CN101864378A - Streptomyces microflavus - Google Patents
Streptomyces microflavus Download PDFInfo
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Abstract
The invention discloses a streptomyces microflavus. The streptomyces microflavus is the streptomyces microflavus NMG2-4-8, and the preservation number thereof is CGMCG No.3442. The streptomyces microflavus is separated and screened from alfalfa healthy tree rhizosphere soil for the first time in the invention, and experiments prove that the streptomyces microflavus has remarkable antagonistic action for 18 pathogenic fungi and 9 bacteria and broad antibacterial spectrum. The streptomyces microflavus has strong inhabiting action for crop pathogenic bacteria, such as alfalfa downy mildew and the like, shows a good disease prevention effect for the alfalfa downy mildew, powdery mildew, brown spots, stemphylium leaf spot disease, root rot disease and the like, and indicates that the bacterial strain has broad application prospect in the biological prevention and cure fields of alfalfa diseases.
Description
Technical field
The present invention relates to a strain streptomyces microflavus.
Background technology
Biocontrol of plant disease is emphasized the benign cycle and the environment protection of the ecosystem, and a series of problems that can avoid chemical pesticide effectively and brought are because its social benefit, ecological benefits, long-term interest significantly and more and more cause people's attention.Along with people more and more pay attention to the eubiosis and green health notion, bio-control method has obtained unprecedented concern in agriculture production.Entered since 21 century, the national governments and the common people pay attention to the development and application of biological pesticide more, and biological pesticide rises year by year in the share in whole agricultural chemicals market.In environmental pollution serious day by day today, biological control is the only way of agricultural development especially.Actinomycetes are the biological and ecological methods to prevent plant disease, pests, and erosion microorganisms that are applied to the earliest in the production, its important economic worth is because the microbiotic that they produced can be prevented and treated human and animal's contagious disease effectively, some disease of particularly bacteroidal disease and farm crop, it has brought into play enormous function in biocontrol of plant disease.In about 8000 kinds of biologically active substances of from microorganism, finding at present, have nearly 70% to be that actinomycetes produce.Utilize the novel pesticide of actinomycetic secondary metabolite one microbiotic preparation because that it has is pollution-free, noresidue, renewable, be easy to characteristics such as industrialization, cost be low and become the main body of public nuisance-free agricultural chemicals and the developing direction of following agricultural chemicals.
Clover is most important leguminous forage, is described as " king of herbage ", and the clover cultivated area of China occupies first of the various herbages at present.In recent years, along with the development and the agricultural structure adjustment of China's grassland agriculture, the cultivated area of clover further enlarged, and the disease problem becomes increasingly conspicuous.Disease not only makes the big spoke degree of alfalfa output reduce, and nutritional components reduces, and also has a strong impact on output and the feeding value of clover.Downy mildew of alfalfa, Powdery Mildew, brown spot, root rot etc. are the bigger several diseases of harm on the current ALFALFA PRODUCTION, when causing the clover underproduction serious even total crop failure.Currently do a lot of work from aspects such as breeding for disease resistance, chemical prevention and agricultural measures both at home and abroad, protection effect is not satisfactory.And because clover as singularity and the factors such as economy, environment and food safety of forage grass, uses chemical pesticide that strict restriction is arranged in its breeding time.Therefore biological control has boundless application prospect equally in sustainable management of clover disease, develop the antagonism bacterium and be effectively to control one of the most potential prophylactico-therapeutic measures of clover disease.
Summary of the invention
An object of the present invention is to provide a strain streptomyces microflavus.
Streptomyces microflavus provided by the present invention is streptomyces microflavus (Streptomyces microflavus) NMG2-4-8, and its deposit number is CGMCC No.3442.
Another object of the present invention provides the antagonist of a kind of bacterium.
The antagonist of bacterium provided by the present invention, its activeconstituents are streptomyces microflavus (Streptomycesmicroflavus) NMG2-4-8 CGMCC No.3442; Described bacterium is gram positive bacterium, gram negative bacterium or fungi.
In the antagonist of above-mentioned bacterium, described gram positive bacterium is that potato encircles rotten excellent bacillus Clavibactermichiganensis subsp.sepedonicus, streptococcus aureus Staphylococcus aureus, subtilis Bacillus subtilis or bacillus cereus Bacillus cereus
Described gram negative bacterium is carrot soft rot Erwinia Erwinia carotovora var.carotovora, blue or green withered Lei Er Salmonella Ralstonia solanacearum, clover Xanthomonas campestris Xanthomonascampestris pv.alfalfae, intestinal bacteria Escherichia coli or Pseudomonas fluorescens Pseudomouasuoresceus
Described fungi is Sclerotinia sclerotiorum Sclerotinia sclerotiorum, cucumber fusarium axysporum Fusariumoxysporum f.sp.cucumerinum, watermelon anthrax bacteria Colletotrichum lagenarium, clover eggplant fusarium Fusarium solani, dry thread Pyrenomycetes Rhizoctonia solani, clover septoria musiva leaf spot fungi Septoria medicaginis, the mould Pythium ultimum of ultimate corruption, big beautiful Verticillium Verticillium dahlia, gaeumannomyce Gaeumannomyces graminis, the clover mould Stemphylium botryosum of handle that crawls, standing grain is bent spore mould Curvularia lunata, Phytophthora capsici Phytophthora capsici, Pyricularia oryzae Pyriculariagrisea, alternaric bacteria Alternaria tenuis, clover point sickle spore bacterium Fusarium oxysporum, tomato gray mould bacterium Botrytis cinerea, candiyeast Candida sp., geotrichum candidum Geotrichum candidum, aspergillus oryzae Aspergillus oryza or Penicilllum expansum Penicillium expansum.
In the antagonist of above-mentioned bacterium, it is that potato encircles rotten excellent bacillus Clavibacter michiganensis subsp.sepedonicusACCC 01233 that described potato encircles rotten excellent bacillus Clavibacter michiganensis subsp.Sepedonicus, described streptococcus aureus Staphylococcus aureus is streptococcus aureus Staphylococcus aureusACCC01336, described subtilis Bacillus subtilis is subtilis Bacillus subtilis CGMCC1.1656, described bacillus cereus Bacillus cereus is bacillus cereus Bacillus cereus CGMCC 1.348
Described carrot soft rot Erwinia Erwinia carotovora var.carotovora is carrot soft rot Erwinia Erwinia carotovora var.carotovora CGMCC1.1000, the withered Lei Er Salmonella of described green grass or young crops Ralstonia solanacearum is blue or green withered Lei Er Salmonella Ralstonia solanacearum ACCC01470, described intestinal bacteria Escherichia coli is intestinal bacteria Escherichia coli ACCC10196, described Pseudomonas fluorescens Pseudomouas uoresceus is Pseudomonas fluorescens Pseudomouas uoresceusACCC01241
Described Sclerotinia sclerotiorum Sclerotinia sclerotiorum is Sclerotinia sclerotiorum SclerotiniaSclerotiorum ACCC36169, described cucumber fusarium axysporum Fusarium oxysporum f.sp.cucumerinum is cucumber fusarium axysporum Fusarium oxysporum f.sp.cucumerinumACCC30442, described watermelon anthrax bacteria Colletotrichum lagenarium is watermelon anthrax bacteria Colletotrichumlagenarium ACCC36152, described dry thread Pyrenomycetes Rhizoctonia solani is dry thread Pyrenomycetes Rhizoctonia solani ACCC30332, the mould Pythium ultimum of described ultimate corruption is the mould Pythium ultimumACCC36075 of ultimate corruption, described big beautiful Verticillium Verticillium dahlia is big beautiful Verticillium Verticillium dahliaACCC36109, described gaeumannomyce Gaeumannomyces graminis is gaeumannomyce Gaeumannomyces graminis ACCC30310, the curved spore mould Curvularia lunata of described standing grain is the curved spore mould Curvularia lunataACCC36580 of standing grain, described Phytophthora capsici Phytophthora capsici is Phytophthora capsici Phytophthora capsiciACCC36279, described Pyricularia oryzae Pyricularia grisea is Pyricularia oryzae Pyricularia griseaACCC30320, described alternaric bacteria Alternaria tenuis is alternaric bacteria Alternaria tenuisACCC36110, described tomato gray mould bacterium Botrytis cinerea is tomato gray mould bacterium Botrytis cinerea ACCC30091, described candiyeast Candida sp. is candiyeast Candida sp.ACCC21131, described geotrichum candidum Geotrichum candidum is geotrichum candidum Geotrichumcandidum CGMCC2.1084, described aspergillus oryzae Aspergillus oryza is aspergillus oryzae Aspergillusoryza ACCC30155, and described Penicilllum expansum Penicillium expansum is Penicilllum expansum PenicilliumexpansumACCC30898.
The application of streptomyces microflavus (Streptomyces microflavus) NMG2-4-8 CGMCC No.3442 in antagonism gram positive bacterium, gram negative bacterium or fungi also belongs to protection scope of the present invention.
In the above-mentioned application, described gram positive bacterium is that potato encircles rotten excellent bacillus Clavibactermichiganensis subsp.sepedonicus, streptococcus aureus Staphylococcus aureus, subtilis Bacillus subtilis or bacillus cereus Bacillus cereus
Described gram negative bacterium is carrot soft rot Erwinia Erwinia carotovora var.carotovora, blue or green withered Lei Er Salmonella Ralstonia solanacearum, clover Xanthomonas campestris Xanthomonascampestris pv.alfalfae, intestinal bacteria Escherichia coli or Pseudomonas fluorescens Pseudomouasuoresceus
Described fungi is Sclerotinia sclerotiorum Sclerotinia sclerotiorum, cucumber fusarium axysporum Fusariumoxysporum f.sp.cucumerinum, watermelon anthrax bacteria Colletotrichum lagenarium, clover eggplant fusarium Fusarium solani, dry thread Pyrenomycetes Rhizoctonia solani, clover septoria musiva leaf spot fungi Septoria medicaginis, the mould Pythium ultimum of ultimate corruption, big beautiful Verticillium Verticillium dahlia, gaeumannomyce Gaeumannomyces graminis, the clover mould Stemphylium botryosum of handle that crawls, standing grain is bent spore mould Curvularia lunata, Phytophthora capsici Phytophthora capsici, Pyricularia oryzae Pyriculariagrisea, alternaric bacteria Alternaria tenuis, clover point sickle spore bacterium Fusarium oxysporum, tomato gray mould bacterium Botrytis cinerea, candiyeast Candida sp., geotrichum candidum Geotrichum candidum, aspergillus oryzae Aspergillus oryza or Penicilllum expansum Penicillium expansum.
In the above-mentioned application, it is that potato encircles rotten excellent bacillus Clavibacter michiganensis subsp.sepedonicusACCC 01233 that described potato encircles rotten excellent bacillus Clavibacter michiganensis subsp.Seoedonicus, described streptococcus aureus Staphylococcus aureus is streptococcus aureus Staphylococcus aureusACCC01336, described subtilis Bacillus subtilis is subtilis Bacillus subtilis CGMCC1.1656, described bacillus cereus Bacillus cereus is bacillus cereus Bacillus cereus CGMCC 1.348
Described carrot soft rot Erwinia Erwinia carotovora var.carotovora is carrot soft rot Erwinia Erwinia carotovora var.carotovora CGMCC1.1000, the withered Lei Er Salmonella of described green grass or young crops Ralstonia solanacearum is blue or green withered Lei Er Salmonella Ralstonia solanacearum ACCC01470, described intestinal bacteria Escherichia coli is intestinal bacteria Escherichia coli ACCC10196, described Pseudomonas fluorescens Pseudomouas uoresceus is Pseudomonas fluorescens Pseudomouas uoresceusACCC01241
Described Sclerotinia sclerotiorum Sclerotinia sclerotiorum is Sclerotinia sclerotiorum SclerotiniaSclerotiorum ACCC36169, described cucumber fusarium axysporum Fusarium oxysporum f.sp.cucumerinum is cucumber fusarium axysporum Fusarium oxysporum f.sp.cucumerinumACCC30442, described watermelon anthrax bacteria Colletotrichum lagenarium is watermelon anthrax bacteria Colletotrichumlagenarium ACCC36152, described dry thread Pyrenomycetes Rhizoctonia solani is dry thread Pyrenomycetes Rhizoctonia solani ACCC30332, the mould Pythium ultimum of described ultimate corruption is the mould Pythium ultimumACCC36075 of ultimate corruption, described big beautiful Verticillium Verticillium dahlia is big beautiful Verticillium Verticillium dahliaACCC36109, described gaeumannomyce Gaeumannomyces graminis is gaeumannomyce Gaeumannomyces graminis ACCC30310, the curved spore mould Curvularia lunata of described standing grain is the curved spore mould Curvularia lunataACCC36580 of standing grain, described Phytophthora capsici Phytophthora capsici is Phytophthora capsici Phytophthora capsiciACCC36279, described Pyricularia oryzae Pyricularia grisea is Pyricularia oryzae Pyricularia griseaACCC30320, described alternaric bacteria Alternaria tenuis is alternaric bacteria Alternaria tenuisACCC36110, described tomato gray mould bacterium Botrytis cinerea is tomato gray mould bacterium Botrytis cinerea ACCC30091, described candiyeast Candida sp. is candiyeast Candida sp.ACCC21131, described geotrichum candidum Geotrichum candidum is geotrichum candidum Geotrichumcandidum CGMCC2.1084, described aspergillus oryzae Aspergillus oryza is aspergillus oryzae Aspergillusoryza ACCC30155, and described Penicilllum expansum Penicillium expansum is Penicilllum expansum PenicilliumexpansumACCC30898.
The present invention first from clover healthy tree rhizosphere soil separation screening to a strain streptomyces microflavus Streptomycesmicroflavus NMG2-4-8, experimental results show that, this streptomyces microflavus Streptomyces microflavus has remarkable antagonistic action, has a broad antifungal spectrum to 18 kinds of pathogenic fungies and 9 kinds of bacteriums.This streptomyces microflavus Streptomyces microflavus has very strong restraining effect to phytopathogens such as downy mildew of alfalfa, as downy mildew of alfalfa, Powdery Mildew, brown spot, the mould leaf spot of handle of crawling etc. demonstrated good protection effect, illustrate that this bacterial strain has broad application prospects in the biological control field of clover disease.
Description of drawings
Fig. 1 is NMG2-4-8 aerial hyphae form (under the opticmicroscope 200 *).
Fig. 2 is bacterial strain NMG2-4-8 spore shape (under the scanning electron microscope 5000 *).
Fig. 3 is bacterial strain NMG2-4-8 fibrillae of spores form (under the scanning electron microscope 10000 *).
Fig. 4 is the phylogenetic tree according to the bacterial strain NMG2-4-8 of 16S rDNA gene order structure.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The separation of embodiment 1, bacterial strain and evaluation
One, separates
For trying soil sample: pick up from the living clover healthy tree of Tongliao, Inner Mongolia area 2-3 rhizosphere soil.
Strains tested: provide by grassland research institute, China Agriculture academy of sciences's herbage disease control group;
Substratum: 1. seed culture medium: every premium on currency contains the 5g peptone, 3g yeast extract paste, 2g glucose, 1g extractum carnis, PH7.0~7.2; 2. basic fermention medium: every premium on currency contains the 25g soyflour, 40g starch, 0.8g (NH
4)
2SO
4, 3g CaCO
3, 2g NaCl, PH7.0~7.2.
The preparation of pedotheque diluent: pedotheque picks up from the living clover healthy tree of Tongliao, Inner Mongolia area 2-3 rhizosphere soil.During sampling, remove the residual body of ground flora, gather the pedotheque at 1~15cm place, put into the special aluminum ware of sterilization with the self-control soil sampler.Soil sample is sieved through natural air drying, weighs, and takes by weighing soil sample 10g, puts into the 100mL sterilized water, shakes 30min on the 120r/min shaking table.Leave standstill and get supernatant liquor 100 μ L and add 900 μ L sterilized waters, serial gradient dilution is prepared into different concns (10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6) the pedotheque diluent.
The separation of bacterial strain, purifying: adopt dilution-plate method to separate and obtain actinomycetes strain.The even coating of diluent of drawing different concns with pipettor is inoculated in Gause I substratum (Zulkovsky starch 20g, agar 20g, KNO
31g, K
2HPO
40.5g, MgSO
47H
2O 0.5g, NaCl 0.5g, FeSO
47H
2O 0.01g, distilled water 1000mL, pH7.2~7.4) planar surface, every concentration repeats for 3 times, places 28 ℃ incubator to cultivate 5~10d, observes the colony growth situation.Picking list bacterium colony dilutes line separation and Culture, purifying.The breeding preservation on the Gause I substratum according to a conventional method of actinomycetes after purified.
The actinomycetes that are separated to are inoculated in the Gause I agar plate, stand-by behind 28 ℃ of following cultivation 5d.9 kinds of bacteriums and 18 kind of plant pathogenic fungies (seeing Table 8) are inoculated in beef extract-peptone nutrient agar (extractum carnis 3g, peptone 10g, sodium-chlor 5g, agar 20g, distilled water 1000mL, pH7.2), PDA nutrient agar (potato (peeling) 200g, sucrose (or glucose) 20g, distilled water 1000mL, pH nature) planar surface, fungi is cultivated 3-5d for 25 ℃, and 2d is stand-by for 28 ℃ of cultivations of bacterium.
Adopt dull and stereotyped face-off method to carry out the primary dcreening operation of thalline bacteriostatic activity for the examination actinomycetes.With cultured actinomycetes with sterilization punch tool cut-off footpath 5mm the bacterium dish place in the middle of the PDA flat board, behind the 3d onesize clover stemphylium botryosum Stemphyllium botryosum bacterium cake face-off is connected to the actinomycetes periphery, cultivate 5d down for 25 ℃, routine observation, write down antibacterial result, and measure the diameter that presses down (molten) bacterium circle.Therefrom filter out strong antagonistic strain according to the size that presses down (molten) bacterium loop diameter with maximum bacteriostatic activity.
The result obtains 15 strains altogether to the stronger antagonism actinomycetes strain of clover stemphylium botryosum Stemphyllium botryosum bacteriostatic action, and it is best that wherein a strain name is called the bacterial strain fungistatic effect of NMG2-4-8, and its antibacterial circle diameter is 35.52mm.
Two, identify
(1) strain culturing feature
Morphological specificity is observed: bacterial strain NMG2-4-8 to be identified is made inserted sheet cultivate, with the photomicrography as a result of viewed form.
Cultural characteristic is observed: with bacterial strain to be identified, be seeded in respectively Gause I, Cha Shi, glucose-asparagine, No. one, Ke Shi, potato soak on juice, the glucose yeast cream agar slant and the potato ball substratum on, place 28 ℃ of cultivations, respectively 7,14 and 28d observe its cultural characteristic and colour-change.Get and stablize sophisticated color characteristic, as identifying the foundation of planting as its cultural characteristic.Observe color, the color of matrix filament and the color of soluble pigment of record aerial hyphae.The result charges to the evaluation table.In addition, observe the upgrowth situation of bacterial strain, as the quality of growing, aerial hyphae presents how appearance-velvet-like, powdery or cotton-shaped etc. as with reference to feature.
(2) bacterial classification physio-biochemical characteristics
The gelatine liquefication ability is measured: with inoculation in cylindricality gelatine culture surface, 22 ℃ of following constant temperature culture, respectively 5,10,20, and 30d, each observes the degree that once liquefies.Should be before observing with the freezing 20~30min of bacterial classification pipe.Not liquefying as gelatin is still solid state, is liquefaction if present liquid.
Milk solidifies and peptonize mensuration: with inoculation to be determined in skimmed milk, 28 ℃ of constant temperature culture, respectively the 3rd, 6,10,20, and 30d respectively observe 1 time.
The starch hydrolysis is measured: after substratum is dissolved, cool off 50 ℃ and fall dull and stereotyped, after solidifying with the bacterial classification dibbling on flat board, thermophilic is cultivated 2~4d, drip road Ge Shi iodine liquid after forming lawn on flat board, be advisable to be paved with periphery of bacterial colonies, it is blue that flat board is, and periphery of bacterial colonies has or not transparent circle to occur illustrating whether starch is hydrolyzed, and the size of transparent circle can illustrate the size of hydrolyzed starch ability.
Cellulose hydrolysis test: preparation is fit to the actinomycetes growth and the synthetic basic culture solution of carbonaceous sources not, behind the packing test tube, make Mierocrystalline cellulose (carbon source) with filter paper of Xinhua, it is cut into wide 1cm, long 6cm filter paper bar, add in the test tube, half is immersed in the liquid, and half is exposed at outside the liquid, to identify inoculation outside liquid on one section filter paper bar, observe after putting 28 ℃ of constant temperature culture 30d.If this bacterium can be resolved into the filter paper bar loose fibres, or makes it to fracture, it is positive to be fragmented into matter shape person, illustrates that this bacterial strain produces cellulase and makes it hydrolysis.Otherwise the person is negative for the filter paper no change.
Hydrogen sulfide produces test: inoculation to be measured to the organic inclined-plane that contains ironic citrate, is put 28 ℃ and cultivated (looking the lawn growth and maturity is advisable) observations about 10d.If occur the chocolate precipitation in the substratum, the expression experiment is positive.Otherwise nondiscoloration person is negative.
Utilization of carbon source test: with 1~2 of the spore suspension of bacterial strain to be identified, be seeded on the no carbon source substratum, be coated with evenly with aseptic spreading rod, the ditching of ruling then is the boundary with the ditch, and plate culture medium is divided into some sub-districts, and every sub-district adds a kind of carbon source respectively.Carbon source commonly used has 9 kinds: pectinose, semi-lactosi, rhamnosyl, wood sugar, fructose, sucrose, sorbose, N.F,USP MANNITOL and inositol etc.Every ware need be established the sugar-free check plot.After putting 28 ℃ of constant temperature culture 7,14d, observe the record growth respectively and utilize situation.
(3) evaluation of bacterium cell wall chemical composition
Paper layer chromatography cell walls amino acid analysis is according to G
+The kind of the 3rd amino acids in the bacteria cell wall peptidoglycan molecule is divided into nine monoids such as tables 1 with actinomycetes.
The main type of table 1 actinomycetes cell walls
Annotate: LL-DAP: racemize diamino acidic group pimelic acid; Meso-DAP: meso diaminopimelic acid; DAB:1,4-dihydroxyl butyric acid.
Key step is:
(1) thalline preparation: scrape and get the centrifuge tube that cultured bacterial strain is put into 1.5mL;
(2) thalline hydrolysis: the HCl 100 μ L at the thalline adding 6mol/L that is used for full cell amino acid analysis, put into 120 ℃ of 15min of Autoclave;
(3) point sample: get hydrolyzed solution point sample on No. 1 filter paper of Xinhua that 4 μ L are used for amino acid analysis, get standard amino acid sample diaminopimelic acid DAP (containing LL-DAP, Meso-DAP and DD-DAP), Methionin, ornithine, aspartic acid, glycine, L-glutamic acid, each 1 μ L of L-Ala interval point sample on filter paper successively more respectively.
(4) exhibition layer: the exhibition layer system that is used for the analysis of full cell amino acid hydrolyticsolution is a methyl alcohol: water: HCl (6N): pyridine=16: 5.2: 0.8: 2, and exhibition layer 2 times;
(5) colour developing: amino acid analysis is with 0.4% triketohydrindene hydrate acetone soln, 100~110 ℃ of heating 2~3min, observation.
The full cell walls sugar of paper layer chromatography is analyzed according to the chemical composition of actinomycetes cell walls and full cell hydrolyzed solution sugar type, actinomycetes is divided into 4 sugared types sees Table 2.
The main sugared type of the full cell of table 2 actinomycetes
Key step is:
(1) thalline preparation: scrape and get the centrifuge tube that cultured bacterial strain is put into 1.5mL;
(2) thalline hydrolysis: add the HCl 100 μ L of 0.25mol/L in the thalline, put into 120 ℃ of 15min of Autoclave;
(3) point sample: get hydrolyzed solution point sample on No. 1 filter paper of Xinhua that 4 μ L are used for amino acid analysis, get the standard sugar sample more respectively: wood sugar, pectinose, semi-lactosi, glucose, rhamnosyl, seminose, each 1 μ L of ribose be interval point sample on filter paper successively.
(4) exhibition layer: the exhibition layer system that is used for the analysis of full cell amino acid hydrolyticsolution is a propyl carbinol: water: pyridine: methyl alcohol=10: 6: 6: 1, and exhibition layer 2 times;
(5) colour developing: developer: phthalic acid: aniline: water saturated propyl carbinol (3.25: 2: 100), 120 ℃ of heating 3min.
(4) bacterial strain 16S rDNA gene order and phylogenetic systematics analysis
The preparation of DNA lamina membranacea
DNA extraction:
Antagonism actinomyces strain NMG2-4-8 behind the purifying is inoculated in the Gause I culture dish, and 28 ℃ are cultured to the mid-term of growing, standby.
The key step of extracting is as follows:
(1) scrapes the thalline that takes a morsel in the ware, add 60 μ L, 2 * CATB damping fluid, be ground to slurries in the ice bath mortar, move into (every bacterium is cooked 3 pipes) in the 1.5mL centrifuge tube.
(2) add 500 μ L N,O-Diacetylmuramidase treatment solutions (N,O-Diacetylmuramidase 2mg/mL, RNase solution 50 μ g/mL, sucrose 0.3M, Tris-HCl 1M, pH8.0) place 37 ℃ the insulation 2h.
(3) add 250 μ L 20%SDS solution, concussion mixes.
(4) 55 ℃ of insulation 60min.
(5) add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) fully shakes up, 4 ℃ of Tris-HCl 1M, centrifugal under the pH8.0 (8000r/min, 5min).
(6) get supernatant liquor and move in another centrifuge tube, add 10%NaAC liquid, 2.5 times of freezing ethanol sedimentations of volume.
(7) 4 ℃ centrifugal, and (10,000r/min 5min), abandons supernatant liquor.
(8) repeating step (5)~(7) is 1 time.
(9) wash 2~3 times with 70% ethanol, dry, add TE damping fluid 60 μ L, fully get 3 μ L after the dissolving and detect also-20 ℃ preservation down.
Pcr amplification 16S rDNA:
(1) PCR instrument: model: ALD-1244, rev, C.B
(2) amplimer: 16S rDNA universal primer (50 μ M) forward Pf is 5 '-AGAGTTTGATCCTGGCTCAG-3 ' (corresponding to E.coli8~27 bit bases), oppositely Pr be 5 '-TACGGCTACCTTGTTACGACTT-3 ' (corresponding to E.coli1492~1514 bit bases), synthetic by Institute of Microorganism, Academia Sinica genetically engineered center.
(3) amplification system:
Table 316S rDNA pcr amplification system
Annotate: 25 μ L systems
Pcr amplification parameter and program:
Add each component successively by amplification system (table 3), instantaneous centrifugal mixing, instantaneous centrifugal mixing behind the adding Taq enzyme.95 ℃ of pre-sex change 5min enter circulation: 94 ℃ of sex change 1min, and 50 ℃ of annealing 1min, 72 ℃ are extended 2min.After 35 circulations, 72 ℃ are extended 10min.Amplified production-20 ℃ storage.
The electrophoretic examinations of pcr amplification product:
Deposition condition is 0.8% sepharose (containing EB 0.5 μ g/mL), 1 * TAE electrophoretic buffer, and 80V voltage electrophoresis 40min, PCR product applied sample amount are 4 μ L, with point sample behind the 2 μ L sample-loading buffer mixings.
Observations under the 254nm ultraviolet is with the DNA Marker[DL2000 of TaKaRa company] be nucleic acid standard molecular weight object of reference, determine expanding fragment length.The amplified production band should be on position, the standard substance 1500bp left and right sides.
16S rDNA PCR product complete sequence determination:
The purifying of PCR product and sequencing are undertaken by Shanghai Ying Jun Bioisystech Co., Ltd.During order-checking with amplimer as forward and reverse primer, by Shanghai Ying Jun Bioisystech Co., Ltd with calculating power traction thing design software, primer and synthetic in the middle of designing according to the sequence search of measured sample.
The structure of systematic evolution tree:
The procaryotic 16S rDNA sequence of having measured in the 16S rDNA sequence surveyed and the GenBank gene pool is compared, access 16S rDNA sequence with the higher bacterial strain of its sequence homology.Adopt CLUSTAL X 1.8 softwares that the homologous sequence of being measured is carried out many couplings and arrange, carry out the structure of systematic evolution tree by Treeview software.
The result:
(1) morphological specificity
Bacterial strain NMG2-4-8 bacterium colony gray suede powdery, drying, the base silk is dark yellow, and cultivate through inserted sheet and observe under opticmicroscope: the aerial hyphae of NMG2-4-8 is longer, and multi-branched is seen Fig. 1; The spore oval is to cylindricality, and fibrillae of spores is straight or crooked or twist, sees Fig. 2, Fig. 3, and the feature of typical streptomyces is arranged.
(2) cultural characteristic
Feature such as the table 4 of NMG2-4-8 on various substratum, as seen this bacterium is obviously different in the cultivation proterties on the different substratum: aerial hyphae is a melon wooden dipper powder on Gause I agar, the base silk is dark yellow, no soluble pigment; Aerial hyphae is the fallen or falling flowers powder on agar of Ke Shi, basic sponge gourd wooden dipper powder; The Czapek's agar aerial hyphae is greyish white, and the base silk is greyish white; Aerial hyphae is the fermented bean drink Huang on the glucose asparagine agar, base silk pumpkin Huang; Aerial hyphae is an Eggshell on the glucose yeast cream agar, the shallow mango palm fibre of base silk; It is the fallen or falling flowers powder that potato is soaked juice agar aerial hyphae, base silk deer horn palm fibre; The potato ball aerial hyphae is the fallen or falling flowers powder.NMG2-4-8 does not all produce soluble pigment on above all confession examination substratum.
The cultural characteristic of table 4 bacterial strain NMG2-4-8
(3) physiological and biochemical property
Bacterial strain NMG2-4-8 liquefy gelatin ability, 10d can make milk solidify, and it is peptonized, and can make the starch hydrolysis, and do not grow on the Mierocrystalline cellulose (not utilizing Mierocrystalline cellulose) do not produce H
2S can utilize pectinose, fructose, sucrose, glucose, rhamnosyl, semi-lactosi, wood sugar, N.F,USP MANNITOL and inositol.
(4) strain cell wall chemical composition is analyzed
1, paper layer chromatography cell walls amino acid analysis result
Bacterial strain NMG2-4-8 is carried out paper layer chromatography cell walls amino acid analysis, the results are shown in following table:
The full cell amino acid analysis of table 5 bacterial strain 2-4-8 result
2, the full cell walls sugar of paper layer chromatography analytical results
Bacterial strain NMG2-4-8 is carried out the analysis of the full cell walls sugar of paper layer chromatography, the results are shown in following table:
The full cell wall sugar part of table 6 bacterial strain NMG2-4-8 analytical results
Consolidated statement 5, table 6 result be as can be known: containing amino acid whose kind in the bacterial strain NMG2-4-8 cell is: LL-DAP, glycine and L-glutamic acid, in the contrast literary composition table 1 as can be known this strain cell wall belong to the I type, belong to streptomyces; Do not contain other sugared kind in the table 2 in the bacterial strain NMG2-4-8 cell except that containing glucose, this bacterial strain sugar type is the C type.
(5) 16S rDNA phylogenetic analysis
16S rDNA base sequence measurement result is measured 1422 effective bases of 16S rDNA of bacterial strain NMG2-4-8 altogether shown in sequence in the sequence table 1.
Phylogenetic tree makes up: choose 11 strain type strains and carry out Phylogenetic Analysis, table 7 is listed in the 16S rDNA sequence number of landing of used streptomyces bacterial strain, and the phylogenetic tree of structure is seen shown in Figure 4.
Table 7 constructing system is grown the used Streptomvces 16S rDNA GenBank number of landing of tree
Be streptomyces with the higher bacterial strain of strains tested homology as can be seen from Table 7, and majority all is streptomyces microflavus S.microflavus, can learn that bacterial strain NMG2-4-8 belongs to streptomyces Streptomyces, this bacterial strain is an independent branch with streptomyces microflavus S.microflavus EU570571 on phylogenetic tree in addition, sibship is nearest, and similarity reaches 99.9%.
Because physiological and biochemical property (the fibrillae of spores regular helix shape on Gause I agar of strains tested and streptomyces microflavus, the pink little purple of aerial hyphae, the base silk is dark yellow, no soluble pigment, suppress positive or negative bacterium and yeast and filamentous fungus, gelatine liquefication is fast, the starch hydrolysis, growth is unstable on the Mierocrystalline cellulose) more similar.Therefore, binding molecule biological assay result is accredited as streptomyces microflavus Streptomyces microflavus with bacterial strain NMG2-4-8.
This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 12nd, 2009 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.3442.The classification called after streptomyces microflavus (Streptomyces microflavus) of this bacterial strain, strain name is NMG2-4-8.
Three, cultivate
Gause I nutrient agar: agar 15g, Zulkovsky starch 20g, NaCl 0.5g, KNO
3Lg, K
2HPO
43H
2O0.5g, MgSO
47H
2O 0.5g, FeSO
47H
2O 0.01g, distilled water 1000mL, pH7.2~7.4.
Seed culture medium: peptone 5g, yeast extract paste 3g, glucose 2g, extractum carnis 1g, distilled water 1000mL, pH7.0~7.2;
Fermention medium: analysis for soybean powder 20g, starch 5g, glucose 20g, peptone 2g, yeast extract paste 5g, NaCl5g, K
2HPO
40.5g, MgSO
47H
2O 0.5g, CaCO
32g, distilled water 1000mL, pH7.5.
Peptone is available from the favour commerce and trade company limited of Inner Mongol letter, and catalog number is SH0010; Yeast extract paste is available from the favour commerce and trade company limited of Inner Mongol letter, and catalog number is SH0348; Analysis for soybean powder is available from the supermarket.
Bacterial strain NMG2-4-8 is inoculated on the Gause I inclined-plane, cultivates 5-6d at 28 ℃; Be inoculated in the 100mL seed culture medium with aseptic inoculation ring picking 2~3 rings from the good actinomycetes inclined-plane of growing, 28 ℃, 220r/min shaking table shaking culture 48h obtain seed culture fluid; Inoculum size by 10% (v/v) is inoculated in seed culture fluid in the fermention medium, and 220r/min, 28 ℃ of shaking culture 120h obtain fermented liquid, and this moment, bacterial strain produced the bacteriostatic activity meta-bolites of high density in fermented liquid; With the centrifugal 10min of fermented liquid 5000r/min that obtains, get supernatant liquor place 4 ℃ standby, this supernatant liquor note is made aseptic ferment filtrate.
The antimicrobial spectrum of embodiment 2, bacterial strain is measured
Adopting cylinder plate method that bacterial strain NMG2-4-8 is carried out antimicrobial spectrum measures.Sterilization Oxford cup is put into (the dull and stereotyped preparation method: the 1mL sterilized water is added each target bacterium culture test tube of the dull and stereotyped central authorities of PDA of mixed target bacterium, with transfering loop bacterium colony, mycelium or spore are scraped in the culture dish of pouring 9cm after making bacteria suspension and PDA substratum 10mL mixing into and to make flat board), add the aseptic ferment filtrate of 200 μ L in the cup, put 25 ℃ respectively and cultivate 4-5d (fungi), 28 ℃ and cultivate 2d (bacterium), cover with culture dish to the target bacterium till.Adopt the right-angled intersection method to measure the diameter of antibacterial (bacteriolyze) circle.Every kind of target bacterium is repeated results averaged 3 times.
PDA nutrient agar: potato (peeling) 200g, sucrose (or glucose) 20g, distilled water 1000mL, pH nature.
Result such as table 8, bacterial strain NMG2-4-8 encircles 4 kinds of gram positive bacteriums such as rotten excellent bacillus, streptococcus aureus to potato, 18 kinds of fungies such as 5 kinds of gram negative bacteriums such as carrot soft rot Erwinia, blue or green withered Lei Er Salmonella and Sclerotinia sclerotiorum, cucumber fusarium axysporum, clover eggplant fusarium, clover crawl that handle is mould, gaeumannomyce, Pyricularia oryzae, tomato gray mould bacterium, candiyeast kind, Penicilllum expansum have the obvious suppression effect.The antimicrobial spectrum of proof bacterial strain NMG2-4-8 is wide.
The antimicrobial spectrum measurement result of table 8-1 bacterial strain NMG2-4-8
Indicator | Antibacterial circle diameter (mm) | Indicator | Antibacterial circle diameter (mm) |
Gram positive bacterium Gram-positive bacteria | Dry thread Pyrenomycetes Rhizoctonia solani | ??35.20 | |
Potato encircles rotten excellent bacillus Clavibacter michiganensis subsp.sepedonicus | ??29.56 | Clover septoria musiva leaf spot fungi Septoria medicaginis | ??28.02 |
Streptococcus aureus Staphylococcus aureus | ??31.02 | The mould Pythium ultimum of ultimate corruption | ??30.55 |
Subtilis Bacillus subtilis | ??18.45 | Big beautiful Verticillium Verticillium dahlia | ??20.36 |
Bacillus cereus Bacillus cereus | ??26.30 | Gaeumannomyce Gaeumannomyces graminis | |
Gram negative bacterium Gram-negative bacteria | The clover mould Stemphylium botryosum of handle that crawls | ??35.52 | |
Carrot soft rot Erwinia Erwinia carotovora var.carotovora | ??29.10 | Standing grain is bent spore mould Curvularia lunata | ??19.97 |
Blue or green withered Lei Er Salmonella Ralstonia solanacearum | ??23.48 | Phytophthora capsici Phytophthora capsici | ??30.14 |
Clover Xanthomonas campestris Xanthomonas campestris pv.Alfalfae | ??30.69 | Pyricularia oryzae Pyricularia grisea | ??17.69 |
Intestinal bacteria Escherichia coli | ??25.78 | Alternaric bacteria Alternaria tenuis | ??4.80 |
Indicator | Antibacterial circle diameter (mm) | Indicator | Antibacterial circle diameter (mm) |
Pseudomonas fluorescens Pseudomouas uoresceus | ??19.36 | Clover point sickle spore bacterium Fusarium oxysporum | ??25.99 |
Fungi Fungi | ??22.55 | Tomato gray mould bacterium Botrytis cinerea | ??20.45 |
Sclerotinia sclerotiorum Sclerotinia sclerotiorum | ??21.33 | Candiyeast Candida sp. | ??15.43 |
Cucumber fusarium axysporum Fusarium oxysporum f.sp.cucumerinum | ??20.80 | Geotrichum candidum Geotrichum candidum | ??16.52 |
Watermelon anthrax bacteria Colletotrichum lagenarium | ??17.92 | Aspergillus oryzae Aspergillus oryza | ??14.38 |
Clover eggplant fusarium Fusarium solani | ??32.40 | Penicilllum expansum Penicillium expansum | ??11.86 |
Each bacterium source of table 8-2
Indicator | Deposit number | Indicator | Deposit number | ||
Gram-positive microorganism Gram-positive bacteria | Dry thread Pyrenomycetes Rhizoctonia solani | ??ACCC30332 | Available from ACCC | ||
Potato encircles rotten excellent bacillus Clavibacter michiganensis subsp. sepedonicus | ??ACCC01233 | Available from ACCC | Clover septoria musiva leaf spot fungi Septoria medicaginis | The letter of guarantee | |
Streptococcus aureus Staphylococcus aureus | ??ACCC01336 | Available from ACCC | The mould Pythium ultimum of ultimate corruption | ??ACCC36075 | Available from ACCC |
Subtilis Bacillus subtilis | ??CGMCC1.1??656 | Available from CGMCC | Big beautiful Verticillium Verticillium dahlia | ??ACCC36109 | Available from ACCC |
Bacillus cereus Bacillus cereus | ??CGMCC??1.348 | Available from CGMCC | Gaeumannomyce Gaeumannomyces graminis | ??ACCC30310 | Available from ACCC |
Gram-negative bacteria Gram-negative bacteria | The clover mould Stemphylium botryosum of handle that crawls | The letter of guarantee |
Indicator | Deposit number | Indicator | Deposit number | ||
Carrot soft rot Erwinia Erwinia carotovora var. carotovora | ??CGMCC??1.1000 | Available from CGMCC | Standing grain is bent spore mould Curvularia lunata | ??ACCC36580 | Available from ACCC |
Blue or green withered Lei Er Salmonella Ralstonia solanacearum | ??ACCC01470 | Available from ACCC | Phytophthora capsici Phytophthora capsici | ??ACCC36279 | Available from ACCC |
Clover Xanthomonas campestris Xanthomonas campestris pv. alfalfae | The letter of guarantee | Pyricularia oryzae Pyricularia grisea | ??ACCC30320 | Available from ACCC | |
Intestinal bacteria Escherichia coli | ??ACCC10196 | Available from ACCC | Alternaric bacteria Alternaria tenuis | ??ACCC36110 | Available from ACCC |
Pseudomonas fluorescens Pseudomouas uoresceus | ??ACCC01241 | Available from ACCC | Clover point sickle spore bacterium Fusarium oxysporum | The letter of guarantee | |
Fungi Fungi | Tomato gray mould bacterium Botrytis cinerea | ??ACCC30091 | Available from ACCC |
Indicator | Deposit number | Indicator | Deposit number | ||
Sclerotinia sclerotiorum Sclerotinia sclerotiorum | ??ACCC36169 | Available from ACCC | Candiyeast Candida sp. | ??ACCC21131 | Available from ACCC |
Cucumber fusarium axysporum Fusarium oxysporum f.sp.cucumerinum | ??ACCC30442 | Available from ACCC | Geotrichum candidum Geotrichum candidum | ??CGMCC??2.1084 | Available from CGMC C |
Watermelon anthrax bacteria Colletotrichum lagenarium | ??ACCC36152 | Available from ACCC | Aspergillus oryzae Aspergillus oryza | ??ACCC30155 | Available from ACCC |
Clover eggplant fusarium Fusarium solani | The letter of guarantee | Penicilllum expansum Penicillium expansum | ??ACCC30898 | Available from ACCC |
Clover Xanthomonas campestris Xanthomonas campestris pv.alfalfae (Hou Tianjue. China's clover disease generation present situation and Preventing Countermeasures [J]. Inner Mongol grass cultivation, 1994,3:4-8.) (provide) by grassland research institute, China Agriculture academy of sciences
Clover septoria musiva leaf spot fungi Septoria medicaginis (Hou Tianjue. China's clover disease generation present situation and Preventing Countermeasures [J]. Inner Mongol grass cultivation, 1994,3:4-8.) (provide) by grassland research institute, China Agriculture academy of sciences
Clover crawl the mould Stemphylium botryosum of handle (Hou Tianjue. China's clover disease generation present situation and Preventing Countermeasures [J]. Inner Mongol grass cultivation, 1994,3:4-8.) (provide) by grassland research institute, China Agriculture academy of sciences
Clover point sickle spore Fusarium oxysporum (Cao Lixia, Zhao Cunhu, Bai Quanjiang, etc. middle of Inner Mongolia area clover root-rot disease pathogen research (English) [J]. North China agronomy newspaper, 2008,23 (6): 105-107.) (provide) by grassland research institute, China Agriculture academy of sciences
Clover eggplant fusarium Fusarium solani (Cao Lixia, Zhao Cunhu, Bai Quanjiang, etc. middle of Inner Mongolia area clover root-rot disease pathogen research (English) [J]. North China agronomy newspaper, 2008,23 (6): 105-107.) (provide) by grassland research institute, China Agriculture academy of sciences
The greenhouse biological control Plant diseases experiment of embodiment 3, bacterial strain NMG2-4-8
(No. 1, middle lucerne) seed is available from national germplasm herbage storehouse in mid-term, and catalog number is 00068.
Clover downy mildew Personspora aestivalis (Hou Tianjue. northern China meadow disease survey and main disease control [J]. Chinese meadow, 1993,3:56-60.) (provide) by grassland research institute, China Agriculture academy of sciences
Clover powdery mildew Erysiphe polygoni (Hou Tianjue. China's clover disease generation present situation and Preventing Countermeasures [J]. Inner Mongol grass cultivation, 1994,3:4-8.) (provide) by grassland research institute, China Agriculture academy of sciences
The false cup fungi Pseudopeziza of clover medicaginis (Hou Tianjue. northern China meadow disease survey and main disease control [J]. Chinese meadow, 1993,3:56-60.) (provide) by grassland research institute, China Agriculture academy of sciences
Clover crawl the mould Stemphylium botryosum of handle (Hou Tianjue. China's clover disease generation present situation and Preventing Countermeasures [J]. Inner Mongol grass cultivation, 1994,3:4-8.) (provide) by grassland research institute, China Agriculture academy of sciences
Clover eggplant fusarium Fusarium solani (Xu Linbo, Di Caixia, Wang Lanying; Deng. ten kinds of medicaments are to the Toxicity Determination [A] of clover eggplant fusarium. Cheng Zhuomin. and grain security and plant protection scientific and technical innovation. grain security and plant protection scientific and technical innovation-Chinese Plants protection association academic nd Annual Meeting collection [C] in 2009. Beijing: Scientia Agricultura Sinica technology press; 2009,804-807.) (provide) by grassland research institute, China Agriculture academy of sciences
Used NMG2-4-8 fermented liquid all is to prepare according to method in the experiment three among the embodiment 1 in the following experiment.
One, to the prevention effect of downy mildew of alfalfa
Adopt indoor seedling stage spray inoculation method that NMG2-4-8 is carried out oidium control test.Downy mildew of alfalfa is caused by clover downy mildew Personspora aestivalis, picks up from the Sha Er of the grassland research institute, China Agriculture academy of sciences susceptible alfalfa plants in testing station that oozes.
(1) provide protection
Cultivate alfalfa seed and obtain the clover seedling, 4 true leaves treating the clover seedling spray medicine when fully launching to be handled, and normal management 48h connects pathogenic bacteria again, meets behind the bacterium dark 24h that preserves moisture, and enters Routine Management.Behind the Routine Management 7-10d, investigation morbidity feelings are wished, classification is put down in writing, calculate disease index and protection effect.
(2) therapeutic action:
Cultivate alfalfa seed and obtain the clover seedling, 4 true leaves treating the clover seedling connect pathogenic bacteria when fully launching, and meet behind the bacterium dark 24h that preserves moisture, and produce to induce sporocyst, spray chemicals treatment then, behind the 24h that preserves moisture again, enter Routine Management.Behind the Routine Management 7-10d, investigation morbidity feelings are wished, classification is put down in writing, calculate disease index and result of treatment.
(3) concrete operations
1, potted plant seeding and growing seedling matrix is the mixture (volume ratio is 1: 1: 1) of soil (through the high temperature dry sterilization), the peat composed of rotten mosses and vermiculite.For trying alfalfa seed behind 0.1% bromogeramine solution disinfection 3min, clean with flushing with clean water, put into the culture dish that is lined with gauze, place 25 ℃ incubator to germinate then, treat that radicle is long during to 0.5cm, it is sowed in the seedling-growing container that above-mentioned matrix is housed (seedling-growing container is in advance through 0.1% formaldehyde solution sealing fumigation).Grow seedlings in 18~20 ℃ of greenhouses, every basin keeps 4 strain clover seedling.
2, inoculation method is got the branch of the alfalfa plants that infects Mu Pseudoperonospora cubensis Peronospora aestivalis, after indoor elder generation brushes away the mould layer of sick leaf back gently with writing brush, place under 18-20 ℃, relative humidity 〉=97% condition dark to preserve moisture and cultivate 24h, after waiting to grow fresh sporocyst, it is brushed in the distilled water, be prepared into sporangia suspension, concentration has 20~30 sporocysts to be advisable to check in average every visual field under low-power microscope.Carry out spray inoculation with the small hand-held atomizer, inoculation position is a blade, inoculum size: with bacterium liquid evenly spray in clover blade positive and negative two to, moistening and do not drip degree of being with the blade face.
3,3 processing of 20,10,5 times of diluents of NMG2-4-8 fermented liquid are established in the application method test, are agricultural chemicals contrast (all diluting with clear water) with the anti-200 times of diluents of-120 aquas of 2% farming, are blank with the clear water, and every processing repeats 5 times.Use handheld atomizer with soup evenly spray in clover blade positive and negative two to, moistening and do not drip degree of being with the blade face.
4, dark processing: with potted plant clover seedling on the kraft bag cover.
5, the processing of preserving moisture: with potted plant clover seedling on the plastic closures bag cover.
State of an illness grade scale:
0 grade: no scab;
1 grade: the scab total area is less than 1/3 of the blade total area in each blade;
2 grades: the scab total area accounts for 1/3~2/3 of leaf area in each blade;
3 grades: the scab total area is greater than 2/3 of the blade total area in each blade.
In the disease index, the level values of representing at different levels refer to 0,1,2,3; The highest level value of representing refers to 3.
Experimental result is as shown in table 9.The fermented liquid of result: a NMG2-4-83 concentration gradient to the protection effect of downy mildew of alfalfa all clearly.Wherein the protection effect of 5 times of diluents is 88.69%, and the protection effect that is better than 86.97%, 10 times, the 20 times diluent of effect of pesticide corrosion 120 is respectively 77.00%, 63.06%; The therapeutic action effect is also remarkable, but all is lower than the preventive effect of farming anti--120.
Table 9NMG2-4-8 fermented liquid is to the quick fruit of the control of downy mildew of alfalfa
Two, to the prevention effect of Alfalfa Powdery Mildew Disease
Adopt indoor seedling stage spray inoculation method that NMG2-4-8 is prevented and treated the Alfalfa Powdery Mildew Disease test.Alfalfa Powdery Mildew Disease is caused by clover powdery mildew Erysiphe polygoni.
(1) provide protection
Cultivate alfalfa seed and obtain the clover seedling, treat that the clover seedling cultivates 3-4 and spray medicine during week and handle, normal management 24h connects pathogenic bacteria again, meets behind the bacterium dark 24h that preserves moisture, and enters Routine Management.Behind the Routine Management 7-10d, investigation morbidity feelings are wished, classification is put down in writing, calculate disease index and protection effect.
(2) therapeutic action:
Cultivate alfalfa seed and obtain the clover seedling, treat that the clover seedling cultivates 3-4 inoculation pathogenic bacteria during week, meet behind the bacterium dark 24h that preserves moisture, spray chemicals treatment then, behind the 24h that preserves moisture again, enter Routine Management.Behind the Routine Management 7-10d, investigation morbidity feelings are wished, classification is put down in writing, calculate disease index and result of treatment.
(3) concrete operations
1, potted plant growing seedlings: method is with identical described in the experiment one.
2, inoculation method adopt the traditional powder method of trembling inoculation Alfalfa Powdery Mildew Disease bacterium (Fang Zhongda. plant disease research method [M]. Beijing: Chinese agriculture press, 1998:366-368.).Shake off lightly by on the clover seedling leaves of inoculating infecting powdery mildew conidium on the clover blade of Alfalfa Powdery Mildew Disease bacterium Erysiphe polygoni intercurrent disease.
3,20,10,5 times of diluents of NMG2-4-8 fermented liquid, the anti-200 times of diluents of-120 aquas of 2% farming and clear water contrast totally 5 processing are established in the application method test, and every processing repeats 5 times.Use handheld atomizer with soup evenly spray in clover blade positive and negative two to, moistening and do not drip degree of being with the blade face.
4, dark processing: with potted plant clover seedling on the kraft bag cover.
5, the processing of preserving moisture: with potted plant clover seedling on the plastic closures bag cover.
State of an illness grade scale:
0 grade: no illness;
1 grade: the scab total area accounts for below 1/3 of leaf area in each blade, and white powder is smudgy;
2 grades: the scab total area accounts for 1/3~2/3 of leaf area in each blade, and white powder is comparatively obvious;
3 grades: the scab total area accounts for more than 2/3 of leaf area in each blade, and the white powder layer is thicker, in flakes.
Disease index, disease refer to that the calculation formula of rate of increase, prevention effect is as testing as described in one.
Experimental result is as shown in table 10.The result: the NMG2-4-8 fermented liquid of different concns has the better protecting effect to Alfalfa Powdery Mildew Disease, uses 5 times of diluents, and the protection effect reaches 90.81%, significantly is better than the protection effect of the anti-200 times of liquid of-120 aquas of 2% farming to Alfalfa Powdery Mildew Disease.Use 10 times of diluents, the protection effect reaches 80.44%, and is suitable with the preventive effect of the anti-200 times of liquid of-120 aquas of 2% farming.The NMG2-4-8 fermented liquid is better to the therapeutic action of Alfalfa Powdery Mildew Disease, and when working concentration was 5 times, result of treatment was 69.69%, is better than the preventive effect 61.36% of farming anti--120.
Table 10NMG2-4-8 fermented liquid is to the pot experiment effect of Alfalfa Powdery Mildew Disease
Three, to the prevention effect of clover brown spot
Adopt indoor seedling stage spray inoculation method that NMG2-4-8 is prevented and treated the test of clover brown spot.The clover brown spot is caused by the false cup fungi Pseudopeziza of clover medicaginis.
(1) provide protection
Cultivate alfalfa seed and obtain the clover seedling, treat that the clover seedling cultivates 3-4 and spray medicine during week and handle, normal management 24h connects pathogenic bacteria again, meets behind the bacterium dark 24h that preserves moisture, and enters Routine Management.Behind the Routine Management 10-15d, investigation morbidity feelings are wished, classification is put down in writing, calculate disease index and protection effect.
(2) therapeutic action:
Cultivate alfalfa seed and obtain the clover seedling, treat that the clover seedling cultivates 3-4 inoculation pathogenic bacteria during week, meet behind the bacterium dark 24h that preserves moisture, spray chemicals treatment then, behind the 24h that preserves moisture again, enter Routine Management.Behind the Routine Management 10-15d, investigation morbidity feelings are wished, classification is put down in writing, calculate disease index and result of treatment.
(3) concrete operations
1, potted plant method for culturing seedlings is with identical described in the experiment one.
2, inoculation method is made suspension with the false cup fungi Pseudopeziza of Tween-20 flush away clover medicaginis thecaspore with thecaspore, and concentration is 10
5~10
6Individual thecaspore/mL.The spore suspension for preparing is sprayed on the alfalfa plants equably with throat spray, moistening and do not drip degree of being with the blade face.The inoculation back will keep relative humidity 100%, temperature 20-25 ℃ for the examination material seal with plastic lousing.Preserving moisture removes plastic lousing behind the 48h, carries out Routine Management in the greenhouse.
3,3 processing of 20,10,5 times of diluents of NMG2-4-8 fermented liquid are established in the application method test, are agricultural chemicals contrast (all diluting with clear water) with the anti-200 times of diluents of-120 aquas of 2% farming, are blank with the clear water, and every processing repeats 5 times.Use handheld atomizer with soup evenly spray in clover blade positive and negative two to, moistening and do not drip degree of being with the blade face.
4, dark processing: with potted plant clover seedling on the kraft bag cover.
5, the processing of preserving moisture: with potted plant clover seedling on the plastic closures bag cover.
State of an illness grade scale: the percentage that covers leaf area according to scab carries out classification:
0 grade: asymptomatic performance;
1: in each blade the scab total area be leaf area 5% or below;
2: the scab total area is 6%~15% of a leaf area in each blade, scab position chlorisis;
3: the scab total area is that 16%~35%, 3/4 blade of leaf area begins chlorisis in each blade;
4: the scab total area is 36%~50% of a leaf area in each blade, and blade is all flavescence almost;
5: the scab total area is 51%~70% of a leaf area in each blade, the whole chlorisis of blade;
6: in each blade the scab total area be leaf area more than 70% or all withered.
Disease index, disease refer to that the calculation formula of rate of increase, prevention effect is as testing as described in one.
Experimental result is as shown in table 11.The result: the NMG2-4-8 fermented liquid of different concns has the better protecting effect to the clover brown spot, uses 5 times of diluents, and the protection effect reaches 80.22%, and is approaching to the protection effect 81.99% of clover brown spot with the anti-200 times of liquid of-120 aquas of 2% farming.Use 10 times of diluents, the protection effect is 66.47%, uses 20 times of diluents, and the protection effect is 43.15%, all is lower than the protection effect of the anti-200 times of liquid of-120 aquas of 2% farming.The NMG2-4-8 fermented liquid is not obvious to the therapeutic action of clover brown spot, and when working concentration was 5 times of diluents, preventive effect only was 58.88%.
Table 11, NMG2-4-8 fermented liquid are to the pot experiment effect of clover brown spot
Annotate: * represents poor effect or to no effect.
Four, to the crawl prevention effect of the mould leaf spot of handle of clover
Adopt indoor seedling stage spray inoculation method that NMG2-4-8 is prevented and treated the clover mould leaf spot test of handle of crawling.The clover mould leaf spot of handle of crawling is caused by the clover mould Stemphylium botryosum of handle that crawls.
(1) provide protection
Cultivate alfalfa seed and obtain the clover seedling, treat that the clover seedling cultivates 3-4 and spray medicine during week and handle, connect pathogenic bacteria behind the 24h again, meet behind the bacterium dark 24h that preserves moisture, enter Routine Management.Behind Routine Management 10~12d, investigation morbidity feelings are wished, classification is put down in writing, calculate disease index and protection effect.
(2) therapeutic action:
Cultivate alfalfa seed and obtain the clover seedling, treat that the clover seedling cultivates 3-4 inoculation pathogenic bacteria during week, meet behind the bacterium dark 24h that preserves moisture, spray chemicals treatment then, behind the 24h that preserves moisture again, enter Routine Management.Behind Routine Management 10~12d, investigation morbidity feelings are wished, classification is put down in writing, calculate disease index and result of treatment.
(3) concrete operations
1, potted plant method for culturing seedlings is with identical described in the experiment one.
2, inoculation method is used for prepare suspension with the crawl conidium of the mould Stemphyllium botryosum of handle of Tween-20 flush away clover with conidium, and concentration is 10
5~10
6Cfu/mL.Adopt the method for blade spraying to inoculate by handheld atomizer, moistening and do not drip degree of being with the blade face.Inoculation back will be for the examination material seal with plastic lousing, and maintenance relative humidity 100% removes plastic lousing behind temperature 20-23 ℃, the 48h that preserves moisture, and plant is moved into place backlight, makes leaf natural air-dry.In the greenhouse, carry out Routine Management.
3,3 processing of 20,10,5 times of diluents of NMG2-4-8 fermented liquid are established in the application method test, are agricultural chemicals contrast (all diluting with clear water) with the anti-200 times of diluents of-120 aquas of 2% farming, are blank with the clear water, and every processing repeats 5 times.Use handheld atomizer with soup evenly spray in clover blade positive and negative two to, moistening and do not drip degree of being with the blade face.
4, dark processing: with potted plant clover seedling on the kraft bag cover.
5, the processing of preserving moisture: with potted plant clover seedling on the plastic closures bag cover.
State of an illness grade scale:
0 grade: plant health, no scab occurs.
1 grade: 1 scab of each blade, the scab total area account for leaf area below 5%.
2 grades: each blade 1-3 scab, the scab total area accounts for leaf area 6%-20% and blade begins chlorisis.
3 grades: each blade 3-10 scab, the scab total area account for leaf area 21%-50%, scab necrosis and yellow leaf.
4 grades: 10 above scabs of each blade, the scab total area account for leaf area more than 50% and yellow leaf come off.
Disease index, disease refer to that the calculation formula of rate of increase, prevention effect is as testing as described in one.
Experimental result is as shown in table 12.The result: different concns NMG2-4-8 fermented liquid has excellent protection and therapeutic action concurrently to the clover mould leaf spot of handle of crawling.Use 5 times of diluents, the protection effect reaches 80.35%, a little less than the anti-200 times of liquid of-120 aquas of 2% farming to the crawl protection effect 85.32% of the mould leaf spot of handle of clover.Use 10,20 times of times of diluents, the protection effect is respectively 72.00%, 59.96%.And the result of treatment of NMG2-4-8 fermented liquid is better, obviously is better than above-mentioned 3 kinds of diseases, and when working concentration was 5 times of diluents, preventive effect reached 73.02%.
Table 12NMG2-4-8 fermented liquid is to the crawl pot experiment effect of the mould leaf spot of handle of clover
Five, to the prevention effect of clover root rot
Adopt and irritate the root inoculation method indoor seedling stage NMG2-4-8 is prevented and treated clover root rot test.The clover root rot is caused by clover eggplant fusarium Fusarium solani.
(1) provide protection
Cultivating alfalfa seed and obtain the clover seedling, get the potted plant clover seedling in 3 weeks after the field planting, irritate root inoculating strain NMG2-4-8 different concns fermented liquid and the anti-200 times of diluents of-120 aquas (agricultural chemicals contrast) of 2% farming earlier, is blank to inoculate clear water; Behind the dispenser 3d, irritate root inoculation clover eggplant sickle spore pine root fungus again.Behind the Routine Management 20d, investigation morbidity feelings are wished, the classification record, calculate disease index and result of treatment.
(2) concrete operations
1, potted plant growing seedlings: method is with identical described in the experiment one.
2, inoculation method is adopted indicator strain clover eggplant fusarium Fusarium solani is cultivated 5-6d with medium oatmeal in 28 ℃ of constant incubator, after mycelia is covered with substratum, light-secretly alternately cultivate 7d, wash conidium with sterilized water, add 0.02%Tween-20, remove mycelia through biofilter sterile filtration, making concentration is 10
6The spore suspension of cfu/mL.The inoculation of filling root, every strain clover seedling inoculation 10mL spore suspension.
3,3 processing of 20,10,5 times of diluents of NMG2-4-8 fermented liquid are established in the application method test, are agricultural chemicals contrast (all diluting with clear water) with the anti-200 times of diluents of-120 aquas of 2% farming, are blank with the clear water, and every processing repeats 5 times.The ferment filtrate of different concns bacterial strain NMG2-4-8 is irritated in clover seedling root every strain 10mL.Behind the 3d clover eggplant sickle spore pine root fungus is irritated the root inoculation, every strain clover seedling is inoculated the 10mL spore suspension.
State of an illness grade scale:
0 grade-healthy tree, apparent asymptomatic;
There is scab 1 grade-rhizome or main root part, but not in flakes;
There are scab and in flakes in 2 grades-main root, lateral root and rhizome portion, but are no more than 1/3;
Variable color is infected by 3 grades-1/3~1/2 root and rhizome portions, and lateral root obviously reduces;
The variable color of 4 grades-root and rhizome portion, partial decomposition, the obvious browning of vascular bundle, plant strain growth is suppressed and short and small withered and yellow;
5 grades-butt rot, vascular bundle blackening and plant are wilted dead.
Experimental result (table 13) shows that the fermented liquid of confession examination antagonism bacterium NMG2-4-8 can significantly reduce the morbidity of clover root rot, uses 5 times of diluents, and relative control effect reaches 90.80%, a little more than the processing preventive effect 87.75% of the anti-200 times of liquid of-120 aquas of 2% farming.Handle back alfalfa plants growing way and obviously be better than contrast, the growth-promoting functions that antagonism bacterium NMG2-4-8 shows has compensating action to the harm of disease to a certain extent.In pot experiment, the antagonism bacterium can effectively alleviate the state of an illness, demonstrates comparatively ideal application potential.
Table 13, NMG2-4-8 fermented liquid are to the pot experiment effect of clover root rot
Sequence table
<110〉grassland research institute, China Agriculture academy of sciences
<120〉streptomyces microflavus
<160>1
<210>1
<211>1422
<212>DNA
<213〉streptomyces microflavus (Streptomyces microflavus)
<400>1
gacggcagct?tacacatgca?gtcgaacgat?gaaccctttc?gggggggatt?agtggcgaac?????60
gggtgagtaa?cacgtgggca?atctgccctt?cactctggga?caagccctgg?aaacggggtc????120
taataccgga?taacactcct?gcctgcatgg?gcgggggttg?aaagctccgg?cggtgaagga????180
tgagcccgcg?gcctatcagc?ttgttggtgg?ggtaatggcc?taccaaggcg?acgacgggta????240
gccggcctga?gagggcgacc?ggccacactg?ggactgagac?acggcccaga?ctcctacggg????300
aggcagcagt?ggggaatatt?gcacaatggg?cgaaagcctg?atgcagcgac?gccgcgtgag????360
ggatgacggc?cttcgggttg?taaacctctt?tcagcaggga?agaagcgaaa?gtgacggtac????420
ctgcagaaga?agcgccggct?aactacgtgc?cagcagccgc?ggtaatacgt?agggcgcaag????480
cgttgtccgg?aattattggg?cgtaaagagc?tcgtaggcgg?cttgtcacgt?cggatgtgaa????540
agcccggggc?ttaaccccgg?gtctgcattc?gatacgggca?ggctagagtg?tggtagggga????600
gatcggaatt?cctggtgtag?cggtgaaatg?cgcagatatc?aggaggaaca?ccggtggcga????660
aggcggatct?ctgggccatt?actgacgctg?aggagcgaaa?gcgtggggag?cgaacaggat????720
tagataccct?ggtagtccac?gccgtaaacg?ttgggaacta?ggtgttggcg?acattccacg????780
tcgtcggtgc?cgcagctaac?gcattaagtt?ccccgcctgg?ggagtacggc?cgcaaggcta????840
aaactcaaag?gaattgacgg?gggcccgcac?aagcagcgga?gcatgtggct?taattcgacg????900
caacgcgaag?aaccttacca?aggcttgaca?tataccggaa?agcatcagag?atggtgcccc????960
ccttgtggtc?ggtatacagg?tggtgcatgg?ctgtcgtcag?ctcgtgtcgt?gagatgttgg???1020
gttaagtccc?gcaacgagcg?caacccttgt?tctgtgttgc?cagcacgccc?ttcggggtgg???1080
tggggactca?caggagactg?ccggggtcaa?ctcggaggaa?ggtggggacg?acgtcaagtc???1140
atcatgcccc?ttatgtcttg?ggctgcacac?gtgctacaat?ggccggtaca?atgagctgcg???1200
atgtcgtgag?gcggagcgaa?tctcaaaaag?ccggtctcag?ttcggattgg?ggtctgcaac???1260
tcgaccccat?gaagtcggag?ttgctagtaa?tcgcagatca?gcattgctgc?ggtgaatacg???1320
ttcccgggcc?ttgtacacac?cgcccgtcac?gtcacgaaag?tcggtaacac?ccgaagccgg???1380
tggcccaacc?ccttgtggga?gggagcttcg?aaggtgacta?tc??????????????????????1422
Claims (7)
1. streptomyces microflavus (Streptomyces microflavus) NMG2-4-8, its deposit number is CGMCC No.3442.
2. the antagonist of bacterium, its activeconstituents is streptomyces microflavus (Streptomyces microflavus) NMG2-4-8CGMCC No.3442; Described bacterium is gram positive bacterium, gram negative bacterium or fungi.
3. antagonist according to claim 2, it is characterized in that: described gram positive bacterium is that potato encircles rotten excellent bacillus Clavibacter michiganensis subsp.sepedonicus, streptococcus aureus Staphylococcus aureus, subtilis Bacillus subtilis or bacillus cereus Bacilluscereus
Described gram negative bacterium is carrot soft rot Erwinia Erwinia carotovora var.carotovora, blue or green withered Lei Er Salmonella Ralstonia solanacearum, clover Xanthomonas campestris Xanthomonascampestris pv.alfalfae, intestinal bacteria Escherichia coli or Pseudomonas fluorescens Pseudomouasuoresceus
Described fungi is Sclerotinia sclerotiorum Sclerotinia sclerotiorum, cucumber fusarium axysporum Fusariumoxysporum f.sp.cucumerinum, watermelon anthrax bacteria Colletotrichum lagenarium, clover eggplant fusarium Fusarium solani, dry thread Pyrenomycetes Rhizoctonia solani, clover septoria musiva leaf spot fungi Septoria medicaginis, the mould Pythium ultimum of ultimate corruption, big beautiful Verticillium Verticillium dahlia, gaeumannomyce Gaeumannomyces graminis, the clover mould Stemphylium botryosum of handle that crawls, standing grain is bent spore mould Curvularia lunata, Phytophthora capsici Phytophthora capsici, Pyricularia oryzae Pyriculariagrisea, alternaric bacteria Alternaria tenuis, clover point sickle spore bacterium Fusarium oxysporum, tomato gray mould bacterium Botrytis cinerea, candiyeast Candida sp., geotrichum candidum Geotrichum candidum, aspergillus oryzae Aspergillus oryza or Penicilllum expansum Penicillium expansum.
4. according to claim 2 or 3 described antagonists, it is characterized in that: it is that potato encircles rotten excellent bacillus Clavibactermichiganensis subsp.sepedonicus ACCC 01233 that described potato encircles rotten excellent bacillus Clavibacter michiganensis subsp.Sepedonicus, described streptococcus aureus Staphylococcus aureus is streptococcus aureus Staphylococcus aureus ACCC01336, described subtilis Bacillus subtilis is subtilis Bacillus subtilisCGMCC1.1656, described bacillus cereus Bacillus cereus is bacillus cereus Bacilluscereus CGMCC 1.348
Described carrot soft rot Erwinia Erwinia carotovora var.carotovora is carrot soft rot Erwinia Erwinia carotovora var.carotovora CGMCC1.1000, the withered Lei Er Salmonella of described green grass or young crops Ralstonia solanacearum is blue or green withered Lei Er Salmonella Ralstonia solanacearum ACCC01470, described intestinal bacteria Escherichia coli is intestinal bacteria Escherichia coli ACCC10196, described Pseudomonas fluorescens Pseudomouas uoresceus is Pseudomonas fluorescens Pseudomouas uoresceusACCC01241
Described Sclerotinia sclerotiorum Sclerotinia sclerotiorum is Sclerotinia sclerotiorum SclerotiniaSclerotiorum ACCC36169, described cucumber fusarium axysporum Fusarium oxysporum f.sp.cucumerinum is cucumber fusarium axysporum Fusarium oxysporum f.sp.cucumerinumACCC30442, described watermelon anthrax bacteria Colletotrichum lagenarium is watermelon anthrax bacteria Colletotrichumlagenarium ACCC36152, described dry thread Pyrenomycetes Rhizoctonia solani is dry thread Pyrenomycetes Rhizoctonia solani ACCC30332, the mould Pythium ultimum of described ultimate corruption is the mould Pythium ultimumACCC36075 of ultimate corruption, described big beautiful Verticillium Verticillium dahlia is big beautiful Verticillium Verticillium dahlia ACCC36109, described gaeumannomyce Gaeumannomyces graminis is gaeumannomyce Gaeumannomyces graminis AGCC30310, the curved spore mould Curvularia lunata of described standing grain is the curved spore mould Curvularia lunata ACCC36580 of standing grain, described Phytophthora capsici Phytophthora capsici is Phytophthora capsici Phytophthora capsici ACCC36279, described Pyricularia oryzae Pyricularia grisea is Pyricularia oryzae Pyricularia grisea ACCC30320, described alternaric bacteria Alternaria tenuis is alternaric bacteria Alternaria tenuis ACCC36110, described tomato gray mould bacterium Botrytis cinerea is tomato gray mould bacterium Botrytis cinerea ACCC30091, described candiyeast Candida sp. is candiyeast Candida sp.ACCC21131, described geotrichum candidum Geo trichum candidum is geotrichum candidum Geotrichumcandidum CGMCC2.1084, described aspergillus oryzae Aspergillus oryza is aspergillus oryzae Aspergillusoryza ACCC30155, and described Penicilllum expansum Penicillium expansum is Penicilllum expansum Penicilliumexpansum ACCC30898.
5. the application of streptomyces microflavus (Streptomyces microflavus) NMG2-4-8 CGMCC No.3442 in antagonism gram positive bacterium, gram negative bacterium or fungi.
6. application according to claim 5, it is characterized in that: described gram positive bacterium is that potato encircles rotten excellent bacillus Clavibacter michiganensis subsp.sepedonicus, streptococcus aureus Staphylococcus aureus, subtilis Bacillus subtilis or bacillus cereus Bacilluscereus
Described gram negative bacterium is carrot soft rot Erwinia Erwinia carotovora var.carotovora, blue or green withered Lei Er Salmonella Ralstonia solanacearum, clover Xanthomonas campestris Xanthomonascampestris pv.alfalfae, intestinal bacteria Escherichia coli or Pseudomonas fluorescens Pseudomouasuoresceus
Described fungi is Sclerotinia sclerotiorum Sclerotinia sclerotiorum, cucumber fusarium axysporum Fusariumoxysporum f.sp.cucumerinum, watermelon anthrax bacteria Colletotrichum lagenarium, clover eggplant fusarium Fusarium solani, dry thread Pyrenomycetes Rhizoctonia solani, clover septoria musiva leaf spot fungi Septoria medicaginis, the mould Pythium ultimum of ultimate corruption, big beautiful Verticillium Verticillium dahlia, gaeumannomyce Gaeumannomyces graminis, the clover mould Stemphylium botryosum of handle that crawls, standing grain is bent spore mould Curvularia lunata, Phytophthora capsici Phytophthora capsici, Pyricularia oryzae Pyriculariagrisea, alternaric bacteria Alternaria tenuis, clover point sickle spore bacterium Fusarium oxysporum, tomato gray mould bacterium Botrytis cinerea, candiyeast Candida sp., geotrichum candidum Geotrichum candidum, aspergillus oryzae Aspergillus oryza or Penicilllum expansum Penicillium expansum.
7. according to claim 5 or 6 described application, it is characterized in that: it is that potato encircles rotten excellent bacillus Clavibactermichiganensis subsp.sepedonicus ACCC 01233 that described potato encircles rotten excellent bacillus Clavibacter michiganensis subsp.Sepedonicus, described streptococcus aureus Staphylococcus aureus is streptococcus aureus Staphylococcus aureus ACCC01336, described subtilis Bacillus subtilis is subtilis Bacillus subtilisCGMCC1.1656, described bacillus cereus Bacillus cereus is bacillus cereus Bacilluscereus CGMCC 1.348
Described carrot soft rot Erwinia Erwinia carotovora var.carotovora is carrot soft rot Erwinia Erwinia carotovora var.carotovora CGMCC1.1000, the withered Lei Er Salmonella of described green grass or young crops Ralstonia solanacearum is blue or green withered Lei Er Salmonella Ralstonia solanacearum ACCC01470, described intestinal bacteria Escherichia coli is intestinal bacteria Escherichia coli ACCC 10196, described Pseudomonas fluorescens Pseudomouas uoresceus is Pseudomonas fluorescens Pseudomouas uoresceusACCC01241
Described Sclerotinia sclerotiorum Sclerotinia sclerotiorum is Sclerotinia sclerotiorum SclerotiniaSclerotiorum ACCC36169, described cucumber fusarium axysporum Fusarium oxysporum f.sp.cucumerinum is cucumber fusarium axysporum Fusarium oxysporum f.sp.cucumerinum ACCC30442, described watermelon anthrax bacteria Colletotrichum lagenarium is watermelon anthrax bacteria Colletotrichumlagenarium ACCC36152, described dry thread Pyrenomycetes Rhizoctonia solani is dry thread Pyrenomycetes Rhizoctonia solani ACCC30332, the mould Pythium ultimum of described ultimate corruption is the mould Pythium ultimum of ultimate corruption ACCC36075, described big beautiful Verticillium Verticillium dahlia is big beautiful Verticillium Verticillium dahlia ACCC36109, described gaeumannomyce Gaeumannomyces graminis is gaeumannomyce Gaeumannomyces graminis ACCC30310, the curved spore mould Curvularia lunata of described standing grain is the curved spore mould Curvularia lunataACCC36580 of standing grain, described Phytophthora capsici Phytophthora capsici is Phytophthora capsici Phytophthora capsici ACCC36279, described Pyricularia oryzae Pyricularia grisea is Pyricularia oryzae Pyricularia grisea ACCC30320, described alternaric bacteria Alternaria tenuis is alternaric bacteria Alternaria tenuis ACCC36110, described tomato gray mould bacterium Botrytis cinerea is tomato gray mould bacterium Botrytis cinerea ACCC30091, described candiyeast Candida sp. is candiyeast Candida sp.ACCC21131, described geotrichum candidum Geo trichum candidum is geotrichum candidum Geotrichumcandidum CGMCC2.1084, described aspergillus oryzae Aspergillus oryza is aspergillus oryzae Aspergillusoryza ACCC30155, and described Penicilllum expansum Penicillium expansum is Penicilllum expansum Penicilliumexpansum ACCC30898.
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN1442477A (en) * | 2003-04-01 | 2003-09-17 | 青岛地恩地生物科技有限公司 | Solid fermentation process of streptomyces flavus and its production equipment |
CN101054568A (en) * | 2007-04-17 | 2007-10-17 | 周文彩 | Microorganism bacterium composition, special biological organic fertilizer for tobacco containing the same, preparation and use method for the microorganism bacterium composition |
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-
2010
- 2010-04-21 CN CN2010101559005A patent/CN101864378B/en not_active Expired - Fee Related
Patent Citations (3)
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---|---|---|---|---|
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