CN104988097B - A kind of streptomycete ER1396 bacterial strains and purposes - Google Patents
A kind of streptomycete ER1396 bacterial strains and purposes Download PDFInfo
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- CN104988097B CN104988097B CN201510454421.6A CN201510454421A CN104988097B CN 104988097 B CN104988097 B CN 104988097B CN 201510454421 A CN201510454421 A CN 201510454421A CN 104988097 B CN104988097 B CN 104988097B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/12—Powders or granules
- A01N25/14—Powders or granules wettable
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
Abstract
The present invention is a kind of streptomycete ER1396 bacterial strains（It is CGMCC No.9871 in the deposit number of CGMCC）And the method that zymotic fluid is prepared into soluble powder.Said preparation can be prevented effectively by colletotrichum category（Colletotrichumspp.）The anthracnose that fungal infection triggers.
Description
Invention field
The present invention relates to a kind of microorganism and purposes, more specifically to a kind of streptomycete (Streptomyces
Angustmyceticus) the preparation method of bacterial strain, purposes and its preparation, belongs to biological pesticide technical field.
Background technology
By colletotrichum category (Colletotrichum spp.) fungal infection trigger anthracnose, be summer strawberry nursery,
The Major Diseases that grape picking time occurs.Currently, the prevention of anthracnose mainly has with chemical agent come prevention and control, common fungicide
Methoxy acrylate (QoI) series bactericidal agent (Fluoxastrobin, kresoxim-methyl), sterol biosynthesis inhibitor (SBIs) series bactericidal agent
(difenoconazole, prochloraz), succinate dehydrogenase inhibitors (SDHI) series bactericidal agent (Boscalid, fluopyram, pyrrole thiophene
Bacterium amine, fluxapyroxad) and suppress cell mitogen benzimidazole (MBC) series bactericidal agent (carbendazim, benomyl, methyl
Thiophanate) etc..Due to peasant household, more frequencys, multiple dose saturation apply chemical agent throughout the year, cause such phytopathogen to chemistry
Medicament all generates different degrees of anti-(resistance to) pharmacological property, when have the example that prevention unsuccessfully causes total crop failure.
The biological pesticide special preparation for being used successfully to prevention anthrax bacteria at present registers application also not in production.It is existing
The biological and ecological methods to prevent plant disease, pests, and erosion Pseudomonas (kind) of document report biological control control disease mainly has in trichoderma fungi (Trichoderma spp.)
Trichoderma viride (Trichoderma viride) and Trichoderma harzianum (Trichoderma harzianum)；Bacillus is thin
Bacillus subtilis (Bacillus subtilis) in bacterium (Bacillus spp.), bacillus polymyxa (Bacillus
Polymyxa), bacillus megaterium (Bacillus megaterium) and bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens) etc.；Pseudomonas bacterium in pseudomonas bacterium (Pseudomonas spp.)
(Pseudomonas fluorescens)；Jute streptomycete (Streptomyces in streptomyces bacterium
Corchorusii) etc..Prevention anthrax bacteria it is relevant authorize patent of invention have ZL201210354630.X and
ZL201210354628.2.At present, the biological prevention and control agent in relation to streptomycete is with its fermentating metabolism product, and directly sharp streptomycete
Live body spore is process being rarely reported for preparation.
Strawberry and grape etc. are the industrial crops that anthracnose mainly causes harm, and such crop is mostly fruits in season class, more
Instant to adopt, the selection in chemical prevention to medicament has significant limitation.Therefore, to the industrial crops disease such as anthracnose
The research and development of biological control agent become active demand.Biocontrol bacteria and its work provided by the invention that anthracnose can effectively be prevented
Spore body preparation, does not refer to and openly in the prior art.
The content of the invention
The present invention solves the shortcomings of the prior art, there is provided one plant of new strepto- for being isolated from goose enteron aisle Bacterial diversity
Bacterium：Streptomycete (Streptomyces angustmyceticus) ER1396 bacterial strains, for preventing by colletotrichum category
(Colletotrichum spp.) infects strawberry and the bitter rot or anthracnose of grape of initiation.The present invention also provides with the streptomycete at the same time
The viable spore of ER1396 bacterial strains be processed into can for a long time room temperature preserve soluble powder preparation method.
The present invention is achieved by the following technical solutions：
Streptomycete (Latin name is Streptomyces angustmyceticus) ER1396 bacterial strains of the present invention,
It is in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, preserving number on October 29th, 2014：
CGMCC No.9871.The strain of the present invention has following feature：
(1) the cell microscopic morphology of ER1396 bacterial strains, culture and physiological and biochemical property (being shown in Table 1,2)
Table 1.ER1396 strain cell cultural characteristics
2. Physiology and biochemistry of table and microscopic features
(2) the 16S rDNA gene orders of streptomycete ER1396 bacterial strains of the invention are (i.e. SEQ ID NO:1)：
TTACACATGCAGTCGAACGATGAACCTCCTTCGGGAGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCT
GCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATACGACCACCGACCGCATGGTCTGGTGGTGG
AAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGG
GTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGG
AATATGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTC
AGCAGGGAAGAAGCGAGAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACG
TAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCCG
GGGCTTAACCCCGGGTCTGCATTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGG
TGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAA
GCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTGGGCGACATTCC
ACGTCGTCCGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAAT
TGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACAT
ACACCGGAAAACCCTGGAGACAGGGTCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTV
GTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGG
ACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGG
CTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCA
GTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAA
TACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCT
GTGGGAGGGAATCGTCGAAGG。
Biocontrol agent prepared by the ER1396 bacterial strains for the prevention anthracnose of the present invention, its preparation method include following step
Suddenly：
(1) preparation of level-one inoculum
On the inclined-plane of ER1396 inoculations to the culture medium containing CYD, under the conditions of 25 DEG C culture to production spore, with 10mL without
The spore of the lower bacterial strain of bacterium distillation washing makes suspension, and spore concentration is adjusted 1 × 10 with sterile distilled water4-105CFU/mL, puts
Saved backup in 4 DEG C of refrigerators, which is level-one inoculum.The composition of the CYD culture mediums is：Add in 1L distilled water
0.5g casamino acids, 0.8g yeast extracts, 0.4g glucose, 2.0g K2HPO4, 18g agar, pH is adjusted to 7.2-7.4
(2) preparation of two-stage inoculation body
Above-mentioned level-one inoculum storage solution 10mL is taken, is inoculated into the volume equipped with 100mL YGM nutrient solutions as the three of 250mL
In the bottle of angle, 32-36h is cultivated under the conditions of 250rpm, 30 DEG C, which is named as two-stage inoculation body, and two-stage inoculation body is store
It is hidden in spare in 4 DEG C of refrigerators.The YGM culture mediums form：Yeast extract 0.6% (W/V), 1.0% (W/ of glucose
), V mineral salt (adds 5.3g Na in 1L YGM nutrient solutions2HPO4,1.98g KH2PO4,0.2g MgSO4.7H2O,0.2g NaCl,
0.05g CaCl2.2H2O) and liquid microelement (in 1L YGM nutrient solutions plus 1.0mL), pH is adjusted to 7.1-7.2.Liquid microelement
Composition be：Dissolved with 6.4g CuSO in 1L sterile distilled waters4.5H2O,1.1g FeSO4.7H2O,7.9g MnCl2.4H2O,1.5g
ZnSO4.7H2O。
(3) preparation of three-level inoculum
Above-mentioned two-stage inoculation body storage solution 100mL is taken, is inoculated in the volume equipped with 1.1L YGM nutrient solutions as the three of 2.0L
In the bottle of angle, 200rpm is placed in, 48h is cultivated under the conditions of 30 DEG C, puts that 4 DEG C of storages are spare, which is named as three-level inoculum.
(4) ferment
Three-level inoculum storage solution 7.2L is taken, the volume equipped with 40L YGM (pH6.5) nutrient solution is inoculated in and ferments for 60L
Tank (Zhenjiang east GUS-60).It is 300rpm under fermentation condition, 30 DEG C, fermentation time 72h.
(5) preparation of high concentration mother bacterium powder
10min is centrifuged under the conditions of above-mentioned zymotic fluid is centrifuged 8000rpm, sediment is resuspended in 5L sterile distilled waters
Solution (dissolved with 750g lactose and 80g ammonium chlorides in solution), freeze-drying is (than bright FD-1D-80) after mixing, product mistake
200 mesh sieves, the bacterium powder of acquisition are high concentration mother's bacterium powder, and content is up to 1012-1014CFU/g, female bacterium powder are put 4 DEG C of low temperature and can be protected for a long time
Deposit.
(6) preparation of commercial soluble powder
Take 1g high concentrations female powder talcum powder sterile with 16kg and the sterile kaolin of 2.0kg to be sufficiently mixed, cross 200 mesh sieves, inspection
The content for surveying viable spore is about 1.0 × 109CFU/g。
The beneficial effects of the invention are as follows：
Bacterial strain provided by the invention is obtained by screening, effectively to suppress various plants from herbivore enteron aisle actinomyces
The biocontrol bacterial strain of disease fungus.In the present invention biocontrol agent used prepare by processing by the tunning of the bacterial strain and
Into.Of the invention provide in detail prepares helping for the processing technology of microbial inoculum, the application process of microbial inoculum and preparation using ER1396 bacterial strains
Agent component content.Viable bacteria body clump count (colony in the commercial pulvis obtained by processing technology provided as the invention
Forming units, CFU) reach 10 after testing8CFU/g.Said preparation can preserve for a long time under low temperature (5 DEG C) storage condition
(3 years), preserve the spore survival rate more than 80% of 12 months under room temperature storage condition.By the streptomycete work spore of the present invention
Preparation, greatly reduces in the past using the cost of streptomycete fermentation metabolite production biological pesticide, with before wide application
Scape.
(Latin name is Streptomyces angustmyceticus to the streptomycete ER1396 of the present invention
ER1396), on October 29th, 2014 in China Committee for Culture Collection of Microorganisms's common micro-organisms center's (abbreviation
CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences) preservation, preserving number is：CGMCC
No.9871。
Embodiment
Embodiment
The streptomycete ER1396 (Latin name is Streptomyces angustmyceticusER1396) of the present invention,
It is in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, preserving number on October 29th, 2014：
CGMCC No.9871.Cellular morphology, culture and the physiological and biochemical property of the bacterial strain of the present invention are shown in 1 He of " content of the invention " part table
Table 2.The 16S rDNA gene orders of the streptomycete ER1396 bacterial strains of the present embodiment are shown in " content of the invention " part.
The biocontrol agent prepared with the ER1396 bacterial strains of the prevention anthracnose of the present invention, its preparation method include following step
Suddenly：
(1) preparation of level-one inoculum
On the inclined-plane of ER1396 inoculations to the culture medium containing CYD, under the conditions of 25 DEG C culture to production spore, with 10mL without
The spore of the lower bacterial strain of bacterium distillation washing makes suspension, and spore concentration is adjusted 1 × 10 with sterile distilled water4-105CFU/mL, puts
Saved backup in 4 DEG C of refrigerators, which is level-one inoculum.The composition of the CYD culture mediums is：Add in 1L distilled water
0.5g casamino acids, 0.8g yeast extracts, 0.4g glucose, 2.0g K2HPO4, 18g agar, pH is adjusted to 7.2-7.4
(2) preparation of two-stage inoculation body
Above-mentioned level-one inoculum storage solution 10mL is taken, is inoculated into the volume equipped with 100mL YGM nutrient solutions as the three of 250mL
In the bottle of angle, 32-36h is cultivated under the conditions of 250rpm, 30 DEG C, which is named as two-stage inoculation body, and two-stage inoculation body is store
It is hidden in spare in 4 DEG C of refrigerators.The YGM culture mediums form：Yeast extract 0.6% (W/V), 1.0% (W/ of glucose
), V mineral salt (adds 5.3g Na in 1L YGM nutrient solutions2HPO4,1.98g KH2PO4,0.2g MgSO4.7H2O,0.2g NaCl,
0.05g CaCl2.2H2O) and liquid microelement (in 1L YGM nutrient solutions plus 1.0mL), pH is adjusted to 7.1-7.2.Liquid microelement
Composition be：Dissolved with 6.4g CuSO in 1L sterile distilled waters4.5H2O,1.1g FeSO4.7H2O,7.9g MnCl2.4H2O,1.5g
ZnSO4.7H2O。
(3) preparation of three-level inoculum
Above-mentioned two-stage inoculation body storage solution 100mL is taken, is inoculated in the volume equipped with 1.1L YGM nutrient solutions as the three of 2.0L
In the bottle of angle, 200rpm is placed in, 48h is cultivated under the conditions of 30 DEG C, puts that 4 DEG C of storages are spare, which is named as three-level inoculum.
(4) ferment
Three-level inoculum storage solution 7.2L is taken, the volume equipped with 40L YGM (pH6.5) nutrient solution is inoculated in and ferments for 60L
Tank (Zhenjiang east GUS-60).It is 300rpm under fermentation condition, 30 DEG C, fermentation time 72h.
(5) preparation of high concentration mother bacterium powder
10min is centrifuged under the conditions of above-mentioned zymotic fluid is centrifuged 8000rpm, sediment is resuspended in 5L sterile distilled waters
Solution (dissolved with 750g lactose and 80g ammonium chlorides in solution), freeze-drying is (than bright FD-1D-80) after mixing, product mistake
200 mesh sieves, the bacterium powder of acquisition are high concentration mother's bacterium powder, and content is up to 1012-1014CFU/g, female bacterium powder are put 4 DEG C of low temperature and can be protected for a long time
Deposit.
(6) preparation of commercial soluble powder
Take 1g high concentrations female powder talcum powder sterile with 16kg and the sterile kaolin of 2.0kg to be sufficiently mixed, cross 200 mesh sieves, inspection
The content for surveying viable spore is about 1.0 × 109CFU/g。
Implementation result
(1) indoor virulence of the ER1396 bacterial strains soluble powder to colletotrichum
The soluble powder that ER1396 bacterial strain fermentation liquors are prepared is as follows to the Toxicity Determination method of colletotrichum：
With ER1396 bacterial strains soluble powder (1.0 × 109CFU/g it is respectively 10) to be prepared into spore content7、106、105、
104With 103The CYD tablets of CFU/mL, and sterile distilled water is set as blank control；Comparison medicament bacillus subtilis wettable
Pulvis (1.0 × 109CFU/g) measured by above-mentioned same concentrations.With the card punch of a diameter of 4mm, put down in the colletotrichum grown
Beaten on the edge of plate and take bacterium piece to be inoculated into the center containing the above-mentioned tablet containing bacterium, each concentration of each bacterial strain sets 4 repetitions.Will be flat
Plate is placed in 25 DEG C of culture 7d.The bacterium colony net growth diameter (net growth diameter=colony diameter-bacterium piece of bacterium colony is measured with crossing method
Diameter), calculate inhibiting rate.Inhibiting rate=(the net growth diameter of the blank control bacterium colony-net growth diameter of drug containing bacterium colony)/blank control
Net growth diameter × 100% of bacterium colony.Virulence regression equation is calculated with 13.0 softwares of DPS.
Toxicity Determination result of the ER1396 soluble powders to colletotrichum：
Growth of the ER1396 soluble powders to colletotrichum mycelia has very strong inhibitory action (table 3,4,5), its EC50Value
For 6.6602 × 103CFU/mL.In contrast, under the conditions of identical extension rate bacillus subtilis wettable powder to pierce disk spore
The EC of bacterium50Value is respectively 1.1452 × 106CFU/mL.The antibacterial virulence pole of ER1396 soluble powders is significantly higher than withered grass gemma
Bacillus wettable powder.
Average inhibition of the table 3.ER1396 soluble powders to colletotrichum
| Concentration (CFU/mL) | 107 | 106 | 105 | 104 | 103 |
| Average inhibition (%) | 86.42 | 71.36 | 62.89 | 52.04 | 41.84 |
Average inhibition of the 4. bacillus subtilis wettable powder of table to colletotrichum
| Concentration (CFU/mL) | 107 | 106 | 105 | 104 | 103 |
| Average inhibition (%) | 64.14 | 49.87 | 31.42 | 24.28 | 11.27 |
Toxicity test result of the table 5.ER1396 soluble powders to colletotrichum
(2) field controling test of the ER1396 soluble powders to Strawberry anthracnose
Test process method：This experiment is in the strawberry Seedlings nursery of cloud rabbitweed certain kind of berries cooperative society of industry, strawberry cultivars
For " the red cheek " of high sense anthracnose.On the June 8th, 2014 of spraying prevention for the first time, is spaced spray in 10 days and once (is subject to postponement in case of rain to next day
Prevention), co-continuous to prevent 8 times, August is investigated on the 28th, records diseased plant number, and calculate prevention effect.Test medicine processing：(A)
Streptomycete ER1396 soluble powders (1.0 × 109CFU/g) 1000 times of liquid；(B) comparison medicament bacillus subtilis wettable
Pulvis (1.0 × 109CFU/g) 1000 times of liquid；(C) blank control is sprayed with clear water.Mu water consumption 50kg, plot area 20m2,
Experiment sets 4 repetitions.
As a result：Field test results (table 6) show that the prevention effect of 1000 times of liquid of ER1396 soluble powders is
92.32%, the prevention effect of 1000 times of liquid of bacillus subtilis wettable powder is only 77.71% in contrast, between two medicaments
Reach statistics level difference.
Field controling test result of the table 6.ER1396 soluble powders to Strawberry anthracnose
Note：Different capitalizations represent P<0.01.
(3) field controling test of the ER1396 soluble powders to bitter rot or anthracnose of grape
Test process method：It is (white behind Jurong City of Jiangsu Province that agricultural science and technology Demonstration Garden is popular in the industry Wanshan Mountain
Town), garden grape contiguous plant is tested, grape variety is huge rich.Transplanted using horizontal rack formula, distance between rows and hills 2m × 4m, the age of tree 7 years
It is raw.Horse liver soil, middle fertility, the content of organic matter 1% or so, orchard management more solito.During experiment (5~August in 2014).Examination
Testing chemicals treatment is：(A) 1000 times of liquid of ER1396 soluble powders；(B) comparison medicament is bacillus subtilis wettable powder
1000 times of liquid；(C) blank control is not prevent.Plot area 200m2, random district's groups arrangement, 4 repetitions.Tested May 16
1st spray, is prevented 5 times, last 1 prevention date is 8 altogether every 10 days (adjustment spray date when running into rainy day) later
The moon 1.It is per 667m with liquid volume2Spray 100kg liquids.(August 15 days) investigates preventive effect 14 days after last 1 dispenser.Investigation
When in each cell between select 4 plants of grapes at random, take 1 fruit ear respectively in upper, middle and lower, left and right 5 orientation, totally 20 fruits
Fringe, records disease fruit number and total fruit grain number, and calculates diseased fruit rate and prevention effect.
As a result：Field test results (table 7) show that the prevention effect of 1000 times of liquid of ER1396 soluble powders is
91.31%, the prevention effect of 1000 times of liquid of bacillus subtilis wettable powder is only 47.47% in contrast.Reached between medicament
To statistics level difference.
Field controling test result of the table 7.ER1396 soluble powders to bitter rot or anthracnose of grape
Note：Different capitalizations represent P<0.01.
Sequence table
<110>Jiangsu Hilly Ground Zhenjiang Agriculture Science Research Institute
<120>A kind of streptomycete ER1396 bacterial strains and purposes
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1407
<212> DNA
<213> Streptomyces angustmyceticus
<400> 1
ttacacatgc agtcgaacga tgaacctcct tcgggagggg attagtggcg aacgggtgag 60
taacacgtgg gcaatctgcc cttcactctg ggacaagccc tggaaacggg gtctaatacc 120
ggatacgacc accgaccgca tggtctggtg gtggaaagct ccggcggtga aggatgagcc 180
cgcggcctat cagcttgttg gtggggtgat ggcctaccaa ggcgacgacg ggtagccggc 240
ctgagagggc gaccggccac actgggactg agacacggcc cagactccta cgggaggcag 300
cagtggggaa tatgcacaat gggcgaaagc ctgatgcagc gacgccgcgt gagggatgac 360
ggccttcggg ttgtaaacct ctttcagcag ggaagaagcg agagtgacgg tacctgcaga 420
agaagcgccg gctaactacg tgccagcagc cgcggtaata cgtagggcgc aagcgttgtc 480
cggaattatt gggcgtaaag agctcgtagg cggcttgtca cgtcggatgt gaaagcccgg 540
ggcttaaccc cgggtctgca ttcgatacgg gcaggctaga gttcggtagg ggagatcgga 600
attcctggtg tagcggtgaa atgcgcagat atcaggagga acaccggtgg cgaaggcgga 660
tctctgggcc gatactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac 720
cctggtagtc cacgccgtaa acgttgggaa ctaggtgtgg gcgacattcc acgtcgtccg 780
tgccgcagct aacgcattaa gttccccgcc tggggagtac ggccgcaagg ctaaaactca 840
aaggaattga cgggggcccg cacaagcagc ggagcatgtg gcttaattcg acgcaacgcg 900
aagaacctta ccaaggcttg acatacaccg gaaaaccctg gagacagggt cccccttgtg 960
gtcggtgtac aggtggtgca tggctgtcgt cagctcgtgt vgtgagatgt tgggttaagt 1020
cccgcaacga gcgcaaccct tgttctgtgt tgccagcatg cccttcgggg tgatggggac 1080
tcacaggaga ctgccggggt caactcggag gaaggtgggg acgacgtcaa gtcatcatgc 1140
cccttatgtc ttgggctgca cacgtgctac aatggccggt acaatgagct gcgataccgc 1200
gaggtggagc gaatctcaaa aagccggtct cagttcggat tggggtctgc aactcgaccc 1260
catgaagtcg gagttgctag taatcgcaga tcagcattgc tgcggtgaat acgttcccgg 1320
gccttgtaca caccgcccgt cacgtcacga aagtcggtaa cacccgaagc cggtggccca 1380
acccctgtgg gagggaatcg tcgaagg 1407
Claims (6)
1. a kind of streptomycete bacterial strain, its Classification And Nomenclature is Streptomyces angustmyceticus ER1396, in being preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, preserving number are：CGMCC No.9871.
2. streptomycete bacterial strain according to claim 1, it is characterised in that the 16S rDNA gene orders of the bacterial strain are as such as
SEQ ID NO:Shown in 1.
3. application of the streptomycete bacterial strain on prevention Strawberry anthracnose and bitter rot or anthracnose of grape described in claim 1.
A kind of 4. soluble powder being prepared using streptomycete bacterial strain described in claim 1.
A kind of 5. method for preparing the soluble powder described in claim 4, it is characterised in that comprise the following steps：
(1) preparation of level-one inoculum
On the inclined-plane of ER1396 inoculations to the culture medium containing CYD, culture is to production spore under the conditions of 25 DEG C, with the sterile steamings of 10mL
The spore that distilled water washes lower bacterial strain makes suspension, and spore concentration is adjusted 1 × 10 with sterile distilled water4-105CFU/mL, is placed in 4
Saved backup in DEG C refrigerator, which is level-one inoculum；
(2) preparation of two-stage inoculation body
The level-one inoculum storage solution 10mL obtained by (1) is taken, it is 250mL's to be inoculated into the volume equipped with 100mL YGM nutrient solutions
In triangular flask, 32-36h is cultivated under the conditions of 250rpm, 30 DEG C, which is named as two-stage inoculation body, by two-stage inoculation body
It is stored in spare in 4 DEG C of refrigerators；
(3) preparation of three-level inoculum
Two-stage inoculation body storage solution 100mL obtained by taking (2), is inoculated in the triangle that the volume equipped with 1.1L YGM nutrient solutions is 2.0L
In bottle, 200rpm is placed in, cultivates 48h under the conditions of 30 DEG C, put that 4 DEG C of storages are spare, which is named as three-level inoculum；
(4) ferment
Three-level inoculum storage solution 7.2L obtained by taking (3), is inoculated in equipped with 40L YGM, the volume of pH=6.5 nutrient solutions is 60L
Fermentation tank, fermentation condition 300rpm, 30 DEG C, fermentation time 72h；
(5) preparation of high concentration mother bacterium powder
10min will be centrifuged under the conditions of (4) zymotic fluid 8000rpm, sediment is resuspended in 5L sterile distilled water solution, mixed
Freeze-drying, product cross 200 mesh sieves after uniformly, and the bacterium powder of acquisition is high concentration mother's bacterium powder, and content is up to 1012-1014CFU/g, it is female
Bacterium powder is put 4 DEG C of low temperature and can be preserved for a long time；
(6) preparation of commercial soluble powder
1g high concentration female powders are often taken, take the sterile talcum powder of 16kg and the sterile kaolin of 2.0kg to be sufficiently mixed, cross 200 mesh sieves, detection
The content of viable spore is about 1.0 × 109CFU/g。
6. preparation method according to claim 5, it is characterised in that the composition of the CYD culture mediums in the step (1)
For：Add 0.5g casamino acids, 0.8g yeast extracts, 0.4g glucose, 2.0g K in 1L distilled water2HPO4, 18g agar,
PH is adjusted to 7.2-7.4；
YGM culture mediums in the step (2), which form, is：Yeast extract 0.6%W/V, glucose 1.0%W/V, mineral salt and
Liquid microelement；PH is adjusted to 7.1-7.2；The mineral salt is in 1L YGM nutrient solutions plus 5.3g Na2HPO4、1.98g
KH2PO4、0.2g MgSO4.7H2O、0.2g NaCl、0.05g CaCl2·2H2O；Add the micro members of 1.0mL in 1L YGM nutrient solutions
Plain liquid, the composition of the liquid microelement are：Dissolved with 6.4g CuSO in 1L sterile distilled waters4.5H2O、1.1g FeSO4.7H2O、
7.9g MnCl2.4H2O、1.5g ZnSO4.7H2O；
Dissolved with 750g lactose and 80g ammonium chlorides in sterile distilled water solution in (5).
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