CN109825456A - A kind of Manas' bacillus marinus E40208a1 and its application - Google Patents
A kind of Manas' bacillus marinus E40208a1 and its application Download PDFInfo
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Abstract
The invention discloses a kind of Manas' bacillus marinus E40208a1 and its applications, Manas' bacillus marinus E40208a1 was deposited in Guangdong Province's Culture Collection on 01 03rd, 2019, deposit number is GDMCC NO:60497, the bacterial strain secondary metabolite fermentation liquid is to marmor upsilon (Potato virus Y, PVY virus disease caused by) has significant antagonism, and material source is extensive, and it is at low cost, it is environmentally safe.
Description
Technical field
The invention belongs to microorganisms technical fields, are related to one plant and answer from the Halophiles of Cha Er Han Salt Lake and its biological and ecological methods to prevent plant disease, pests, and erosion
With specially a kind of Manas' bacillus marinus E40208a1 and its application.
Background technique
Marmor upsilon (Potato virus Y, PVY) is marmor upsilon section (Potyviridae) potato Y disease
Poison belongs to the prototypical member of (Potyvirus), and virion is in micro-bend curve-like, long 680-900nm, wide 11-12nm.The base of PVY
Because group is single RNA normal chain, relative molecular mass is 3.1 × 106-3.5×106, it is about 10000 amino acid.In genome
5 ' ends is with the genome binding protein (VPg) and one section of non-coding region of Covalent bonding together, about 180 bases；Genome
3 ' ends have poly (A) tail, only one reading frame of whole gene group generates a big polyprotein, passes through after translation
The protease of itself coding is processed to form mature coat protein and at least seven kinds of non-structural proteins.
Potyvirus functional genomics achieves impressive progress in recent years, obtains to known viral gene function research
The host range of remarkable progress, PVY is very wide, is important worldwide distributing virus, can infect 34 and belong to more than 170 plants,
Based on plant of Solanaceae, followed by Chenopodiaceae and leguminous plant.In China, PVY is in addition to tobacco of causing harm, also serious harm Ma Ling
The crops such as potato, tomato and capsicum.Potato infects PVY, the general underproduction 50% or so, when itself and potato virus X (Potato
Virus X, PVX) or when marmor solani (Potato virus A, PVA) Combined Infection, potato is made to generate serious wrinkle
Contracting flower leaf paresthesia, plant is short and small, and leaf-shrinkage, the underproduction is up to 80%.The current research in relation to marmor upsilon disease is mainly needle
To tobacco potato Y virus disease.Tobacco potato Y virus disease is also known as arteries and veins necrosis disease, brown arteries and veins disease, macula lutea necrosis disease etc., is by PVY
A kind of disease of caused systemic infection, is distributed widely in all over the world, is one of the important disease in World tobacco producing region.PVY
The tobacco from seedling to Adult plant can be infected, macroscopical symptom is roughly divided into floral leaf disease, and arteries and veins necrosis, stippled streak disease and stem are bad
Four seed type of incruable disease.
The major measure of prevention and treatment tobacco potato Y virus disease has the following at present: cultivating and utilize disease-resistant variety；It is planting
Cigarette front shovel is except viral wild host, to reduce primary source of infection；It avoids for tobacco field being arranged near solanaceous crops, in tobacco field and poison
Plantation isolation crop between the plant of source, such as corn, sunflower, to prevent aphid to tobacco field transmitted virus；It enhances field management,
Reasonable top dressing, in time ridging are watered to promote cigarette strain robust growth, and disease resistance is improved, and shade and low-lying land is avoided to plant cigarette, agriculture
Must carry out disinfection hand and tool when thing operation processing；Aphid diseases prevention is driven in seedbed and tobacco field silvery reflection film, before planting cigarette
The solanaceous crops near tobacco field and the aphid on weeds are sprayed using medicament, avoids alatae from migrating and passes poison.
Halophiles is a kind of microbe groups for having important researching value and wide application prospect.Its application value
Mainly including the use of Halophiles production food additives, production enzyme preparation, applied to the neck such as food, packaging, adhesive and medicine
Domain is applied to processing industrial wastewater etc. in terms of environmental organism is administered.Applicant further found that one plant of Halophiles bacterial strain is to Phytophthora capsici
Bacterium inhibitory activity with higher.But in relation to the biological control for carrying out potato virus disease using Halophiles bacterial strain, especially
Using such microbial resources carry out marmor upsilon anti-virus formulation research and development and the mechanism of action in terms of research still belong to sky
White collar domain.
Summary of the invention
In view of this, it is an object of the invention to provide a kind of Manas' bacillus marinus E40208a1 and its application, the bacterium
Strain isolates and purifies to obtain from Cha Er Han Salt Lake lake water, and is applied to the research of marmor upsilon biological control of diseases, makes
It obtains biological control marmor upsilon disease to be possibly realized, while being the extreme environments resource such as salt lake using foundation is provided, establish
Basis is determined.
In order to achieve the above technical purposes, the present invention is realized especially by following technical scheme:
The present invention provides bacterial strain E40208a1, which uses dilution plate enrichment culture from Cha Er Han Salt Lake lake water
Method carries out separation and purifying obtains.
The molecules characteristic of bacterial strain E40208a1 of the present invention extracts bacterial strain DNA and carries out PCR amplification, is somebody's turn to do
The 16S rRNA gene of bacterium, nucleotide sequence is as shown in SEQ ID NO.1.By GenBank nucleic acid sequence library and obtained sequence
Column carry out sequence analysis, find the homology highest of bacterial strain E40208a1 and Oceanobacillus manasiensis, reach
To 99%.It by the dientification of bacteria is Manas' bacillus marinus according to 16S rDNA sequence homology and morphological analysis
(Oceanobacillus manasiensis), therefore obtained bacterial strain E40208a1 of the invention is named as Manas ocean
Bacillus (Oceanobacillus manasiensis) E40208a1.
This bacillus marinus E40208a1 that receives preservation is subjected to, depositary institution: Guangdong Province microorganism fungus kind
Collection, preservation address: preservation date: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 on 01 03rd, 2019, is protected
Hiding number is GDMCC NO:60497.
Application of the Manas' bacillus marinus E40208a1 of the present invention in biocontrol of plant disease, it is described
Plant disease be disease caused by PVY, the disease is specially marmor upsilon disease, and the plant is solanaceous crops,
Preferably tobacco or potato.
Preferably, Manas' bacillus marinus (Oceanobacillus manasiensis) E40208a1
Application of the secondary metabolite fermentation liquid in biocontrol of plant disease is also within the scope of the present invention.
Described Manas' bacillus marinus (Oceanobacillus manasiensis) the E40208a1 fermentation liquid
The preparation method comprises the following steps: the colony edge after cultivating 3d takes a little lawn to be inoculated into the culture of 100mlATCC213 Media modified
In bottle, every bottle connects an acicula, 28 DEG C, 200r/min-1Cultivate 7d.
The ATCC213 Media modified are as follows: MgSO4·7H2O 10g, CaCl2·2H2O0.2g, KCl 5g, albumen
Peptone 2.5g, yeast extract 10g, NaCl 30g, agar powder 12g, distilled water to 1000ml, pH 7.2-7.4.
Manas' bacillus marinus (Oceanobacillus manasiensis) E40208a1 of the present invention exists
As the application in preparation prevention and treatment crop disease drug.
By conventional liquid or solid culture, obtaining includes microorganism culture, raw by conventional liquid fermentation
Produce, be then added surfactant such as dispersing agent, stabilizer, wetting agent, binder, defoaming agent, disintegrating agent, in antifreeze
After one or more or absorption carrier are mixed in a certain ratio, it is prepared into wettable powder, water dispersible granules, suspending agent, outstanding cream
Agent, aqueous emulsion or microemulsion；The crop includes solanaceous crops, preferably tobacco and potato.
Certain Manas' bacillus marinus (Oceanobacillus manasiensis) of the present invention
The secondary metabolite fermentation liquid of E40208a1 is preventing and treating the application in crop disease drug also in guarantor of the invention as preparation
It protects in range.
Manas' bacillus marinus (Oceanobacillus manasiensis) E40208a1 of the present invention prevents and treats crop
The method of disease also belongs to protection scope of the present invention, and this method is that spraying treatment is carried out in growing process；The spray
Mist is the bacteria suspension or fermentation liquid of Manas' bacillus marinus (Oceanobacillus manasiensis) E40208a1
Or the pesticidal preparations of metabolite preparation.
The invention has the benefit that
The present invention is for seriously affecting tobacco and Potato Quality and yield in tobacco and potato agricultural production process
PVY is target, and separation screening is to a kind of Manas' bacillus marinus (Oceanobacillus from Cha Er Han Salt Lake lake water
Manasiensis) E40208a1, itself can significant antagonism marmor upsilon, and secondary metabolite fermentation liquid is to potato
Y virus has preferable preventive effect, while to tobacco safety.The present invention being capable of effectively antagonism and control marmor upsilon disease, and original
Expect it is from a wealth of sources, it is at low cost, it is environmentally safe.
Detailed description of the invention
Fig. 1 is the phylogenetic tree of Manas' bacillus marinus E40208a1 of the present invention；
Fig. 2 is that host's three lives cigarette total serum IgE mentions after Manas' bacillus marinus E40208a1 of embodiment of the present invention processing
Take result；
Fig. 3 is expression quantity of target gene of the embodiment of the present invention PVY in different disposal；A, b respectively represents positive control
With bacterial strain E40208a1 processing；
Fig. 4 is expression quantity of target gene of the embodiment of the present invention CPIP in different disposal；A, b, c respectively represent blank pair
According to, positive control and bacterial strain E40208a1 processing；
Fig. 5 is expression quantity of target gene of the embodiment of the present invention Cullin in different disposal；A, b, c respectively represent blank
Control, positive control and bacterial strain E40208a1 processing；
Fig. 6 is expression quantity of target gene of the embodiment of the present invention GST in different disposal；A, b, c respectively represent blank pair
According to, positive control and bacterial strain E40208a1 processing；
Fig. 7 is expression quantity of target gene of the embodiment of the present invention PSII in different disposal；A, b, c respectively represent blank pair
According to, positive control and bacterial strain E40208a1 processing；
Fig. 8 is expression quantity of target gene of the embodiment of the present invention OEE3 in different disposal；A, b, c respectively represent blank pair
According to, positive control and bacterial strain E40208a1 processing；
Fig. 9 is the expression of Potyvirus functional protein related gene of the embodiment of the present invention.
Specific embodiment
Technical solution of the present invention is further described below with reference to embodiment, it is as described below, it is only to of the invention
Preferred embodiment not limits the present invention, any person skilled in the art possibly also with
The technology contents of the disclosure above are changed or are modified as the equivalent embodiment changed on an equal basis.It is all without departing from the present invention program
Hold, according to the technical essence of the invention any simple modification, equivalent change and modification made to the above embodiment, all falls within this
In the protection scope of invention.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The separation and identification of 1 bacterial strain E40208a1 of embodiment
1) separation of bacterial strain E40208a1
Appropriate salt lake lake mud sample is taken, is air-dried (7d or so), 120 DEG C of processing 1h.The lake mud sample that 10g is handled well is weighed,
90m L antiseptic sea water is added, is put into the triangular flask equipped with glass marble to have sterilized, sufficiently oscillation 30min, is drawn after standing
Supernatant, this supernatant are 10-1Soil sample.By above-mentioned 10-1Sample is successively diluted to 10-2With 10-3Times, draw each gradient sample of 150 μ L
Product are respectively coated on each culture medium flat plate, and each sample is repeated 3 times.Plate after coating is inverted in incubator, respectively at
It is inverted culture at 28 DEG C and 37 DEG C, has observed bacterium colony appearance, picking single colonie, purifying.Bacterial strain after purification is stored in test tube
Inclined-plane, and be placed in 4 DEG C of refrigerators.Culture medium is ATCC213 improved culture medium: MgSO4·7H2O10g, CaCl2·2H2O
0.2g, KCl 5g, peptone 2.5g, yeast extract 10g, NaCl 30g, agar powder 12g, distilled water to 1000ml, pH 7.2-
7.4.It is placed in picking single colonie culture in 37 DEG C of illumination boxs after 3d.
2) identification of bacterial strain E40208a1
The DNA for extracting bacterial strain E40208a1 carries out PCR amplification, PCR amplification system: 10 × Buffer, 2.5 μ L, template DNA
1 μ L, Taq enzyme (5U/ μ L) 0.2 μ L, dNTP (2.5mmol/ μ L) 2 μ L, 27 (5'-AGAGTTTGATCCTGGCTCAGG- of primers F
3') and primer P1541 (5'-AAGGAGGTGGTGATCCAGCCGCA-3') (10pmol/ μ L) each 1 μ L, moisturizing to 25 μ L.Reaction
Condition are as follows: 94 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 80s, totally 30 recycle；72℃10min.PCR reaction condition: 94 DEG C
It is denaturalized 45s, 50 DEG C of annealing 45s, 72 DEG C of extension 75s, 50 μ L reaction systems 30 recycle.PCR product is through agarose gel electrophoresis
Detection, obtains sequence results through cloning and sequencing.
The 16S rRNA gene of the bacterium is obtained, nucleotide sequence is as shown in SEQ ID NO.1.By GenBank nucleic acid sequence
It arranges library and obtained sequence carries out sequence analysis, discovery bacterial strain E40208a1 and Oceanobacillus manasiensis's
Homology highest, reaches 99%.Obtained 16S rDNA complete sequence and the 16S rDNA obtained from the databases such as GenBank
Sequence is compared, and carries out the building (Fig. 1) of phylogenetic tree.According to 16S rDNA sequence homology and systematic growth by the bacterium
It is accredited as Manas' bacillus marinus (Oceanobacillus manasiensis), therefore by obtained bacterial strain of the invention
E40208a1 is named as Manas' bacillus marinus (Oceanobacillus manasiensis) E40208a1.
Obtained Manas' bacillus marinus E40208a1 preservation is subjected to, depositary institution: Guangdong Province's microbial bacteria
Kind collection, preservation address: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, preservation date: on 01 03rd, 2019,
Deposit number is GDMCC NO:60497.
The activation of 2 bacterial strain of embodiment, fermentation liquid preparation and inhibition PVY evaluation of resistance
1.1 prepare for examination culture medium, bacterial strain activation and fermentation liquid
1.1.1 Halophiles activation fermentation medium is as follows:
ATCC213 improved culture medium: MgSO4·7H2O 10g, CaCl2·2H2O 0.2g, KCl 5g, peptone 2.5g,
Yeast extract 10g, NaCl 30g, agar powder 12g, distilled water to 1000mL, pH 7.2-7.4.
1.1.2 Halophiles is separated from Qinghai Chaerhan salt lakes by key lab, the Qinghai-Tibet biotechnology Ministry of Education
Purification storage.Bacterial strain will be saved to cross in the solid medium of ATCC213 improved culture medium, and in 37 DEG C of constant incubators
It cultivates spare for 24 hours.
1.1.3 prepared by fermentation liquid: the Halophiles bacterial strain that activation is completed, which is inoculated in liquid amount with 20% inoculum concentration, is
In 80% 1000mL conical flask, and under conditions of 28 DEG C, 180r/min carries out shake culture 7d to obtain fermentation liquid spare.
1.2 bacterial strains inhibit PVY evaluation of resistance
1.2.1 Real-time PCR method is evaluated
Three lives cigarette carries out seedling basin plantation in heliogreenhouse, when plant 4-5 piece leaf extends completely, according to passivation mode,
It is sun with phosphate buffer mixing PVY virus liquid using bacterial strain fermentation liquor with isometric PVY virus liquid mixing 30min as processing
Property control, to be only inoculated with phosphate buffer as blank control, using Ningnanmycin mixing PVY virus liquid as reference, with tradition
Frictional inoculation method be inoculated with three lives cigarette, experiment is repeated 3 times.
1.2.2 Real-time PCR quantitative detection PVY content Establishing
(1) extraction of total serum IgE
The extraction that tender top vane carries out total serum IgE, the extraction reference of tobacco leaf total serum IgE are acquired after being inoculated with 1 week
TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa) specification carries out.To guarantee quantitative fluorescent PCR
The accuracy of detection need to detect the quality and concentration for extracting tobacco total serum IgE.
1% agarose gel electrophoresis results of the extracted sample RNA of this test are as shown in Figure 2: 28S rRNA and 18S
Two master tapes of rRNA are apparent, and the former is bright compared with the latter.Meanwhile the concentration and quality of RNA, OD are detected with instrument260/OD280
Ratio meets reverse transcription requirement of experiment between 1.8~2.0.
(2) reverse transcription
Reverse transcription is referring to PrimeScriptTM RT Master Mix (Perfect Real Time) (TaKaRa) explanation
Book carries out.Reaction system is as follows: 5 × PrimeScript RT Master Mix (Perfect Real Time) 2 μ l, Total
RNA volume is equal to 500 divided by RNA concentration, with RNase Free ddH2O is supplied to 10 μ l, 10 μ l of total volume.Reaction condition: 37
DEG C 15min, 55 DEG C of 5sec, 4 DEG C of Infinite Cyclics.
According to standard fluorescence quantification PCR primer design principle, using DNAMAN6.0 software (LynnonBiosoft,
Quebec, Canada) sequence alignment is carried out to PVY existing in GenBank and tobacco Actin gene complete sequence, selection is each
Sequence region the most conservative carries out design of primers, then utilizes Oligo6.0 software and on-line analysis software OligoCalc (h
Ttp: //www.basic.northwestern.edu/biotools/oligocalc.html) designed primer is commented
Valence, and verified by online BLSAT program (http://www.ncbi.nlm.nih.gov/blast/), to avoid virus
Specific primer sequence and any sequence homology of tobacco gene group.Primer synthesizes (table 1) by Shanghai bioengineering Co., Ltd.
1 primer sequence of table
(3) PVY inhibiting rate calculation method
According to the original testing result of Real Time qPCR, according to 2-△△ctRelative quantification calculation formula:
F=2{ [the house-keeping gene mean CT-number of to be measured group of target gene mean CT-number-to be measured group]-[control group target gene mean CT-number-control group house-keeping gene mean CT-number] }, calculate each processing
The expression quantity of middle target gene PVY.Bacterial strain to PVY inhibiting rate=(expression quantity of PVY in expression quantity-processing of PVY in control)/
Expression quantity × 100% of PVY in control.As a result as shown in Table 2 and Fig. 3.
2 bacterial strain E40208a1 of table has effect to the inhibition of PVY
The response expression of related adversity gene in 3 three lives of embodiment cigarette
1) template prepares
Extracted total serum IgE and the obtained cDNA of reversion are directly as template in embodiment 2.
2) related adversity gene design of primers in three lives cigarette
Referring to primer design method in embodiment 2, the fluorescent primer (table of related resistance genes in host's three lives cigarette is designed
3)。
3 adversity gene sequence of table
3) response of related adversity gene is expressed in three lives cigarette
PVY infects three lives cigarette after a week, by Real-time PCR to 5 kinds of adversity gene expression quantity of host's three lives cigarette into
Row quantitative detection.Wherein, glutathione-S-transferase (GST), the capsid proteins PVY Interaction Protein -3 (CpIp), Cullin
It is albumen -1 (Cullin), related to PVY stress response；Photosynthetical system II (PSII) is related to photosynthesis；Put oxygen enhancing albumen-
3 (OEE3) are related to respiration.
Compared with blank control, after PVY infects system host's three lives cigarette, turn to degeneration-resistant relevant gene glutathione-S-
Move enzyme (GST), the capsid proteins PVY Interaction Protein -3 (C pIp), Cullin albumen -1 (Cullin), photosynthetical system II
(PSII) and put oxygen enhancing protein-3 (OEE3) expression up-regulation (such as Fig. 4-8).Through Manas' bacillus marinus (Oce
Anobacillus manasiensis) after E40208a1 fermentation liquor treatment, the expression quantity of adversity gene is compared with positive control in the three lives
Respectively up-regulated expression 1.06,2.82,3.16,1.15 and 2.47 (table 4).
The expression quantity of related adversity gene in 4 three lives of table cigarette
The expression of 4 Potyvirus functional protein related gene of embodiment
1) extraction of total serum IgE
2) design of primers of Potyvirus functional protein related gene
Referring to primer design method in embodiment 1, the general primer of Potyvirus functional protein related gene is designed
(table 5).
Major protein related gene primer sequence coded by 5 marmor upsilon of table (PVY)
3) expression of Potyvirus functional gene
Three lives cigarette is infected after a week to PVY by conventional RT-PCR, the encoded major protein related gene of PVY virus
Expression quantity carries out half-quantitative detection.
Testing result shows PVY through Manas' bacillus marinus (Oceanobacillus m anasiensis)
After E40208a1 Passivation Treatment, amplification breeding of the PVY in host's three lives cigarette is inhibited, PVY protein related gene CP, P1,
The expression of P3, N1a, N1b, HC-pro are compared compared with positive control is obviously inhibited (such as Fig. 9).
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understand without departing from the principles and spirit of the present invention can to these examples carry out it is a variety of variation, modification, replacement and
Modification, the scope of the present invention is defined by the appended.
Sequence table
<110>Qinghai Academy of Agriculture and Forestry Sciences
<120>a kind of Manas' bacillus marinus E40208a1 and its application
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<170> SIPOSequenceListing 1.0
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tgacctcgcg gttttgcttc cctttgttcc atccattgta gcacgtgtgt agcccaggtc 240
ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca ccggcagtca 300
ccttagagtg cccaacttaa tgctggcaac taagatcaag ggttgcgctc gttgcgggac 360
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ccgaagggaa cttcctatct ctaggaatag cagaggatgt caagacctgg taaggttctt 480
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ccactctcct cttctgtact caagtcctcc agtttccaat gaccgttcgc ggttgagccg 840
cgagatttca catcagactt aaaggaccgc ctgcgagcgc tttacgccca ataattccgg 900
acaacgcttg ccccctacgt attaccgcgg ctgctggcac gtagttagcc ggggctttct 960
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gtggccgatc accctctcag gtcggctacg catcgtcgcc ttggtgagcc gttacctcac 1200
caactagcta atgcgccgcg ggcccatctg taagtggcag ccgaaaccgc ctttcaactt 1260
tcctttatgc aaagaaaagt attatccggt attagccccg gtttcccggg gttatcccag 1320
tcttacaggc aggttgccca cgtgttactc acccgtccgc cgctcattcc acaagcgtca 1380
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ctgatgccgc acattatatt cttc 24
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
gccaaatcac tcatgagagg tttaa 25
<210> 23
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
ttgctctaca acaacatcat gatcaa 26
<210> 24
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
gctaaacatt ctgcgtggat gtat 24
<210> 25
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
ttgatggtgc acttcataag tatcg 25
<210> 26
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
ggaaatgaca caatcgatgc ag 22
<210> 27
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
cagttcttga ctccaagtag agtatg 26
<210> 28
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
agagtttgat cctggctcag g 21
<210> 29
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
aaggaggtgg tgatccagcc gca 23
Claims (9)
1. a kind of Manas' bacillus marinus E40208a1, which is characterized in that Manas' bacillus marinus
E40208a1 was deposited in Guangdong Province's Culture Collection on 01 03rd, 2019, and deposit number is GDMCC NO:
60497。
2. a kind of Manas' bacillus marinus E40208a1 according to claim 1, which is characterized in that the 16S of the bacterium
RRNA gene, nucleotide sequence is as shown in SEQ ID NO.1.
3. application of the Manas' bacillus marinus E40208a1 described in claim 1 in biocontrol of plant disease.
4. application according to claim 4, which is characterized in that the plant disease is disease caused by PVY, described
Plant is tobacco or potato.
5. the secondary metabolite fermentation liquid of Manas' bacillus marinus E40208a1 described in claim 1 is in plant disease
Application in biological control.
6. application according to claim 5, which is characterized in that the fermentation liquid the preparation method comprises the following steps: after cultivating 3d
Colony edge take a little lawn to be inoculated into the culture bottle of 100ml ATCC213 Media modified, every bottle connects an acicula, 28
℃、200r/min-1Cultivate 7d.
7. application according to claim 6, which is characterized in that the ATCC213 Media modified are as follows: MgSO4·7H2O
10g, CaCl2·2H2O 0.2g, KCl 5g, peptone 2.5g, yeast extract 10g, NaCl 30g, agar powder 12g, distilled water is extremely
1000ml, pH 7.2-7.4.
8. Manas' bacillus marinus E40208a1 described in claim 1 is in as preparation prevention and treatment crop disease drug
Using.
9. the secondary metabolite fermentation liquid of Manas' bacillus marinus E40208a1 described in claim 1 is as preparation
Prevent and treat the application in crop disease drug.
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