CN109825457A - A kind of salt tolerant bacillus E40207a2 and its application - Google Patents
A kind of salt tolerant bacillus E40207a2 and its application Download PDFInfo
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Abstract
The invention discloses a kind of salt tolerant bacillus E40207a2 and its applications, the salt tolerant bacillus E40207a2 was deposited in Guangdong Province's Culture Collection on 01 03rd, 2019, deposit number is GDMCC NO:60498, 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 salt tolerant bacillus E40207a2 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 salt tolerant bacillus E40207a2 and its application, the bacterial strain is from examining
It isolates and purifies to obtain in your sweat salt lake water, and is applied to the research of marmor upsilon biological control of diseases, so that biological
Prevention and treatment marmor upsilon disease is possibly realized, while providing foundation for the utilization of the extreme environments resource such as salt lake, has established base
Plinth.
In order to achieve the above technical purposes, the present invention is realized especially by following technical scheme:
The present invention provides bacterial strain E40207a2, which uses dilution plate enrichment culture from Cha Er Han Salt Lake lake water
Method carries out separation and purifying obtains, which is Gram-positive, strictly aerobic, rodlike, wide 0.5-0.6mm, long 1.8-
2.0mm, can be mobile by subpolar flagellum.
The molecules characteristic of bacterial strain E40207a2 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 IDNO.1.By GenBank nucleic acid sequence library and obtained sequence
Sequence analysis is carried out, discovery bacterial strain E40207a2 and Bacillus category relationship is nearest, wherein with Bacillus
The homology highest of halotolerans, reaches 99%.According to 16S rDNA sequence homology and morphological analysis by the dientification of bacteria
For salt tolerant bacillus (Bacillus halotolerans), therefore obtained bacterial strain E40207a2 of the invention is named as
Salt tolerant bacillus (Bacillus halotolerans) E40207a2.
The salt tolerant bacillus E40207a2 preservation is subjected to, depositary institution: Guangdong Province's Microbiological Culture Collection
Center, preservation address: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, preservation date: on 01 03rd, 2019, preservation was compiled
Number be GDMCC NO:60498.
Salt tolerant bacillus (Bacillus halotolerans) E40207a2 of the present invention is in plant disease biology
Application in prevention and treatment, the plant disease are disease caused by PVY, and the disease is specially marmor upsilon disease, described
Plant be solanaceous crops, preferably tobacco or potato.
Preferably, the cometabolism of the salt tolerant bacillus (Bacillus halotolerans) E40207a2 produces
Application of the object fermentation liquid in biocontrol of plant disease is also within the scope of the present invention.
Described salt tolerant bacillus (Bacillus halotolerans) the E40207a2 fermentation liquid the preparation method comprises the following steps:
Colony edge after cultivating 3d takes a little lawn to be inoculated into the culture bottle of 100ml ATCC213 Media modified, and every bottle
Connect an acicula, 28 DEG C, 200r/min-1Cultivate 7d.
The ATCC213 Media modified are as follows: MgSO4·7H2O 10g, CaCl2·2H2O 0.2g, KCl 5g, albumen
Peptone 2.5g, yeast extract 10g, NaCl 30g, agar powder 12g, distilled water to 1000ml, pH 7.2-7.4.
Salt tolerant bacillus (Bacillus halotolerans) E40207a2 of the present invention is prevented and treated as preparation
Application in 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.
The cometabolism of certain salt tolerant bacillus (Bacillus halotolerans) E40207a2 of the present invention
Product fermentations liquid is also within the scope of the present invention as the application in preparation prevention and treatment crop disease drug.
The method of salt tolerant bacillus (Bacillus halotolerans) E40207a2 prevention and treatment crop disease of the present invention
It belongs to the scope of protection of the present invention, this method is that spraying treatment is carried out in growing process；Described be the salt tolerant bud by spraying
The pesticide system of bacteria suspension or fermentation liquid or the metabolite preparation of spore bacillus (Bacillus halotolerans) E40207a2
Agent.
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 salt tolerant bacillus (Bacillus halotolerans) from Cha Er Han Salt Lake lake water
E40207a2, itself can significant antagonism marmor upsilon, and secondary metabolite fermentation liquid has preferably marmor upsilon
Preventive effect, while to tobacco safety.The present invention can effectively antagonism and control marmor upsilon disease, and raw material sources are extensive,
It is at low cost, it is environmentally safe.
Detailed description of the invention
Fig. 1 is the phylogenetic tree of salt tolerant bacillus E40207a2 of the present invention；
Fig. 2 is the extraction result of host's three lives cigarette total serum IgE after salt tolerant bacillus E40207a2 of embodiment of the present invention processing；
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 E40207a2 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 E40207a2 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 E40207a2 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 E40207a2 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 E40207a2 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 E40207a2 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 E40207a2 of embodiment
1) separation of bacterial strain E40207a2
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: MgSO4·7H2O 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.It is placed in picking single colonie culture in 37 DEG C of illumination boxs after 3d.
2) identification of bacterial strain E40207a2
The DNA for extracting bacterial strain E40207a2 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.
PCR reaction condition: 94 DEG C of denaturation 45s, 50 DEG C of annealing 45s, 72 DEG C of extension 75s, 50 μ L reaction systems 30 circulation.
PCR product is detected through agarose gel electrophoresis, 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 arranging library and obtained sequence carries out sequence analysis, discovery bacterial strain E40207a2 and Bacillus category relationship is nearest, wherein with
The homology highest of Bacillus halotolerans, reaches 99%.Obtained 16S rDNA complete sequence with from GenBank
The 16S rDNA sequence obtained in equal databases is compared, and carries out the building (Fig. 1) of phylogenetic tree.According to 16S rDNA sequence
The dientification of bacteria is salt tolerant bacillus (Bacillus halotolerans) by column homology and systematic growth, therefore this is sent out
Bright obtained bacterial strain E40207a2 is named as salt tolerant bacillus (Bacillus halotolerans) E40207a2.
Obtained salt tolerant bacillus E40207a2 preservation is subjected to, depositary institution: Guangdong Province's Microbiological Culture Collection
Center, preservation address: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, preservation date: on 01 03rd, 2019, preservation was compiled
Number be GDMCC NO:60498.
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 r RNA and 18S r
Two master tapes of RNA are apparent, and the former is bright compared with the latter.Meanwhile the concentration and quality of RNA, OD are detected with instrument260/OD280Than
Value 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 the principle that standard fluorescence quantification PCR primer designs, to PVY existing in GenBank and tobacco Actin gene
Complete sequence carries out sequence alignment, and the region for selecting each sequence the most conservative carries out design of primers, then soft using Oligo6.0
Part and on-line analysis software OligoCalc (http://www.basic.northwestern.edu/biotools/
Oligocalc.html) designed primer is evaluated, and by online BLSAT program (http: //
Www.ncbi.nlm.nih.gov/blast/ it) is verified, it is any to avoid virus specific primers sequence and tobacco gene group
Sequence homology.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:
Calculate the expression quantity of target gene PVY in each processing.Bacterial strain is to PVY inhibiting rate=(in control in expression quantity-processing of PVY
The expression quantity of PVY)/control in PVY expression quantity × 100%.As a result as shown in Table 2 and Fig. 3.
2 bacterial strain E40207a2 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 9 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
Albumen -1 (Cullin), cassette family albumen (F-Box) and eukaryotic translation initiation factor 5A (eIF5A) and biology and non-life
Object stress response is related；Glucanotransferase hydrolase (XTH) is related to plant cell wall synthesis；Photosynthetical system II (PSII) and
Chlorophyll a/b binding protein (LHC) is related to photosynthesis；It is related to respiration to put oxygen enhancing protein-3 (OEE3).
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 (CpIp), Cullin albumen -1 (Cullin), photosynthetical system II
(PSII) and put oxygen enhancing protein-3 (OEE3) expression up-regulation (such as Fig. 4-8).Through salt tolerant bacillus (Bacillus
Halotolerans) after E40207a2 fermentation liquor treatment, the expression quantity of adversity gene is compared with positive control upper mileometer adjustment respectively in the three lives
Up to 1.08,1.04,1.04,1.35 and 1.05 (tables 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 2, 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 salt tolerant bacillus (Bacillus halotolerans) E40207a2 Passivation Treatment
Afterwards, amplification breeding of the PVY in host's three lives cigarette is inhibited, PVY protein related gene CP, P1, P3, N1a, N1b, HC-pro
Expression compared with positive control compared to being 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 salt tolerant bacillus E40207a2 and its application
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<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 salt tolerant bacillus E40207a2, which is characterized in that the salt tolerant bacillus E40207a2 is in 2019
It is deposited within 03 day 01 month Guangdong Province's Culture Collection year, deposit number is GDMCC NO:60498.
2. a kind of salt tolerant bacillus E40207a2 according to claim 1, which is characterized in that the 16S rRNA base of the bacterium
Cause, nucleotide sequence is as shown in SEQ ID NO.1.
3. application of the salt tolerant bacillus E40207a2 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 salt tolerant bacillus E40207a2 described in claim 1 is anti-in plant disease biology
Application in controlling.
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. salt tolerant bacillus E40207a2 described in claim 1 is as the application in preparation prevention and treatment crop disease drug.
9. the secondary metabolite fermentation liquid of salt tolerant bacillus E40207a2 described in claim 1 is made as preparation prevention and treatment
Application in object disease drug.
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| CN110343641A (en) * | 2019-07-26 | 2019-10-18 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | A kind of salt bacillus SYW-1 and the application in cotton verticillium wilt prevention and treatment |
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| CN110669693A (en) * | 2019-10-24 | 2020-01-10 | 海南大学 | Bacillus halophilus for improving salt tolerance of nile tilapia and application thereof |
| WO2021117034A1 (en) * | 2019-12-08 | 2021-06-17 | The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) | Bacteria-associated volatile organic compounds |
| CN111979149A (en) * | 2020-08-17 | 2020-11-24 | 甘肃省农业科学院植物保护研究所 | Salt-tolerant bacillus SY1836 and application thereof |
| CN111849844A (en) * | 2020-09-02 | 2020-10-30 | 青海省农林科学院 | Bacillus licheniformis A10201 and application thereof |
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