CN1831116A - Pantoea agglomerans and fermentation culture method and application thereof - Google Patents
Pantoea agglomerans and fermentation culture method and application thereof Download PDFInfo
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- CN1831116A CN1831116A CN 200610066981 CN200610066981A CN1831116A CN 1831116 A CN1831116 A CN 1831116A CN 200610066981 CN200610066981 CN 200610066981 CN 200610066981 A CN200610066981 A CN 200610066981A CN 1831116 A CN1831116 A CN 1831116A
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- pantoea agglomerans
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- agglomerans
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Abstract
The invention discloses Pantoea agglomerans B50l CGMCC No.1655 and a fermentation culture method and application thereof. The fermentation culture method comprises the following steps: 1) inoculating Pantoea agglomerans B50l CGMCC No.1655 into a special culture medium for triangular bottles and seed tanks, and culturing at 28-35 ℃ and pH of 7.0-8.0 for 24-48 hours to obtain seed liquid; 2) inoculating the seed solution prepared in the step 1) into a special culture medium for a fermentation tank of the strain, and culturing for 48-96 hours at the temperature of 28-35 ℃ and the pH value of 7.0-8.0. The fungus and the microbial inoculum taking the fungus as an active ingredient can be used for preventing and treating fungal diseases of fruits and vegetables, and have the following advantages: 1) the antibacterial spectrum is wide, and the disease prevention effect is good; 2) the action efficiency is high; 3) the biological safety is high; 4) the fermentation period is short, the preparation process of the microbial inoculum is simple, and the possibility of industrial production is realized.
Description
Technical field
The present invention relates to a stain of Pantoea agglomerans and fermentation culture method thereof and application.
Background technology
1989, (Gavini such as Gavini, F.et al.Transfer of Enterobacter agglomerans (Beijerinck 1888) Ewing and Fife 1972 to Pantoea gen.nov.as Pantoeaagglomerans comb.nov.and description of Pantoea dispersa sp.nov.[J] .Int.J.Syst.Bacteriol, 1989,39:337-345.) founded general Pseudomonas (Genus Pantoea), this genus comprises pantoea agglomerans (Pantoea agglomerans) and disperses two kinds of general bacterium (Pantoea dispersa), wherein pantoea agglomerans kind (Pantoea agglomerans) and enterobacter agglomerans (Enterobacter agglomerans), grass living Erwinia (Erwinia herbicola) and Yunnan Caulis Spatholobi Erwinia (Erwinia milletiae) are alias, it is the yellow flora that produces carotenoid, the earliest from plant, separate obtaining in the seed, in multiple environment, all found the existence of this flora afterwards.The researchist has carried out a large amount of research work to pantoea agglomerans, finds that it all has vital role on medical science, agricultural and genetics, the main performance in the following areas:
1) biological metabolism aspect: have dehalogenate, degraded glyceryl trinitrate and be 1, the ability of ammediol with transformation of glycerol.
2) the short aspect of giving birth to: not only have molten phosphorus, biological nitrogen fixation ability, also can secrete plant hormone and various enzyme, have the potential using value.
3) biological control aspect: in recent years, the researchist finds that this bacterium can suppress effectively by different bacterium, fungus-caused Plant diseases.For example, the pantoea agglomerans that is separated to from the corn rhizosphere soil is inhibited to a kind of indigenous pathomycete (Fusarium moniliforme) that causes Plant diseases; To also having certain prevention effect by separating the fruit fire blast that starch Erwinia (Erwiniaamylovora) causes; In the barley field test, pantoea agglomerans can reach 45%-70% to the prevention effect of the grain eqpidemic disease that the mutation (Pseudomonas syringae pv.syringae) of being caused a disease by the pseudomonas syringae cloves causes; The interior living pantoea agglomerans that is separated to from paddy rice has antagonistic action to dry thread Pyrenomycetes (Rhizoctonia solani); The main fungal disease that can effectively suppress apple, pears and citrus from the isolating pantoea agglomerans CPA-2 of apple surface.
At present, the biocontrol effect of most of bacteriums is relevant with antibiotic generation.The frequent microbiotic that uses can make pathogen develop immunity to drugs, and reduces protection effect, and can threaten to human health.
Summary of the invention
The purpose of this invention is to provide a strain has preventive and therapeutic effect and does not produce antibiotic pantoea agglomerans and fermentation culture method thereof the fruits and vegetables fungal disease.
Pantoea agglomerans bacterial strain provided by the present invention is (Pantoea agglomerans) B501, and this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 03 20th, 2006, and deposit number is CGMCC No.1655.
That pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 is Gram-negative bacteria (0.5-0.8 * 1-3 μ m), after line on the LB substratum, behind 28 ℃ of cultivation 1-2d, can produce smooth, circular, protruding yellow bacterium colony.It is positive that D-seminose, D-wood sugar, D-N.F,USP MANNITOL, L-arabinose, L-rhamnosyl, sucrose, lactose, bigcatkin willow are sweet, maltose and trehalose produce acid; Galactitol ,-inositol (slowly), D-ribitol, Alpha-Methyl-D-glucose is sweet and D-sorbyl alcohol (slowly) to produce acid negative.Arginine depickling enzyme, ornithine depickling enzyme, arginine dihydrolase, phenylalanine deaminase, urea hydrolysis, oxydase, indoles generation, gelatine liquefication, lipase, D-glucose aerogenesis, H
2S produces and KCN is grown to feminine gender; It is positive that mobility, polychrom hydrolysis, malonate utilization, Citrate trianion and D-glucose produce acid.Growth temperature range: 3-42 ℃, optimum growth temperature: 28-32 ℃; Growth potential of hydrogen scope: pH=5-8.6, optimal pH=7.2-7.5.
Second purpose of the present invention provides the fermentation culture method of a kind of pantoea agglomerans (Pantoea agglomerans) B501 CGMCCNo.1655.
The fermentation culture method of pantoea agglomerans provided by the present invention (Pantoea agglomerans) B501 CGMCC No.1655 may further comprise the steps:
1) pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 being inoculated in triangular flask, the seeding tank special culture media, is to cultivate 24-48 hour under the 7.0-8.0 at 28-35 ℃, pH, obtains seed liquor; Triangular flask, the seeding tank special culture media prescription of described pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 are: Tryptones 8-12g, and yeast extract 4-6g, sucrose 10-20g, NaCl 8-12g, water is settled to 1000mL;
2) seed liquor with step 1) preparation is inoculated in the fermentor tank special culture media of this bacterium, cultivates 48-96 hour under the 7.0-8.0 at 28-35 ℃, pH; The prescription of the fermentor tank special culture media of described pantoea agglomerans (Pantoea agglomerans) B501 CGMCCNo.1655 is: sucrose 8-12g, Tryptones 4-6g, Semen Maydis powder 15-25g, soyflour 15-25g, wheat bran 15-25g, Na
2HPO
41.5-2.5g, NaH
2PO
40.15-0.25g, MgSO
40.2-0.4g, ZnSO
40.4-0.6g, CaCO
31.5-2.5g, bubble enemy 0.08-0.12g, water is settled to 1000mL.
In above-mentioned fermentation culture method, inoculative proportion is 5-8%, and culture condition is preferably: at 28-32 ℃, pH is aerated culture under the 7.2-7.5, and air flow is 26-53mM O
2L
-1h
-1
Preferred pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 triangular flask, seeding tank special culture media prescription are: Tryptones 10g, and yeast extract 5g, sucrose 15g, NaCl 10g, water is settled to 1000mL.
Preferred pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 fermentor tank special culture media prescription is: sucrose 10g, Tryptones 5g, Semen Maydis powder 20g, soyflour 20g, wheat bran 20g, Na
2HPO
42g, NaH
2PO
40.2g, MgSO
40.3g, ZnSO
40.5g, CaCO
32g, bubble enemy 0.1g, water is settled to 1000mL.
In addition, for obtaining better culture effect, can carry out triangular flask before carrying out seed tank culture earlier cultivates, concrete grammar is: pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 is inoculated in its triangular flask special culture media, is to cultivate 24-48 hour under the 7.0-8.0 at 28-35 ℃, pH.
In the triangular flask cultural method, inoculative proportion is 5-8%, and culture condition is preferably: at 28-32 ℃, pH is shaking culture under the 7.2-7.5, and vibration velocity is 100-250rpm.
Another object of the present invention provides a kind of pantoea agglomerans (Pantoea agglomerans) B501 CGMCCNo.1655 microbial inoculum.
Pantoea agglomerans provided by the present invention (Pantoea agglomerans) B501 CGMCC No.1655 microbial inoculum, its activeconstituents are pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655.
The fermented liquid that obtains with above-mentioned fermentation culture method can directly use as pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 microbial inoculum, and can adjust fermentation condition as required, obtains the microbial inoculum of different concns.Fermented liquid after low temperature (32-35 ℃) drying, can be obtained pantoea agglomerans (Pantoeaagglomerans) the B501 CGMCC No.1655 microbial inoculum of therapy in dry powder form.In addition, on the basis of above-mentioned fermented liquid, add one or more additives again, as chitin, collagen protein and Mierocrystalline cellulose etc. can obtain being applicable to the microbial inoculum of different diseases degree; The ratio of weight and number of chitin and microbial inoculum is 3-5: 1000, and the ratio of weight and number of collagen protein and microbial inoculum is 5-7: 1000.
The 4th purpose of the present invention provides a kind of method of preventing and treating of fruits and vegetables fungal disease.
The method of preventing and treating of fruits and vegetables fungal disease provided by the present invention is that the last week of gathering is 5 * 10 at the fruit and vegetable surfaces spraying concentration
8Cfu mL
-1-1 * 10
9Cfu mL
-1Pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 microbial inoculum, it is 5 * 10 that the fruit after maybe will plucking is soaked in concentration
8Cfu mL
-1-1 * 10
9Cfu mL
-1Pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 microbial inoculum in handled 1-3 minute, the fruits and vegetables fungal disease is is effectively prevented and treated.
The invention provides pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655.This bacterium and be that the microbial inoculum of activeconstituents can be used for preventing and treating the fruits and vegetables fungal disease with this bacterium, and have the following advantages: 1) antimicrobial spectrum is wider, and protection effect is good.Indoor and field test results shows, this bacterium and microbial inoculum thereof all have good inhibitory effect to multiple fruits and vegetables fungal disease, comprise the jujube fruit black spot that causes by chain lattice spore, the grey mold disease of jujube fruit, apple and pears, mould disease, soft rot evil and brown rot disease, the dark green mildew of citrus, ring rot of apple, soft rot of cabbage and early blight of tomato etc.; Wherein the field disease-preventing effect to jujube fruit black spot reaches 62-78%; 2) functioning efficiency height is at lower concentration (10
7-10
9Cfu mL
-1) the following generation that can effectively suppress various fruits and vegetables fungal diseases; 3) this bacterium does not produce microbiotic, the biological safety height; 4) fermentation period is short, and fungicide preparation technology is simple, has the possibility of suitability for industrialized production.The present invention has broad application prospects in the control of fruits and vegetables fungal disease.
The present invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 is the cluster analysis dendrogram of pantoea agglomerans B501
Fig. 2 dynamically schemes for the growth of pantoea agglomerans B501 on jujube fruit wound
Fig. 3 is the inhibition effect detection result of pantoea agglomerans B501 different treatment liquid to jujube fruit chain lattice spore
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and all percentage concentrations are mass percent concentration, and the solvent in all substratum is water.
Separation, evaluation and the preservation of embodiment 1, pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655
One, the bacterial strain that separate, screening is good to the pathogen antagonistic effect
Pathogen:
Chain lattice spore is from field winter jujube black spot fruit surface isolation, is seeded in PDA substratum (potato 200g after purified, glucose 15g, agar-agar 15g, water is settled to 1000mL) on, after cultivating 10-14d under 28 ℃,, be made into concentration with sterilized water and be about 1 * 10 by hemocytometer
6Spore mL
-1Spore suspension.
Fruit:
The winter jujube is gathered in the Zhanhua of Chinese Shandong Province, and the PVC bag of packing into behind the precooling 2d is hidden in-2 ℃ of refrigerator and cooled.Jujube fruit hardness 12.53kg/cm
2, soluble solid content is 36.8%, total acid is 1.2%.Before the inoculation, that the winter jujube is with the 2%NaOCl 2min that sterilizes, air-dry behind distilled water flushing.
1, the separation of bacterial strain
Jujube fruit face exists in a large number can be to the inhibiting microorganism of the former deposits yields of jujube fruit disease.Now from winter jujube surface and wound isolated strains, concrete grammar is as follows:
1) from winter jujube surface isolation
Get 10 winter jujubes, place it in and contain in the 0.2M phosphoric acid buffer liquid container, in the 100rpm gyrate shaker, wash 10min, abandon washing lotion.Carry out secondary cleaning (before the secondary cleaning, earlier the winter jujube being carried out 30 seconds supersound process) with same procedure again, keep washing lotion.After then secondary cleaning water being carried out serial dilution, diluent is coated on Luria-Bertani (LB) solid medium, every ware 0.1mL, cultivate 24-48h down to growing single bacterium colony at 28 ℃, according to the single bacterium colony of different colony characteristics picked at random, after repeating line on the LB solid medium, the chain lattice spore spore suspension of single bacterium colony of picking purifying and preparation is cooked the face-off test on the PDA plate, abandon the bacterium colony of clear antibacterial band, the bacterium colony of reservation further screens on the winter jujube.
2) separate from winter jujube wound
Get 10 winter jujubes, it is immersed in the triangular flask that fills 150mL sterilization deionized water, in the 100rpm gyrate shaker, clean secondary, keep washing lotion twice.Picking jujube fruit, one of every fruit thorn 3mm * 3mm wound handles three fruits at every turn.Put once and each 20 μ L of secondary cleaning water at each wound, inoculation 20 μ L chain lattice spore spore suspensions behind the 2h, behind the cultivation 7d that preserves moisture under 25 ℃, observe the wound that does not show illness on the winter jujube, scrape with the disinfection inoculation pin and to get the top layer, after serial dilution, getting this diluent 100 μ L is coated on the LB solid medium, after cultivating 1d under 28 ℃, select the single bacterium colony with different shape feature, rule on the LB plate, the chain lattice spore spore suspension of single bacterium colony behind the picking purifying and preparation is cooked the face-off test on the PDA plate, abandon the bacterium colony of antibacterial band, the bacterium colony of reservation further screens on the winter jujube.
Finally from winter jujube surface and two approach of wound, be divided into from screen 9 strains and do not produce antibiotic bacterium, number consecutively is B101-B901.
2, on the winter jujube, screen
Picking ripening degree unanimity, anosis, winter jujube examination material of uniform size.After surface sterilization, one of every fruit thorn 3mm * 3mm wound moves into the suspension (1 * 10 that is made into from the bacterium colonies of 9 strain bacteriums of winter jujube surface, wound separation with step 1 respectively
9Cfu/mL) 50 μ L, inoculation 20 μ L chain lattice spore spore suspensions behind the 2h, preserve moisture under 25 ℃ cultivate 7d after, the sickness rate and the scab diameter of statistics fruit are with aqua sterilisa (Control) in contrast.Each isolated strains is handled 24 fruits, every group of 8 fruits.The test triplicate.Statistics is as shown in table 1, the sickness rate of test strain and scab diameter all significantly are lower than contrast (P=0.05), wherein bacterial strain B501 and B701, the sickness rate of comparing photograph has respectively descended 87% and 68%, also reaches significant degree (P=0.05) with the difference of other bacterial strain.Enlarge amount then for examination jujube fruit colony, to antagonistic effect preferably B501 and B701 proceed test: 40 fruits of every inoculation, every group of 8 fruits, cultivate 5d, 7d, 9d respectively after, the sickness rate and the scab diameter of statistics winter jujube, test triplicate.Statistics is as shown in table 2, when B501 and B701 inoculation culture 5d, 7d, sickness rate and scab diameter are compared with the control, all significantly be lower than contrast (P=0.05), when cultivating 9d, the sickness rate comparison of B501 and B701 is shone and has been descended 86% and 62% respectively, and B501 is all lower than sickness rate and the scab diameter of B701, and both are remarkable difference (P=0.05).The bacterial strain B501 that selection is best to the pathogen antagonistic effect identifies with following method it.
Table 1B101-B901 bacterial isolates is to adopting the inhibition effect of back jujube fruit chain lattice spore
| Bacterial isolates | Sickness rate (%) | Scab diameter (mm) |
| B101 B201 B301 B401 B501 | 100a 100a 100a 100a 13f | 17.4bc 17.9b 17.6b 18.2b 12.3e |
| B601 B701 B801 B901 Control | 73d 32e 95b 84c 100a | 16.8cd 16.1d 17.6b 17.2bcd 19.8a |
Annotate: the same column same letter represents that the multiple disparity range of Deng Kenshi detects difference not remarkable (P=0.05); Numeral is three multiple mean values in the table
Table 2 bacterial isolates B501 and B701 are to adopting the inhibition effect of back jujube fruit chain lattice spore
| Inoculation time | Bacterial isolates | Sickness rate (%) | Scab diameter (mm) |
| 5 days 7 days 9 days | B501 B701 Control B501 B701 Control B501 B701 Control | 0c 12b 52a 11c 29b 93a 14c 38b 100a | 0c 9.2b 13.8a 4.5c 13.2b 17.9a 11.3c 16.7b 19.5a |
Annotate: the same column same letter represents that the multiple disparity range of Deng Kenshi detects difference not remarkable (P=0.05); Numeral is three multiple mean values in the table
Two, the evaluation of bacterial strain B501 and preservation
1, the homology analysis of 16S rDNA sequence
Genomic dna with bacterial strain B501 is a template, with 63F (5 '-CAGGCCTAACACATGCAAGTC-3 ') and 1387R (5 '-GGGCGGTGATGTACAAGGC-3 ') be primer, and its 16S rDNA partial sequence of pcr amplification .PCR reaction conditions is: first 94 ℃ of sex change 5min; 94 ℃ of sex change 40S then, 55 ℃ of renaturation 40S, 72 ℃ are extended 1min, carry out 35 circulations altogether, and last 72 ℃ are extended 10min.After reaction finishes, pcr amplification product is carried out 0.8% sepharose electricity arteries and veins detect, pcr amplification goes out the fragment that size is about 1.3kb as a result.With test kit E.Z.N.A. (OmegaBio-tec, USA) this fragment is reclaimed and purifying after, connect through cloning vector pBluescript SK II (+) that restriction enzyme EcoR V enzyme is cut (Stratagene) in, to connect product transformed into escherichia coli (Escherichia coli) DH5 α competent cell again, screening positive clone, the upgrading grain, order-checking, sequencing result shows the nucleotide sequence that the 16S rDNA fragment of the bacterial strain B501 that is cloned has sequence 1 in the sequence table.Adopt BLAST software that the segmental nucleotide sequence of this 16S rDNA is carried out homology relatively, and use DNAMAN (LynnonBiosoft) software and carry out the genetic distance analysis, set up the cluster analysis dendrogram.By BLAST comparison and DNAMAN software analysis, the pantoea agglomerans (Pantoeaagglomerans) of the 16S rDNA sequence of bacterial strain B501 and general Pseudomonas (Genus Pantoea), enterobacter agglomerans (Enterobacter agglomerans), Yunnan Caulis Spatholobi Erwinia (Erwiniamilletiae), the genetic distance of the living Erwinia of grass (Erwinia herbicola) is nearest, homology reaches more than 99%, because of the homology of the 16S rDNA sequence of bacterium reach can be classified as more than 97% of the same race.Therefore, according to 16S rDNA sequence alignment result, B501 is included into pantoea agglomerans (Pantoea agglomerans), its cluster analysis dendrogram as shown in Figure 1.
2, analysis of physio biochemical characteristics
According to " Bergey ' s Manual of Determinative Bacteriology " the 9th edition pantoea agglomerans bacterial strain B501 is carried out analysis of physio biochemical characteristics, the result is as follows:
Bacterial strain B501 is Gram-negative bacteria (0.5-0.8 * 1-3 μ m), after line on the LB substratum, behind 28 ℃ of cultivation 1-2d, can produce smooth, circular, protruding yellow bacterium colony.It is positive that D-seminose, D-wood sugar, D-N.F,USP MANNITOL, L-arabinose, L-rhamnosyl, sucrose, lactose, bigcatkin willow are sweet, maltose and trehalose produce acid; Galactitol ,-inositol (slowly), D-ribitol, Alpha-Methyl-D-glucose is sweet and D-sorbyl alcohol (slowly) to produce acid negative.Arginine depickling enzyme, ornithine depickling enzyme, arginine dihydrolase, phenylalanine deaminase, urea hydrolysis, oxydase, indoles generation, gelatine liquefication, lipase, D-glucose aerogenesis, H
2S produces and KCN is grown to feminine gender; It is positive that mobility, polychrom hydrolysis, malonate utilization, Citrate trianion and D-glucose produce acid.Growth temperature range: 3-42 ℃, optimum growth temperature: 28-32 ℃; Growth potential of hydrogen scope: pH=5-8.6, optimal pH=7.2-7.5.
16S rDNA sequential analysis and the analysis of physio biochemical characteristics result of comprehensive above-mentioned bacterial strains B501 are accredited as bacterial strain B501 the pantoea agglomerans kind (Pantoea agglomerans) of general Pseudomonas (Genus Pantoea).This bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 03 20th, 2006, and deposit number is CGMCC No.1655.
Embodiment 2, pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 fermentation culture and fungicide preparation thereof
The prescription of pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 triangular flask, seeding tank dedicated liquid substratum is: Tryptones 10g, and yeast extract 5g, sucrose 15g, NaCl 10g, water is settled to 1000mL.
The prescription of pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 fermentor tank dedicated liquid substratum is: sucrose 10g, Tryptones 5g, Semen Maydis powder 20g, soyflour 20g, wheat bran 20g, Na
2HPO
42g, NaH
2PO
40.2g, MgSO
40.3g, ZnSO
40.5g, CaCO
32g, bubble enemy 0.1g, water is settled to 1000mL.
1, the activation of bacterial classification
Picking pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 original seed is seeded in it in LB liquid nutrient medium, cultivates 1-2d at 28 ℃ of following shaking tables.
2, triangular flask is cultivated
Step 1 activatory pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 is inoculated in 5% ratio in the triangular flask that fills triangular flask dedicated liquid substratum, under 28 ℃, 150rpm, cultivated 24 hours.
3, seed tank culture
Getting the bacterium liquid that 4L step 2 is cultivated through triangular flask, insert in the 100L seeding tank (containing 50L seeding tank dedicated liquid substratum) and carry out seed culture, is 7.2 times stir culture 48 hours at 30 ℃, pH, and air flow is 26-53mMO
2L
-1h
-1
4, fermentor cultivation
In 6% ratio the seed liquor of step 2 being inserted in the 1000L fermentor tank (containing 600L fermentor tank dedicated liquid substratum) and carry out fermentation culture, is 7.5 times stir culture 72 hours at 32 ℃, pH, and air flow is 26-53mM O
2L
-1h
-1
The gained fermented liquid can directly use as pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 microbial inoculum, and can adjust fermentation condition as required, obtains the microbial inoculum of different concns.Fermented liquid after low temperature (32-35 ℃) drying, is obtained pantoea agglomerans (Pantoea agglomerans) the B501 CGMCCNo.1655 microbial inoculum of therapy in dry powder form.In addition, on the basis of above-mentioned fermented liquid, add chitin again, one or more in collagen protein and the Mierocrystalline cellulose obtain being applicable to the microbial inoculum of different diseases degree; The ratio of weight and number of described chitin and microbial inoculum is 3-5: 1000, and the ratio of weight and number of collagen protein and microbial inoculum is 5-7: 1000.
The biocontrol effect of embodiment 3, pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 detects
Now with jujube fruit black spot, apple gray mold, penicilliosis, the dark green mildew of the operatic circle soft rot and citrus is an example, detects the prevention effect of pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 (hereinafter to be referred as pantoea agglomerans B501) to Plant diseases, and concrete detection method is as follows:
One, detects the inhibition effect of pantoea agglomerans B501 to jujube fruit black spot
1, the growth of detection pantoea agglomerans B501 on winter jujube wound is dynamic
Many diseases of fruit are from the wound invasion, so the effective colonization ability of pantoea agglomerans B501 on winter jujube wound, are the prerequisites of its prevention effect of decision.Four kinds of processing of this test design: pantoea agglomerans B501 (1 * 10
9CfumL
-1)+chain lattice spore (jujube fruit black spot pathogen, 1 * 10
6Spore mL
-1); Pantoea agglomerans B501 (1 * 10
9Cfu mL
-1)+iprodione (50 μ g mL
-1) (Bayer Crop Science, pre-stage test prove that it can effectively suppress jujube fruit chain lattice spore); CaCl
2(2%)+pantoea agglomerans B501 (1 * 10
9Cfu mL
-1); Pantoea agglomerans B501 suspension (1 * 10
9CfumL
-1).Jujube fruit is after surface sterilization, and one of every fruit thorn 3mm * 3mm wound inoculates 20 μ L in the wound respectively with above four kinds of treatment solutions, the cultivation of preserving moisture under 25 ℃, and the bacterium number of measuring behind the 1h is made as initial value (0h), surveys every day 1 time, surveys 6d altogether.Measuring method is all to be 5mm pulp tissue with punch tool from wound cut-off footpath and height, places in the mortar of the phosphoric acid buffer that adds 1mL 0.2M pH6.5, grinds the back and counts with dilution-plate method.3 fruits of each processing, test repeats 3 times.Cfu is converted into the Log10 logarithm with the gained bacterium.
Draw the growth of pantoea agglomerans B501 on winter jujube wound according to the test-results data and dynamically scheme, as shown in Figure 2 (A: chain lattice spore+pantoea agglomerans B501 group; B: iprodione+pantoea agglomerans B501 group; C:CaCl
2+ pantoea agglomerans B501 group; D: pantoea agglomerans B501 suspension group.Little vertical line is represented standard error), pantoea agglomerans B501 suspension presents quick growth after the inoculation, and 72h is 21 times of 0h, shows that pantoea agglomerans B501 grows at winter jujube wound before 72h effectively surely.CaCl
2Help the increase of pantoea agglomerans B501 colony quantity with having of chain lattice spore, behind the inoculation 48h, the flora quantity of pantoea agglomerans B501 is respectively 5.4 times and 8.8 times of corresponding suspension, shows CaCl
2Help to improve the flora quantity of pantoea agglomerans B501 on wound in early stage, improved the biological and ecological methods to prevent plant disease, pests, and erosion ability of pantoea agglomerans B501, the speed of growth that chain lattice spore quickens pantoea agglomerans B501 is because nutrition and spatial competition have promoted pantoea agglomerans B501 to breed at faster speed.The existence of lower concentration iprodione has suppressed the population quantity of pantoea agglomerans B501 on winter jujube wound significantly, but also has been zooming trend, and when cultivating 6d, the bacterium number has reached 2.3 * 10
7Cfu mL
-1, show that pantoea agglomerans B501 can grow surely at winter jujube wound, can be used in combination with the lower concentration iprodione and prevent and treat winter jujube black spot under the situation that the lower concentration iprodione exists.
This test-results shows that the growth tendency of pantoea agglomerans B501 on winter jujube wound is: increase early stage fast, and the later stage shows slightly the situation of steady rising.Show that this bacterium can grow effectively surely, has the potentiality of the disease of preventing and treating on winter jujube wound.
2, the inoculation time of pantoea agglomerans B501 and chain lattice spore is to the influence of prevention effect
Test is established three groups, one group with pantoea agglomerans B501 at inoculation pathogen 6h, inoculate behind the 12h, another group with pantoea agglomerans B501 at inoculation pathogen 0h, 6h, inoculation before the 24h, 48h, control group is for only inoculating the pathogen group.The inoculum density of pantoea agglomerans B501 is 1 * 10
9Cfu mL
-1, the inoculum density of pathogen one chain lattice spore is 1 * 10
6Spore mL
-1Incidence is measured in the back cultivation of preserving moisture under 25 ℃ of inoculation behind the 7d, 10 fruits of every processing, test triplicate.
The morbidity statistics result is as shown in table 3, and the result shows that the inoculation time of pantoea agglomerans B501 and chain lattice spore is very big to the prevention effect influence.Wherein, 1 * 10
7Cfu mL
-1With 1 * 10
9Cfu mL
-1The B501 of two kinds of concentration all exists and presses down that sick effect significantly is better than simultaneously or the postpone inoculation prior to pathogen chain lattice spore inoculation, especially inoculate chain lattice spore after inoculating B50124h, obtain to press down sick effect best, with contrast and other inoculation time press down sick difference on effect significantly (P=0.05).
The table 3 pantoea agglomerans B501 different vaccination time press down sick effect
| B501 inoculation time (h) | Pantoea agglomerans B501 concentration (cfu mL -1) | |
| 1×10 7 | 1×10 9 | |
| Sickness rate (%) | Sickness rate (%) | |
| 24h behind the inoculation pathogen | 52b | 43b |
| 24h contrast before the 6h inoculation pathogen before the 0h inoculation pathogen before the 6h inoculation pathogen behind the inoculation pathogen | 37c 28d 19e 15f 100a | 32c 21d 12e 10e 100a |
Annotate: the same column same letter represents that the multiple disparity range of Deng Kenshi detects difference not remarkable (P=0.05); Numeral is three multiple mean values in the table
3, the biocontrol effect of pantoea agglomerans B501 different vaccination concentration
Compound concentration is respectively 0 (contrast), 1 * 10
5Cfu mL
-1, 1 * 10
7Cfu mL
-1With 1 * 10
9Cfu mL
-1Pantoea agglomerans B501 suspension, jujube fruit is after surface sterilization, one of every fruit thorn 3mm * 3mm wound inoculates 50 μ L in the wound respectively with the pantoea agglomerans B501 suspension of above four kinds of concentration, inoculates 1 * 10 respectively again behind the 2h
4Spore mL
-1, 1 * 10
5Spore mL
-1And 1 * 10
6Spore mL
-1Chain lattice spore spore suspension 20 μ L, above-mentioned treated fruit is preserved moisture under 25 ℃, add up sickness rate behind the 7d.10 fruits of every processing, this tests triplicate.
The morbidity statistics result is as shown in table 4, and the result shows that the different vaccination concentration of pantoea agglomerans B501 and pathogen influences the effect that presses down disease significantly.The winter jujube wound of inoculation pantoea agglomerans B501, its sickness rate significantly is lower than contrast (P=0.05).When pathogen concentration is 1 * 10
4Spore mL
-1The time, 1 * 10
5Cfu mL
-1With 1 * 10
7Cfu mL
-1Low and both no significant differences (P=0.05) of the sickness rate of pantoea agglomerans B501 inoculum density group, 1 * 10
9Cfu mL
-1Pantoea agglomerans B501 inoculum density group does not have scab and occurs.When pathogen concentration is 1 * 10
5Spore mL
-1The time, 1 * 10
7CfumL
-1With 1 * 10
9Cfu mL
-1The sickness rate difference of pantoea agglomerans B501 inoculum density group is remarkable (P=0.05) not, and both all are markedly inferior to 1 * 10
5Cfu mL
-1The sickness rate (P=0.05) of pantoea agglomerans B501 inoculum density group.When pathogen concentration is 1 * 10
6Spore mL
-1The time, 1 * 10
5Cfu mL
-1, 1 * 10
7Cfu mL
-1With 1 * 10
9Cfu mL
-1The sickness rate of pantoea agglomerans B501 inoculum density group all is remarkable difference each other, and comparison is according to having descended 71%, 80% and 89% respectively.Three kinds of concentration (1 * 10 of pantoea agglomerans B501
5Cfu mL
-1, 1 * 10
7Cfu mL
-1With 1 * 10
9CfumL
-1) sickness rate of inoculation group all surpasses 30%, show pantoea agglomerans B501 to jujube fruit black spot to press down sick effect remarkable.
The different vaccination concentration of table 4 pantoea agglomerans B501 is to the inhibition effect of jujube fruit chain lattice spore
| Pathogen concentration (Alternaria alternata) (spore mL -1) | Pantoea agglomerans B501 concentration (cfu mL -1) | |||
| 0 | 1×10 5 | 1×10 7 | 1×10 9 | |
| Sickness rate (%) | Sickness rate (%) | Sickness rate (%) | Sickness rate (%) | |
| 1×10 4 1×10 5 1×10 6 | 84a 96a 100a | 7b 22b 29b | 5b 13c 20c | 0c 9c 11d |
Annotate: colleague's same letter represents that the multiple disparity range of Deng Kenshi detects difference not remarkable (P=0.05); Numeral is three multiple mean values in the table
4, pantoea agglomerans B501 microbial inoculum is to the field control effect of jujube fruit black spot
The concentration of taking the preparation of embodiment 2 methods is respectively 5 * 10
8Cfu mL
-1With 1 * 10
9Cfu mL
-1Pantoea agglomerans B501 microbial inoculum.Picking fruit sprays the pantoea agglomerans B501 microbial inoculum of above-mentioned two kinds of concentration the last week respectively, and the microbial inoculum of every kind of concentration sprays 5 trees, room temperature storage after the fruit picking, statistics incidence.The pantoea agglomerans B501 microbial inoculum of the above-mentioned two kinds of different concns of result all has the better prevention effect to the black spot that is caused by chain lattice spore, and the high more protection effect of concentration is good more, and preventive effect reaches 62%-78%.
5, pantoea agglomerans B501 microbial inoculum is to adopting the prevention effect of back jujube fruit black spot
Take the pantoea agglomerans B501 dry powder type microbial inoculum of embodiment 2 methods preparation, be mixed with concentration and be respectively (5 * 10
8Cfu mL
-1With 1 * 10
9Cfu mL
-1) bacteria suspension.After jujube is really plucked, fruit was dipped in the pantoea agglomerans B501 bacterium liquid of above-mentioned different concns room temperature storage, statistics incidence 3 minutes.The result is after pantoea agglomerans B501 microbial inoculum is handled, and fruit has black spot prevention effect preferably, and the high more protection effect of concentration is good more, and preventive effect reaches 65%-83%.
Two, detect pantoea agglomerans B501 to adopting the inhibition effect of back green mold of apple and gray mold
Compound concentration is respectively 0 (contrast), 5 * 10
7Cfu mL
-1, 1 * 10
8Cfu mL
-1With 1 * 10
9Cfu mL
-1Pantoea agglomerans B501 suspension, fruit is after surface sterilization, one of every fruit thorn 3mm * 3mm wound inoculates 50 μ L in the wound respectively with the pantoea agglomerans B501 suspension of above four kinds of concentration, inoculates 1 * 10 respectively again behind the 2h
3Spore mL
-1, 1 * 10
4Spore mL
-1And 1 * 10
5Spore mL
-1Mould germ and ash arrhizus bacteria spore suspension 20 μ L, above-mentioned treated fruit is preserved moisture under 25 ℃, add up sickness rate behind the 7d.10 fruits of every processing, this tests triplicate.
Morbidity statistics result behind inoculation mould germ spore and the ash arrhizus bacteria spore shown in table 5, table 6, inoculates lower concentration (1 * 10 respectively on the apple wound
3Spore mL
-1) during the mould germ, only 1 * 10
9Cfu mL
-1Under the inoculum density of pantoea agglomerans B501, sickness rate just is controlled at 10%.And ash arrhizus bacteria is at this lower concentration (1 * 10
3Spore mL
-1) when inoculating, 5 * 10
7, 1 * 10
8, 1 * 10
9Cfu mL
-1Sickness rate under three kinds of pantoea agglomerans B501 inoculum densities all is lower than 10%.When the inoculum density of pantoea agglomerans B501 is 1 * 10
9Cfu mL
-1, pathogen is 1 * 10
3, 1 * 10
4, 1 * 10
5Spore mL
-1During three kinds of different concns, the inhibition effect of penicilliosis is respectively 90%, 64%, 44%, and the inhibition effect of gray mold is respectively 100%, 77%, 58%.The above results shows at room temperature, and pantoea agglomerans B501 can suppress to adopt the generation of back green mold of apple and gray mold effectively, and pantoea agglomerans B501 is better than penicilliosis to the inhibition effect of gray mold.
Table 5 pantoea agglomerans B501 is to adopting the inhibition effect of back green mold of apple
| Pathogen concentration (Penicillium | Pantoea agglomerans B501 concentration (cfu mL -1) | |||
| 0 | 5×10 7 | 1×10 8 | 1×10 9 | |
| Sickness rate (%) | Sickness rate (%) | Sickness rate (%) | Sickness rate (%) | |
| 1×10 3 | 100a | 31b | 30b | 10c |
| 1×10 4 | 100a | 52b | 39c | 36c |
| 1×10 5 | 100a | 65b | 62b | 56b |
Annotate: colleague's same letter represents that the multiple disparity range of Deng Kenshi detects difference not remarkable (P=0.05); Numeral is three multiple mean values in the table
Table 6 pantoea agglomerans B501 is to adopting the inhibition effect of back apple gray mold
| Pathogen concentration (Botrytis cinerea) (spore mL -1) | Pantoea agglomerans B501 concentration (cfu mL -1) | |||
| 0 | 5×10 7 | 1×10 8 | 1×10 9 | |
| Sickness rate (%) | Sickness rate (%) | Sickness rate (%) | Sickness rate (%) | |
| 1×10 3 | 98a | 5b | 2b | 0b |
| 1×10 4 | 100a | 36b | 35b | 23c |
| 1×10 5 | 100a | 63b | 61b | 42c |
Annotate: colleague's same letter represents that the multiple disparity range of Deng Kenshi detects difference not remarkable (P=0.05); Numeral is three multiple mean values in the table
Three, detect pantoea agglomerans B501 to adopting the inhibition effect of back the operatic circle soft rot
Compound concentration is respectively 0 (contrast), 5 * 10
7Cfu mL
-1, 1 * 10
8Cfu mL
-1With 1 * 10
9Cfu mL
-1Pantoea agglomerans B501 suspension, fruit is after surface sterilization, one of every fruit thorn 3mm * 3mm wound inoculates 50 μ L with the pantoea agglomerans B501 suspension of above four kinds of concentration respectively in the wound, inoculates 1 * 10 respectively again behind the 2h
3Spore mL
-1, 1 * 10
4Spore mL
-1And 1 * 10
5Spore mL
-1Bacteria erwinia spore suspension 20 μ L, above-mentioned treated fruit is preserved moisture under 25 ℃, add up sickness rate behind the 7d.10 fruits of every processing, this tests triplicate.
The morbidity statistics result is respectively as shown in table 7, when pantoea agglomerans B501 with high density 1 * 10
9Cfu mL
-1Use, and pathogen is 1 * 10
3, 1 * 10
4, 1 * 10
5Spore mL
-1Under three kinds of different concns, the inhibition effect of soft rot is respectively 98%, 87%, 72%.Show that pantoea agglomerans B501 can effectively suppress the operatic circle soft rot.
Table 7 pantoea agglomerans B501 is to adopting the inhibition effect of back the operatic circle soft rot
| Pathogen concentration (Rhizopus stolonifer) (spore mL -1) | Pantoea agglomerans B501 concentration (cfu mL -1) | |||
| 0 | 5×10 7 | 1×10 8 | 1×10 9 | |
| Sickness rate (%) | Sickness rate (%) | Sickness rate (%) | Sickness rate (%) | |
| 1×10 3 1×10 4 1×10 5 | 96a 100a 100a | 9b 26b 57b | 5b 23b 35c | 2b 13c 28d |
Annotate: colleague's same letter represents that the multiple disparity range of Deng Kenshi detects difference not remarkable (P=0.05); Numeral is three multiple mean values in the table
Four, detect the inhibition effect of pantoea agglomerans B501 to the dark green mildew of citrus
Compound concentration is respectively 0 (contrast), 5 * 10
7Cfu mL
-1, 1 * 10
8Cfu mL
-1With 1 * 10
9Cfu mL
-1Pantoea agglomerans B501 suspension, fruit is after surface sterilization, one of every fruit thorn 3mm * 3mm wound inoculates 50 μ L in the wound respectively with the pantoea agglomerans B501 suspension of above four kinds of concentration, inoculates 1 * 10 respectively again behind the 2h
3Spore mL
-1, 1 * 10
4Spore mL
-1And 1 * 10
5Spore mL
-1Dark green mildew pathogenic bacterium P.italicum and the spore suspension 20 μ L of P.digitatum, above-mentioned treated fruit is preserved moisture under 25 ℃, add up sickness rate behind the 7d.10 fruits of every processing, this tests triplicate.
As a result 1 * 10
8Cfu mL
-1The pantoea agglomerans B501 of concentration can significantly suppress by pathogenic bacterium P.italicum (1 * 10
5Spore mL
-1) and P.digitatum (1 * 10
5Spore mL
-1) generation of the dark green mildew of citrus that causes, the sickness rate comparison is according to having descended 67% and 85% respectively.When the concentration of pantoea agglomerans B501 rises to 1 * 10
9Cfu mL
-1The time, by P.italicum (1 * 10
5Spore mL
-1) and P.digitatum (1 * 10
5Spore mL
-1) sickness rate of the dark green mildew that causes compares respectively according to having descended 75% and 92%.Show that pantoea agglomerans B501 can effectively suppress the generation of the dark green mildew of citrus.
Embodiment 4, the viable cell that detects pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 and metabolite thereof are to the influence of winter jujube black spot
The treatment solution of following four kinds of pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 is adopted in this experiment:
Cultivate stoste (A): be made into 1 * 10 with blood counting chamber
9Cfu mL
-1Concentration;
Filtrate (B): will cultivate stoste with 0.2 μ m membrane filtration, and collect filtrate;
Suspension (C): will cultivate stoste centrifugal 5min under 12000rpm, and abandon supernatant, and add sterilized water, repeated centrifugation behind the mixing is abandoned supernatant, adds sterilized water again, is made into 1 * 10
9Cfu mL
-1The suspension of concentration;
The dead liquid of heat kill (D): will cultivate stoste at 121 ℃ of following autoclaving 20min.
Above-mentioned four kinds of treatment solutions are got 50 μ L respectively to be inoculated in winter jujube wound (3mm * 3mm), be contrast with the aqua sterilisa inoculates 20 μ L 1 * 10 more respectively behind the 2h
6Spore mL
-1Chain lattice spore spore suspension, preserve moisture under 25 ℃, add up incidence behind the 7d, this tests triplicate.
The morbidity statistics result is (A: stoste as shown in Figure 3; B: filtered liquid; C: suspension; D: the dead liquid of heat kill; Contrast: aqua sterilisa.Little vertical line is represented standard error), concentration is 1 * 10
9Cfu mL
-1Pantoea agglomerans (Pantoeaagglomerans) B501 CGMCC No.1655 suspension the inhibition effect of winter jujube chain lattice spore is better than stoste, both sickness rate are remarkable difference (P=0.05).And filtrate is the same with contrast with sterilized solution, and sickness rate all reaches 100%.This result and pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 do not produce antibiotic result on plate consistent, the biocontrol effect that shows pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 depends on viable cell rather than its meta-bolites, has higher security.
Sequence table
<160>1
<210>1
<211>1323
<212>DNA
＜213〉pantoea agglomerans (Pantoea agglomerans)
<400>1
ggacggtagc acagaggagc ttgctccttg ggtgacgagt ggcggacggg tgagtaatgt 60
ctggggatct gcccgataga gggggataac cactggaaac ggtggctaat accgcataac 120
gtcgcaagac caaagagggg gaccttcggg cctctcacta tcggatgaac ccagatggga 180
ttagctagta ggcggggtaa tggcccacct aggcgacgat ccctagctgg tctgagagga 240
tgaccagcca cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga 300
atattgcaca atgggcgcaa gcctgatgca gccatgccgc gtgtatgaag aaggccttcg 360
ggttgtaaag tactttcagc ggggaggaag gcgatggggt taataaccct gtcgattgac 420
gttacccgca gaagaagcac cggctaactc cgtgccagca gccgcggtaa tacggagggt 480
gcaagcgtta atcggaatta ctgggcgtaa agcgcatgca ggcggtctgt taagtcagat 540
gtgaaatccc cgggcttaac ctgggaactg catttgaaac tggcaggctt gagtcttgta 600
gaggggggta gaattccagg tgtagcggtg aaatgcgtag agatctggag gaataccggt 660
ggcgaaggcg gccccctgga caaagactga cgctcaggtg cgaaagcgtg gggagcaaac 720
aggattagat accctggtag tccacgccgt aaacgatgtc gacttggagg ttgttccctt 780
gaggagtggc ttccggagct aacgcgttaa gtcgaccgcc tggggagtac ggccgcaagg 840
ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 900
atgcaacgcg aagaacctta cctactcttg acatccagcg aacttagcag agatgctttg 960
gtgccttcgg gaacgctgag acaggtgctg catggctgtc gtcagctcgt gttgtgaaat 1020
gttgggttaa gtcccgcaac gagcgcaacc cttatccttt gttgccagcg attcggtcgg 1080
gaactcaaag gagactgccg gtgataaacc ggaggaaggt ggggatgacg tcaagtcatc 1140
atggccctta cgagtagggc tacacacgtg ctacaatggc gcatacaaag agaagcgacc 1200
tcgcgagagc aagcggacct cacaaagtgc gtcgtagtcc ggatcggagt ctgcaactcg 1260
actccgtgaa gtcggaatcg ctagtaatcg tggatcagaa tgccacggtg aatacgttcc 1320
cgg 1323
Claims (10)
1, pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655.
2, the method for a kind of fermentation culture pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 may further comprise the steps:
1) pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 being inoculated in its triangular flask, the seeding tank special culture media, is to cultivate 24-48 hour under the 7.0-8.0 at 28-35 ℃, pH, obtains seed liquor; Triangular flask, the seeding tank special culture media prescription of described pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 are: Tryptones 8-12g, and yeast extract 4-6g, sucrose 10-20g, NaCl 8-12g, water is settled to 1000mL;
2) seed liquor with step 1) preparation is inoculated in the fermentor tank special culture media of this bacterium, cultivates 48-96 hour under the 7.0-8.0 at 28-35 ℃, pH; The prescription of the fermentor tank special culture media of described pantoea agglomerans (Pantoea agglomerans) B501 CGMCCNo.1655 is: sucrose 8-12g, Tryptones 4-6g, Semen Maydis powder 15-25g, soyflour 15-25g, wheat bran 15-25g, Na
2HPO
41.5-2.5g, NaH
2PO
40.15-0.25g, MgSO
40.2-0.4g, ZnSO
40.4-0.6g, CaCO
31.5-2.5g, bubble enemy 0.08-0.12g, water is settled to 1000mL.
3, fermentation culture method according to claim 2, it is characterized in that: described step 1) and step 2) in the inoculative proportion of pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 and seed liquor thereof be 5-8%, culture condition is: at 28-32 ℃, pH is aerated culture under the 7.2-7.5, and air flow is 26-53mM O
2L
-1h
-1
4, according to claim 2 or 3 described fermentation culture methods, it is characterized in that: the pantoea agglomerans in the described step 1) (Pantoea agglomerans) B501 CGMCC No.1655 triangular flask, seeding tank special culture media prescription are: Tryptones 10g, yeast extract 5g, sucrose 15g, NaCl 10g, water is settled to 1000mL; Step 2) pantoea agglomerans in (Pantoea agglomerans) B501 CGMCC No.1655 fermentor tank special culture media prescription is: sucrose 10g, Tryptones 5g, Semen Maydis powder 20g, soyflour 20g, wheat bran 20g, Na
2HPO
42g, NaH
2PO
40.2g, MgSO
40.3g, ZnSO
40.5g, CaCO
32g, bubble enemy 0.1g, water is settled to 1000mL.
5, according to claim 2 or 3 described fermentation culture methods, it is characterized in that: earlier pantoea agglomerans (Pantoeaagglomerans) B501 CGMCC No.1655 is carried out triangular flask and cultivate, to be inoculated in the seeding tank through the bacterium liquid that triangular flask is cultivated again, carry out step 1) and step 2 successively) cultivation; Described triangular flask cultural method is: pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 is inoculated in its triangular flask, the seeding tank special culture media, is to cultivate 24-48 hour under the 7.0-8.0 at 28-35 ℃, pH.
6, be the microbial inoculum of activeconstituents with pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655.
7, microbial inoculum according to claim 6 is characterized in that: contain chitin in the described microbial inoculum, one or more in collagen protein and the Mierocrystalline cellulose; The ratio of weight and number of described chitin and microbial inoculum is 3-5: 1000, and the ratio of weight and number of collagen protein and microbial inoculum is 5-7: 1000.
8, the application of the described pantoea agglomerans of claim 1 (Pantoea agglomerans) B501 CGMCC No.1655 bacterium in control fruits and vegetables fungal disease.
9, the application of the described pantoea agglomerans of claim 6 (Pantoea agglomerans) B501 CGMCC No.1655 microbial inoculum in control fruits and vegetables fungal disease.
10, application according to claim 9 is characterized in that: gathering preceding is 5 * 10 at the fruit and vegetable surfaces spraying concentration
8Cfu mL
-1With 1 * 10
9Cfu mL
-1Pantoea agglomerans (Pantoea agglomerans) B501 CGMCCNo.1655 microbial inoculum, it is 5 * 10 that the fruit after maybe will plucking is soaked in concentration
8Cfu mL
-1With 1 * 10
9Cfu mL
-1Pantoea agglomerans (Pantoea agglomerans) B501 CGMCC No.1655 microbial inoculum in handled 1-3 minute.
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