN 201410019910 A CN201410019910 A CN 201410019910A CN 103704274 B CN103704274 B CN 103704274B
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- pseudomonas aeruginosa
- ethyl acetate
- bacterial strain
- acetate extract
- extract
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- 241000589517 Pseudomonas aeruginosa Species 0.000 title claims abstract description 16
- 239000002024 ethyl acetate extract Substances 0.000 claims abstract description 16
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- 239000012530 fluid Substances 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 abstract description 19
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- 241000626572 Xanthomonas oryzae pv. oryzicola Species 0.000 abstract description 7
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- 238000000855 fermentation Methods 0.000 abstract description 5
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- 240000007594 Oryza sativa Species 0.000 abstract description 4
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to the technical field of biopesticides and in particular relates to antagonistic bacteria SU8 (Pseudomonas aeruginosa). The Pseudomonas aeruginosa has been preserved at China center for type culture collection in May 7th, 2013, and the preservation number is CCTCC NO: M2013178. An ethyl acetate extract in the SU8 fermentation broth is compounded with validamycin to achieve the synergetic effect on preventing rice sheath blight disease; the ethyl acetate extract has an inhibiting effect on Xanthomonas oryzae pv. oryzicola and X. axonopodis pv. Citri.
Description
The application is application number is 201310250195.0, the applying date is on June 21st, 2013, and denomination of invention is a kind of divisional application of P. aeruginosa bacterial 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, the normal generation using chemical pesticide control crop pest.Although chemical pesticide has desirable control efficiency to crop pest, the negative issue brought by chemical pesticide becomes increasingly conspicuous, such as: contaminated environment, destruction natural enemy, Long-Time Service also easily brings out pathogen and develops immunity to drugs.Secondly, excessive chemical pesticide of using not only increases drug cost, also can cause residue of pesticide.Rely on modern biotechnology Prevention Technique, what adopt Antagonistic Fungi to prevent and treat crop pest plays very important effect, the active substance particularly utilizing Antagonistic Fungi to produce prevents and treats crop pest, has 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 the one of pseudomonad, phenazine-1-carboxylic acid, pyoluteorin, 2 can be produced, 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 it is less to the research of plant pathogenetic bacteria, its metabolite pyo rarely seen is inhibited to bacterium, and the research of other metabolites to plant pathogenetic bacteria also has no report.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, provides a kind of pseudomonas aeruginosa (Pseudomonas aeruginosa) SU8.
Pseudomonas aeruginosa of the present invention (Pseudomonas aeruginosa) SU8, on May 7th, 2013 be deposited in China typical 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 DEG C 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 μm, and paired or short catenation, is shown in Fig. 1 sometimes.Thalline after Gram’s staining takes on a red color, and indicates this bacterial strain to be Gram-negative bacteria.
2. on various medium, grow the feature of (temperature 28-30 DEG C cultivates 30h)
Potato dextrose medium: bacterium colony is mountain range shape, moistening, rough surface the smooth of the edge, in faint yellow without metallic luster, long-time cultivate produce blackish green accumulation thing, uviol lamp under have fluorescence.Soluble pigment has.
Beef-protein medium: bacterium colony is rounded, moistening, rough surface has little spot-like projections, be unstressed configuration under bronzing, uviol lamp in yellow green, later stage in early days.Soluble pigment has.
Gause I medium: bacterium colony is rounded, moistening, surperficial and edge is all smooth, water white transparency shape.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
D-wood sugar can be utilized, D-Glucose, mannose, erythrose, arabitol, melezitose, glycerine, citrate, methyl red, gelatin liquefaction can be made, lipolysis, nitrate reduction, oxidase positive, pyocyanin is positive, catalase is positive, catalase positive, ornithine decarboxylase is positive, but can not lactose be utilized, 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, to grow at 41 DEG C but can not growth at 4 DEG C again.
Reference literature " common bacteria system identification handbook ", in conjunction with the cultural colony of this bacterial strain SU8 on various medium and physiological and biochemical property, shows the relevant identification mark that this bacterial strain meets pseudomonas aeruginosa (P.aeruginosa).Checked order by the pcr amplification product of bacterial strain SU816S rDNA, sequence, through BLAST comparison display, reaches 99% with the autoploidy of pseudomonas aeruginosa strains P.aeruginosa SRDchr3 (EU714901) sequence.Built by systematic evolution tree, show this bacterial strain SU8 and P. aeruginosa type strain 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 this bacterial strain belongs to P. aeruginosa Pseudomonas (P.aeruginosa) with " common bacteria system identification handbook ", and called after pseudomonas aeruginosa (P.aeruginosa) SU8.
Pressed by bacterial strain SU8 of the present invention in 8-12% inoculum concentration access beef extract-peptone liquid nutrient medium, magnetic agitation rotor speed is 100-120r/min, and putting in 28-30 DEG C of shaken cultivation case continuously ferments cultivates 72-96h, can obtain zymotic fluid.Optimal conditions of fermentation is: inoculum concentration 10%, rotating speed 115r/min, and temperature is 29 DEG C, time 85h.
The fermentation liquor extraction into ethyl acetate of bacterial strain SU8 of the present invention is also after concentrated, obtain ethyl acetate extract, after this extract and composite jinggangmycin, to Rhizoctonia solani Kuhn, there is synergistic effect, and it is inhibited to the plant pathogenetic bacteria such as xanthomonas oryzae pv. oryzicola and citrus processing, providing theoretical foundation for developing novel pesticide further, providing fundamental basis for controlling plant disease.
Compared with prior art, the advantage that the present invention has is:
1, zymotechnique is simple, quick, method of operating is convenient.
2, the present invention is separated the extract of acquisition and the compound of jinggangmeisu has synergistic effect to water prevention Rhizoctonia solani Kuhn.
3, the present invention be separated obtain extract to xanthomonas oryzae pv. oryzicola and citrus processing inhibited, expand 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 citrus processing inhibitory action figure.Wherein: 1 represents ethyl acetate extract, and 2 represent ethyl acetate solvent.
Embodiment
Be described further technical scheme of the present invention below in conjunction with concrete test and embodiment, the percentage related in test of the present invention and embodiment is mass percent.
The Rhizoctonia solani Kuhn (Rhizoctonia solani) used in following examples, xanthomonas oryzae pv. oryzicola (Xanthomonoasoryzaepv.oryzicola), citrus processing (XanthomonasCampestris 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 Tanaka altogether from the rice duck of Liuyang City and screen Antagonistic Fungi.Accurately take 10g duck excrement and put into the sterile water that 90mL is with bead, vibration 30min, gets suspension after leaving standstill 10min, is diluted to 1 × 10 by gradient concentration method
-7doubly, then dilution is coated on beef extract-peptone agar medium flat board and cultivates, obtain single bacterium colony.The single bacterium colony sterile water obtained is made suspension and made filter paper, leave in and cultivate on the potato agar culture medium flat plate at central water Rhizoctonia solani Kuhn bacterium cake (5mm) bilateral symmetry 2cm place, after indicator bacteria bacterium colony covers with flat board, picking has the bacterial clump of inhibition zone, purifying repeatedly, obtain single bacterium colony, be pseudomonas aeruginosa SU8 of the present invention.
The activation of example 2 bacterial strain of the present invention and fermentation
Adopt oese that bacterial strain SU8 of the present invention is accessed beef extract-peptone slant medium (beef extract 3-6g, peptone 8-10g, sodium chloride 4-5g, glucose 15-20g, water 1000mL, pH are 7.2-7.3), at 28 DEG C, cultivate 24h.Again by 10% inoculum concentration access beef extract-peptone liquid nutrient medium, magnetic agitation rotor speed is 1000r/min, puts continuous culture 84h in 28-30 DEG C of shaken cultivation case, obtains zymotic fluid.
The extraction of example 3 antibacterial substance
Get zymotic fluid and ethyl acetate and fully mix dipping extraction 48h with the ratio of 1:2, extract is inserted Rotary Evaporators temperature 40 DEG C, rotating speed 110r/min rotary evaporation, to paste, obtains antibacterial substance.
Example 4 antagonistic activity detects
The assay method of Antagonistic Fungus: citrus processing is inoculated in PDA solid culture medium central authorities, mycelia two side perforating (φ=5mm) newly grown up to after cultivating 24h at 28 DEG C, active substance to be measured (bacteriostatic extractive mentioned in embodiment 3) is injected in a hole, corresponding solvent is injected in another side opening, every hole 50 μ L, and make blank with corresponding solvent, cultivate 24-48h for 28 DEG C, observe antibacterial situation and inhibition zone size, each process repeats for 3 times.
The assay method of antagonistic bacterium: adopt plate punch method, by xanthomonas oryzae pv. oryzicola suspension (1 × 10
8cfu/mL) 60 μ L are spread evenly across NA flat board, then (φ=5mm) is punched, active substance to be measured (bacteriostatic extractive mentioned in embodiment 3) is injected in a hole, corresponding solvent is injected in another side opening, every hole 50 μ L, and compare with corresponding solvent, cultivate 24-48h for 28 DEG C, observe antibacterial situation and inhibition zone size, each process repeats for 3 times.
Result shows: this ethyl acetate extract has inhibit activities to xanthomonas oryzae pv. oryzicola and citrus processing, and control solvent unrestraint is active, sees Fig. 3 and Fig. 4.
Example 5 ethyl acetate extract and composite jinggangmycin thing are to the toxicity test of Rhizoctonia solani Kuhn
Determine each medicament Valid concentration through prerun, each medicament establishes 5 dosage process respectively by active constituent content, if corresponding solvent is contrast (water: ethyl acetate: acetone).Carry out with reference to " farm-chemical indoor determination test rule bactericide ", adopt mycelial growth rate method to measure medicament to the virulence of target fungus.Cultivate 72h right-angled intersection method and measure colony diameter, calculate each process mycelial growth inhibition rate, draw the parameter such as EC50 and virulence regression equation, calculate two medicament different ratio synergies ratio (SR) according to Wadley method simultaneously, SR<0.5 is antagonism, 0.5≤SR≤1.5 are synergy, and SR>1.5 is synergistic effect.
Result of the test shows: contrast on Rhizoctonia solani Kuhn without impact, and the Toxicity Determination result of compound to Rhizoctonia solani Kuhn is the EC of ethyl acetate extract and jinggangmeisu
50be respectively 4120.17 μ g/mL and 404.81 μ g/mL, the virulence of ethyl acetate extract is only jinggangmeisu 0.1 times, illustrates that the virulence of ethyl acetate extract is lower than jinggangmeisu; Ethyl acetate extract and jinggangmeisu had after composite within the scope of 1-10 and 10-1 collaborative and synergistic effect, wherein to press 9:1 synergy the most obvious for ethyl acetate extract and jinggangmeisu, other proportioning 1:9,1:1,4:1 also have synergistic effect, 1:4 proportioning then has synergy, sees the following form 1.
Table 1 ethyl acetate extract, jinggangmeisu and compound thereof are to the toxicity test of Rhizoctonia solani Kuhn
Claims (1)
1. a preserving number is that the ethyl acetate extract of the zymotic fluid of pseudomonas aeruginosa (Pseudomonasaeruginosa) the SU8 bacterial strain of CCTCC NO.2013178 is suppressing the application in citrus processing.
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| CN101984044A (en) * | 2010-01-19 | 2011-03-09 | 四川农业大学 | Streptomyces strain for preventing rice sheath disease and rice blast disease |
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| CN101984044A (en) * | 2010-01-19 | 2011-03-09 | 四川农业大学 | Streptomyces strain for preventing rice sheath disease and rice blast disease |
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