CN100532542C - Goffer pseudomonas P94 and application thereof - Google Patents
Goffer pseudomonas P94 and application thereof Download PDFInfo
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- CN100532542C CN100532542C CNB2007100635194A CN200710063519A CN100532542C CN 100532542 C CN100532542 C CN 100532542C CN B2007100635194 A CNB2007100635194 A CN B2007100635194A CN 200710063519 A CN200710063519 A CN 200710063519A CN 100532542 C CN100532542 C CN 100532542C
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Abstract
The invention discloses a new Pseudomonas corrugate strain (P94 GMCC No.1895), which is characterized by the following: manufacturing relative HCN to prevent bacteria, proteinase and phospholipase and IAA; inhibiting partial plant pathogenic fungus (Botrytis cinerea, Ceratocystis fimbriata, Monilinia laxa, Magnaporthe grisea, Pythium aphanidermatum and Phytophthora capsici) and partial pathogenic bacteria (Pseudomonas syringae, Acidovorax avenae and Ralstonia solanacearum); accelerating the growth of tomato and cucumber to improve production by 22%-28%.
Description
Technical field
The present invention relates to new bacterial strain of a strain microorganism and application thereof, specifically Goffer pseudomonas P 94 and the application in plant growth-promoting, biological control of diseases thereof.
Background technology
Infect the important Plant diseases that the eliminating vegetable botrytis that causes is a kind of global distribution by Botrytis cinerea.Solanberry class, berry fruit, melon output are caused heavy losses.Since the eighties in 20th century, along with the continuous expansion of China's protection ground area, eliminating vegetable botrytis harm is on the rise.The microclimate environment of booth extremely helps the generation of eliminating vegetable botrytis, becomes one of most important disease of China protection ground vegetables.Pathogenic bacteria grape spore is many invades from open residual flower, causes that petal rots, and grows the mould layer of one deck beige, and to the young fruit expansion, sick portion is water soaking mode, rots to be tawny gradually again, and the close layer that mildews in surface causes a fruit rot mostly when serious.Low temperature, high humidity, illumination deficiency, poor aeration all help the generation of gray mold.
Producing upward at present, eliminating vegetable botrytis mainly relies on chemical prevention control.The sterilant that is used to prevent and treat eliminating vegetable botrytis mainly contains three classes: benzimidazoles, diformamide class, N-phenyl urethan class.Since the seventies in 20th century, benzimidazoles such as derosal, thiophanate methyl, benomyl etc. have been adopted in succession; Diformamide class such as procymidone, RP-26019, dimetachlone etc.; Various sterilization agent such as amino formate such as the mould prestige of second prevent and treat gray mold.But because frequent heavy dose of these sterilant of use, and the heritable variation of grape spore is big, and adaptability is strong, and the grape spore has produced resistance in various degree to various medicaments commonly used, causes prevention effect to descend.On vegetable crop, except drug-fast problem, the use of chemical bactericide has also caused various problems such as environment, food safety, residual, poisoning.Benzimidazoles has limited use in many countries and regions, and people begin to pay close attention to and utilize beneficial microorganism control eliminating vegetable botrytis since the seventies in last century.At present, be used to prevent and treat the nearly kind more than ten of biocontrol fungi of eliminating vegetable botrytis, research and to use wider mainly be the mould and yeast of wood etc. is as viride (Trichodermaviride), trichoderma harziarum (Trichoderma harzianum), intend Kang Mu mould (Trichodermapseudokoningii), lignin wood mould (Trichoderma lignorum), stalk short mould (Aureobasidium pullulans) sprouts, light white latent ball yeast (Cryptococcus albidus), gluing rhodotorula (Rhodotorula glutinis), sticking broom mould (Gliocladium sp.), the sticking broom mould (Gliocladium catenulatum) of chain spore etc.; Research and the biocontrol bacteria that is applied to eliminating vegetable botrytis have kind more than ten, mainly contain genus bacillus (Bacillus sp.), subtilis (Bacillus subtilis), how sticking bud bacillus (Bacillus polymyxa), Bacillus licheniformis (Bacillus licheniformis Bacillus), newborn genus bacillus (Lactobacillus sp.), short body genus bacillus (Bacillus pumilus), xanthomonas maltophilia (Xanthomonasmaltophilia), Pseudomonas fluorescens (Pseudomonas fluorescens) etc.; Mycin microbiotic such as (ezomycin S) all has the better prevention effect to gray mold when the Wuyiencin (wuyiencin) that actinomycetes Streptomycetaceae streptomyces produces, phosphorus azomycin (phosphazomyzin), albopeptin (albopeptin), allosteric mycin (tautomycin), allosteric rhzomorph (tautomycein) and fish.But the report that utilizes the false monospore of gauffer to be applied to the eliminating vegetable botrytis control there is no report both at home and abroad.
Summary of the invention
The object of the present invention is to provide false new bacterial strain P94 of unit cell of a kind of gauffer and the application in plant growth-promoting and biological control of diseases thereof.
Bacterial strain P94 of the present invention separates the new bacterial strain of the false unit cell of gauffer (Pseudomonas corrugata) that obtains from the tomato warmhouse booth soil of outer suburbs, Beijing.China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC), preserving number CGMCC NO.1895 have been preserved in on December 22nd, 2006.
P94 specifically separates by the following method and obtains: gather soil sample from outer suburbs, Beijing tomato warmhouse booth, get in the triangular flask of dress 100ml stroke-physiological saline solution in the 10g adding, 28 ℃, 120rpm shaking table concussion 15min, behind the static 10min, get 500 μ l and add in the 4.5ml stroke-physiological saline solution, dilute 10 successively again
-2, 10
-3, 10
-4Doubly, get above soil suspension 100 μ l respectively at NA substratum (extractum carnis 5g/l, peptone 10g/l, NaCl 5g/l, agar 15g/l) even coated plate on the flat board, dry up in the super quiet worktable, each concentration repeats 3 times, cultivates 36h in 28 ℃ of incubators, behind the single bacterium colony line of aseptic toothpick picking bacterium purifying, with the ash arrhizus bacteria is the target bacterium, utilizes dull and stereotyped face-off method to carry out antimicrobial preliminary screening short of money; To detecting the greenhouse protection effect behind the antagonism bacterium shake flask fermentation that obtains, finishing screen is selected false unit cell (Pseudomonas corrugata) P94 of the good biocontrol strain gauffer of proterties.
The result that morphological feature, physio-biochemical characteristics, 16SrDNA sequence and the Auele Specific Primer of false unit cell (Pseudomonas corrugata) P94 of comprehensive gauffer analyzed is accredited as gauffer pseudomonas gene II type with it.
Studies show that, P94 can produce and antibiotic relevant HCN, proteolytic enzyme and phospholipase and the growth hormone (IAA) relevant with plant growth-promoting, inhibited to part plant pathogenic fungi and bacterium, gray mold had good preventive and therapeutic effect, and have the plant growth-promoting effect, can improve plant biomass.
The present invention also provides the fermentation culture method of P94, comprises step:
1, actication of culture
Use the KB substratum, culture medium prescription is: peptone 20g/l, glycerine 10ml/l, MgSO
47H
2O 1.5g/l, K
2HPO
41.5g/l, agar 15g/l.False unit cell P94 is inoculated on the KB medium slant with gauffer, cultivates 48h for 28 ℃.
2, culture of seed liquid
Seed culture AY substratum, the composition of AY substratum: sucrose 8-10g/L, yeast extract paste 2-4g/L, NaCl 0.5-1g/L, KH
2PO
40.3-0.5g/L, MgSO
47H
2O 1-1.5g/L, (NH
4)
2SO
43-5g/L, the pH value is 7.0-7.2.The false unit cell P94 of the gauffer that activation is good is mixed with 10 with stroke-physiological saline solution
8The bacteria suspension of cfu/ml, the inoculum size with 0.1% are inoculated in the AY liquid nutrient medium, and 28 ℃ of shaking table concussions are cultivated, and rotating speed is 150-200rpm, and incubation time is 44-48h.
3, ferment tank
Fermention medium consists of: glucose 4-6g/L, starch 5-8g/L, analysis for soybean powder 5-8g/L, (NH
4)
2SO
45-10g/L, KH
2PO
40.5-1g/L, MgSO
47H
2O 0.5-0.8g/L, CaCO
31-1.5g/L the pH value is 7.0-7.5.Per minute air flow volume), tank pressure 1.5-2.0F/cm cultured seed liquid is inserted in the fermentor tank with 2% inoculum size, and 28 ℃, stirring velocity is 180rpm, and air flow is a 1:0.6-0.8 (fermentating liquid volume:
2Fermentation 48h bacterium amount reaches 5-10 * 10
8Cfu/ml obtains the false unit cell P94 of gauffer bacterium liquid.
The present invention is in fermentation culture, and the AY substratum is best suited for the substratum of strain growth of the present invention, and fermentation is considered fermentation costs bacterium amount and produce the antibiont quality to filter out with substratum.
The present invention also comprises the microbial inoculum that contains described bacterial strain.
Bacterial strain P94 of the present invention can produce and antibiotic relevant HCN, proteolytic enzyme and phospholipase and the growth hormone (IAA) relevant with plant growth-promoting, inhibited to part plant pathogenic fungi and bacterium, gray mold is had good preventive and therapeutic effect, the preventive effect of graw mold of tomato and gray mold of cucumber is respectively 86.3% and 78.4%; And have good plant growth-promoting effect, can improve plant biomass, can improve tomato and cucumber yield 22%～28%.
Description of drawings
Fig. 1 is P94 electromicroscopic photograph (growth 18hr; * 20000);
Fig. 2 is with the genomic result of Auele Specific Primer pcr amplification P.corrugata P94.1, Marker; 2, negative control; 3, be template with P.corrugata P94 genome;
Fig. 3 is that the P94 protease activity detects;
Fig. 4 is the detection that P94 produces HCN, and a left side is the right negative contrast of P94;
Fig. 5 is that the activity of P94 phospholipase detects.
Embodiment
Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.
The evaluation of embodiment 1 P94
The result that morphological feature, physio-biochemical characteristics, 16SrDNA sequence and the Auele Specific Primer of false unit cell (Pseudomonas corrugata) the P94 CGMCC of comprehensive gauffer NO.1895 analyzed is accredited as gauffer pseudomonas gene II type with it.Concrete qualification result is as follows:
1, the morphological specificity of thalline
Gram-negative produces yellow-green colour solubility non-fluorescent colour element on the PDA substratum, bacterium colony is blue green; On the KB substratum pigment produce less, the bacterium colony pistac.Electron microscope observation, the thalline oblong, size 0.7 * 1.8 μ m is no less than two polar flagella (see figure 1)s.
2, physio-biochemical characteristics
The false unit cell P94 of gauffer physio-biochemical characteristics are as shown in table 1.
Table 1.P94 physio-biochemical characteristics
Annotate :+, positive reaction;-, negative reaction; W is a little less than the reaction.
3,16SrDNA sequential analysis
Method is: extract the genome of P94, with 16S amplification universal primer 63F5 '-CAGGCCTAACACATGCAAGTC-3 ' and 1494R5 '-GGYTACCTTGTTACGACTT-3 '.In following PCR system, increase: 50 μ l amplification systems, 10pmol primer, 0.2mM dNTP, 1.5mM MgCl
2, 2.5U Taq archaeal dna polymerase (TaKaRa), 1 * PCR buffer (TaKaRa), the 25ng genomic dna is made template.The PCR reaction conditions is: 94 ℃ of pre-sex change of 5min, and 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, and 30 circulations are fully extended 5min for back 72 ℃.Pcr amplification product is with 1% agarose gel electrophoresis analysis, reclaims the T carrier pMD18-T that the back connects TaKaRa company with the purification kit Gel Extraction Kit purifying of OMEGA company.Check order with ABI 3730 dna sequencing instrument in Invitrogen company, nucleotide sequence is shown in sequence table SEQ ID NO.1, and gene order has been submitted GenBank to, and the gene number of including is: EF153018.With blast program GenBank (
Http:// www.ncbi.nlm.nih.gov) to compare similarity the highest for the result of the corresponding sequence comparison 16SrDNA sequence that shows P94 and the relevant bacterial strain of the false unit cell of the gauffer of delivering at present, reaches 99%.
4, Auele Specific Primer analysis
With Auele Specific Primer PC 1/1 (5 '-GGATATGAGCCAGGTCTTCG-3 ') and PC 1/2 (5 '-CGCTCAAGCGCGACTTCAG-3 '), PC 5/1 5 '-CCACAGGACAACATGTCCAC-3 ') and PC 5/2 (5 '-CAGGCGCTTTCTGGAACATG-3 '), the false unit cell Pseudomonas of gauffer corrugata can be divided into two gene groups (group I or group II), amplified band is that 1100bp is group I, and amplified band is that 600bp is group II.The PCR reaction system is: 25 μ l reaction systems, 20ng genomic dna, 1 * PCR buffer (TaKaRa), 1.5mM MgCl
2, 20mM of dNTP (TaKaRa), four Auele Specific Primers (every primer concentration is 10pmol) and 2.5U Taq archaeal dna polymerase (TaKaRa).The PCR reaction conditions is: 94 ℃ of pre-sex change of 5min, and 94 ℃ of sex change 30s, 62 ℃ of annealing 1min, 72 ℃ are extended 90s, and 30 circulations are fully extended 5min for back 72 ℃.Pcr amplification product is with 1% agarose gel electrophoresis analysis, and stripe size is 600bp (as Fig. 2).The result shows that the false unit cell P94 of gauffer belongs to the false unit cell gene group of gauffer II.
Embodiment 2 antimicrobial spectrum analyses
Flat board to pathogenic fungi suppresses detection method: beat the target pathogenic fungi of getting the 5mm diameter with the device that fans the air and be inoculated in the dull and stereotyped central authorities of PDA, P94 becomes 180 ° to be inoculated in target pathogenic fungi two survey 2.5cm places with the target bacterium, cultivate for 25 ℃ and measure the pathogenic fungi colony diameter after 4 to 6 days.The flat board of not inoculating P94 with inoculation target fungi is contrast.Suppressing growth rate calculates according to following formula:
Inhibition growth rate=(C-T)/C * 100%
C: contrast fungal growth diameter
T: fungal growth diameter behind the inoculation P94
The pathogenetic bacteria flat board is suppressed detection method: detect the inhibition situation of the false unit cell P94 of gauffer to pathogenetic bacteria with double-deck culture method.With being made into 10 with physiological saline behind the KB substratum activation P94
8The bacteria suspension of cfu/ml is drawn 5 μ l with liquid-transfering gun and is inoculated in the dull and stereotyped central authorities of KB, and the chloroform with 3ml behind the cultivation 48h kills thalline with its steam.The target bacterium that activation is good is made into 10
8The bacteria suspension of cfu/ml, drawing 50 μ l bacteria suspensions joins and adds water agar (0.7% agar that 3ml melts postcooling to 50 ℃, pH7.0) in, rapid mixing, pour into immediately on the substratum that trichloromethane kills the P94 thalline, be paved into uniform thin layer, cultivated 36 hours for 28 ℃, observe the situation of inhibition zone.
P94 is as shown in table 2 to the dull and stereotyped inhibition of pathogenic fungi result.
Table 2.P94 suppresses pathogenic fungi is dull and stereotyped
Annotate:
aThe expression standard deviation;
b-expression does not have restraining effect
P94 is as shown in table 3 to the dull and stereotyped inhibition of pathogenetic bacteria result.
Table 3.P94 suppresses pathogenetic bacteria is dull and stereotyped
Annotate: a represents standard deviation.
Embodiment 3 biological and ecological methods to prevent plant disease, pests, and erosion correlated character detect
Proteolytic enzyme detects: and usefulness skimmed milk flat board (Tryptones 5g, yeast extract 2.5g, glucose 1g, 7% skimmed milk 250ml, agar 15g, water complements to 1000ml) detection proteolytic enzyme.P94 is inoculated into dull and stereotyped central authorities, cultivates 72h for 28 ℃, periphery of bacterial colonies has transparent circle the positive (see figure 3) of expression proteolytic enzyme to occur.
HCN detects: HCN detects test paper and prepares, 10mg copper (II) ethylacetoacetate and 10mg 4,4 '-methylenebis-(N, N-dimethylaniline) be dissolved in the chloroform of 4ml, shear Whatman filter paper bar, be soaked in the solution for preparing, just be prepared into HCN after the chloroform volatilization fully and detected test paper.Percutaneous puncture-inoculation P94 in the centrifuge tube that contains 1ml KB substratum, HCN detects test paper and is suspended from the centrifuge tube, cultivates 36h for 28 ℃, and HCN detects test paper and shows blueness, shows that P94 produces the HCN (see figure 4).
IAA detects: P94 is inoculated in NA substratum (peptone 5g/l, yeast extract 1.5g/l, extractum carnis 1.5g/l, NaCl5g/l) and add in the 0.5g/l tryptophane NA substratum 28 ℃ shake training 4d after, sampling 5ml, the centrifugal 10min of 12000rpm gets the 2ml supernatant, the 10mM ortho-phosphoric acid that adds 100 μ l, 4ml detects liquid (the 0.5M FeCl of 1ml
3Be dissolved in 50ml of 35% HClO
4) behind the room temperature reaction 25min with spectrophotometric instrumentation 530nm place's photoabsorption.With pure product IAA obtain solution measurement standard absorption curve, and calculate the amount that P94 produces IAA.Result: P94 produces IAA in containing the NA substratum of tryptophane concentration is 15.97 μ g/ml, and the concentration that produces IAA in not containing the NA substratum of tryptophane is 6.97 μ g/ml.
Phosphate esterase active is measured: use the dull and stereotyped phosphate esterase active that detects of Pikovskaya ' s.The dull and stereotyped compound method of Pikovskaya ' s is: yeast extract 0.5g/l, glucose 10g/l, calcium phosphate 5g/l, ammonium sulfate 0.5g/l, Repone K 0.2g/l, magnesium chloride 0.1g/l, manganous sulfate 0.0001g/l, ferrous sulfate 0.0001g/l, agar 15g/l.P94 is inoculated in the dull and stereotyped central authorities of Pikovskaya ' s, cultivates 7d for 28 ℃, transparent circle occurs in periphery of bacterial colonies, this shows that P94 has the phosphate esterase active (see figure 5).
Embodiment 4 fermentative production
1, actication of culture
Use the KB substratum, culture medium prescription is: peptone 20g/l, glycerine 10ml/l, MgSO
47H
2O 1.5g/l, K
2HPO
41.5g/l, agar 15g/l.P94 is inoculated on the KB medium slant, cultivates 48h for 28 ℃.
2, culture of seed liquid
Seed culture AY substratum, the composition of AY substratum: sucrose 9g/L, yeast extract paste 3g/L, NaCl 0.8g/L, KH
2PO
40.4g/L, MgSO
47H
2O 1.2g/L, (NH
4)
2SO
43-5g/L, the pH value is 7.1.The false unit cell P94 of the gauffer that activation is good is mixed with 10 with stroke-physiological saline solution
8The bacteria suspension of cfu/ml, the inoculum size with 0.1% are inoculated in the AY liquid nutrient medium, and 28 ℃ of shaking table concussions are cultivated, and rotating speed is 150-200rpm, and incubation time is 44-48h.
3, ferment tank
Fermention medium consists of: glucose 5g/l, starch 6.5g/l, analysis for soybean powder 7g/l, (NH
4)
2SO
47g/L, KH
2PO
40.8g/L, MgSO
47H
2O 0.65g/L, CaCO
31.3g/L the pH value is 7.2.Per minute air flow volume), tank pressure 0.05MPa cultured seed liquid is inserted in the fermentor tank with 2% inoculum size, and 28 ℃, stirring velocity is 180rpm, and air flow is a 1:0.6-0.8 (fermentating liquid volume:.Fermentation 48h bacterium amount reaches 5-10 * 10
8Cfu/ml obtains the false unit cell P94 of gauffer bacterium liquid.
Embodiment 5 preventive effects are given birth to short
With greenhouse tomato and greenhouse cucumber is that vegetable material detects the prevention effect of P94 to graw mold of tomato and gray mold of cucumber.Botrytis cinerea and botrytis cinerea pers be 28 ℃ of cultivation 7d on the PDA flat board, and black lamp induces 7d to produce spore, with stroke-physiological saline solution wash-out spore, adjust spore concentration to 10 with blood counting chamber
7Every milliliter in individual spore.5 processing of test design.The P94 fermented liquid that ferments dilution twice is sprayed on tomato and cucumber, spray the gray mold spore after waiting the blade face drying, plastics film is preserved moisture, and humidity is controlled more than 80%, and envrionment temperature is controlled to be 21 ℃～23 ℃.In back 7 days investigation results of inoculation.Test-results shows that P94 reaches 86.3% and 78.4% respectively to graw mold of tomato and gray mold of cucumber prevention effect.The tomato and the cucumber growth that spray the false unit cell P94 of gauffer microbial inoculum obviously are better than adjoining tree, and the fresh weight of weighing tomato and cucumber finds that the tomato and the cucumber growth amount that spray P94 improve 22%～28%.P94 is mould to the melon and fruit corruption, the blue or green vegetable disease such as withered of Phytophthora capsici, Solanaceae has potential biological and ecological methods to prevent plant disease, pests, and erosion effect.
Sequence table
＜110〉China Agricultural University
＜120〉Goffer pseudomonas P 94 and application thereof
<130>
<160>1
<170>PatentIn?version?3.3
<210>1
<211>1462
<212>DNA
<213>Pseudomonas?corrugata?P94
<400>1
Claims (8)
1, false unit cell (Pseudomonas corrugata) the P94 CGMCC of gauffer NO.1895.
2, the biological pesticide that contains the described bacterial strain of claim 1.
3, the microbial inoculum that contains the described bacterial strain of claim 1.
4, the application of the described bacterial strain of claim 1 in the control eliminating vegetable botrytis.
5, application as claimed in claim 4 is characterized in that described vegetables are tomato or cucumber.
6, the application of the described bacterial strain of claim 1 in plant growth-promoting.
7, the application of the described bacterial strain of claim 1 in vegetable growing.
8, application as claimed in claim 7 is characterized in that described vegetables are tomato or cucumber.
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| CN102827793B (en) * | 2012-08-29 | 2014-04-16 | 哈尔滨师范大学 | Medicago pseudomonas strain capable of producing ACC (1-aminocyclopropane-1-carboxylate) deaminase and application of medicago pseudomonas strain |
| CN103109870B (en) * | 2013-01-08 | 2014-06-11 | 华中农业大学 | Application of fermentation supernatant liquid of pseudomonas |
| CN104962496B (en) * | 2015-07-08 | 2017-12-19 | 青岛农业大学 | One plant of Pseudomonas Maltophilia bacterial strain QBA 5 for having inhibitory action to ash arrhizus bacteria and its application |
| CN108148782B (en) * | 2018-01-29 | 2021-03-26 | 江苏师范大学 | Bacterial strain for producing volatile gas and antagonizing sweet potato black spot pathogen and application thereof |
| CN109906703B (en) * | 2019-02-15 | 2022-03-18 | 中国环境科学研究院 | Method for increasing urease and phosphatase content in farmland |
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Non-Patent Citations (2)
| Title |
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| 灰霉病生物防治研究进展. 童蕴慧，纪兆林，徐敬友，陈夕军.中国生物防治，第19卷第3期. 2003 |
| 灰霉病生物防治研究进展. 童蕴慧，纪兆林，徐敬友，陈夕军.中国生物防治，第19卷第3期. 2003 * |
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