CN115747104B - Application of Brevundimonas in inducing Arthropoda shapefaciens to produce predatory organs - Google Patents
Application of Brevundimonas in inducing Arthropoda shapefaciens to produce predatory organs Download PDFInfo
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- 210000000056 organ Anatomy 0.000 title claims abstract description 39
- 241000131407 Brevundimonas Species 0.000 title claims abstract description 31
- 230000001939 inductive effect Effects 0.000 title claims abstract description 14
- 241000238421 Arthropoda Species 0.000 title description 6
- 238000000855 fermentation Methods 0.000 claims abstract description 40
- 230000004151 fermentation Effects 0.000 claims abstract description 40
- 239000000725 suspension Substances 0.000 claims abstract description 31
- 241001661277 Moelleriella libera Species 0.000 claims abstract description 14
- 238000009629 microbiological culture Methods 0.000 claims abstract description 5
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- 238000000034 method Methods 0.000 claims description 19
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- 238000009630 liquid culture Methods 0.000 claims description 9
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- 241000132542 Conyza Species 0.000 claims description 7
- 239000011324 bead Substances 0.000 claims description 6
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- 239000011521 glass Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000008223 sterile water Substances 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 241000008376 Paucispora Species 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims 1
- 241000244206 Nematoda Species 0.000 abstract description 10
- 230000003071 parasitic effect Effects 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 2
- 239000002609 medium Substances 0.000 description 28
- 241000722807 Arthrobotrys oligospora Species 0.000 description 16
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- 241000061154 Brevundimonas sp. Species 0.000 description 8
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- 241000186312 Brevibacterium sp. Species 0.000 description 2
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000012271 agricultural production Methods 0.000 description 2
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 241001300819 Brevibacterium sp. 2 Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention provides an application of Brevundimonas in inducing the generation of predatory organs by using the aschersonia aleyrodis, belonging to the technical field of microorganisms, wherein the Brevundimonas is preserved in China general microbiological culture collection center (CGMCC) No.19122 at 12-10 of 2019. The mixed culture of the fermentation liquor of the shortwave monad and the spore suspension of the sarcomeria less spore can induce the sarcomeria less spore to produce a large number of predatory organs, thereby providing a new way for preventing and controlling plant parasitic nematodes. The invention also provides application of the shortwave monad in inducing the aschersonia aleyrodis to produce predatory organs.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to application of Brevundimonas in inducing Arthropoda shapefaciens to produce predatory organs.
Background
The plant parasitic nematode caused disease is one of the main diseases in agricultural production, and causes serious loss to agricultural production. With the continuous manifestation of chemical control drawbacks, biological control is increasingly gaining importance. Nematophagous fungi are important natural enemies of nematodes and they catch nematodes by producing predatory organs, the formation or absence and number of which play a decisive role in the ability of the nematophagous fungi to kill nematodes. Therefore, it is of great importance to mine the induction factors that promote the formation of prey organs by nematode-trapping fungi. Currently, the literature indicates that some factors, including nematodes, nematode secretions, and small molecules such as amino acids, can induce the production of predatory organs by nematode-predating fungi.
The aschersonia aleyrodis (Arthrobotrys oligospora) is a representative model species of the nematode-trapping fungi, at present, the aschersonia aleyrodis is usually induced to generate predatory organs by nematode extracts, amino acids and the like, but the preparation process of the nematode extracts is complicated, and the induction effect of the amino acids is poor, so that an efficient and convenient method for inducing the aschersonia aleyrodis to generate the predatory organs is needed.
Disclosure of Invention
In order to obtain a novel method for inducing the aschersonia aleyrodis to produce predatory organs, the invention provides an application of the Brevibacterium aleyrodis in inducing the aschersonia aleyrodis to produce predatory organs, and a novel approach is provided for preventing and controlling plant parasitic nematodes by carrying out mixed culture on fermentation liquor of the Brevibacterium and spore suspension of the aschersonia aleyrodis to induce the aschersonia aleyrodis to produce a large number of predatory organs.
The invention is realized by the following technical scheme:
the application provides an application of Brevundimonas sp to inducing the generation of predatory organs by using the aschersonia aleyrodis, wherein the Brevundimonas is preserved in China general microbiological culture Collection center (CGMCC) No.19122 at 12-10-2019.
Further, the application includes:
mixing the fermentation liquor of Brevundimonas with the suspension of the aschersonia with the volume ratio of 1 (1-10), and culturing to obtain the aschersonia with predatory organs.
Further, the fermentation broth of Brevundimonas is prepared by the following method:
streaking a single bacterial colony of Brevundimonas on an NB solid plate, and culturing at 28 ℃ for 24 hours;
picking single colony from the NB solid flat plate into NB liquid culture medium, culturing at 25-35 ℃ and 180rpm for 12-24 h, inoculating into a container filled with NB liquid culture medium according to 1-5% of inoculum size, and culturing in a shaking table at constant temperature of 25-35 ℃ for 3-6 days to obtain fermentation mixed liquor;
and centrifuging the fermentation mixed solution at 10000-12000rpm for 10min, and sucking the supernatant to obtain the fermentation solution of Brevundimonas.
Further, the aschersonia oligosporin spore suspension is prepared by the following method:
inoculating the Arthrospora shapessary strain on a PDA or CMA solid plate, and culturing the activated strain in an incubator at 28 ℃ for 5-8 days in an inverted manner;
transferring the activated strain into a container filled with CMA solid culture medium, culturing at 28 ℃ for 8-12 days, adding a proper amount of sterile water and glass beads into the container, shaking and washing all mycelia, and filtering with 6 layers of mirror-cleaning paper to obtain the Conyza shapewear spore suspension.
Further, the fermentation liquor of the shortwave monad and the aschersonia oligospora spore suspension are uniformly mixed according to the volume ratio of 1 (1-10) and then are cultured, and the method specifically comprises the following steps:
uniformly mixing fermentation liquor of Brevundimonas and the aschersonia paucispora spore suspension according to the volume ratio of 1 (1-10), coating the mixture on a WA plate, and standing the WA plate for 2 days at the temperature of 25-28 ℃.
Alternatively, the application includes:
in the process of culturing mycelium from the aschersonia aleyrodis spore suspension, introducing volatile matters of a fermentation liquid of Brevibacterium into a culture container to obtain aschersonia aleyrodis with predatory organs.
Based on the same inventive concept, the application also provides an application of the fermentation liquor of the shortwave monad in inducing the aschersonia aleyrodis to produce predatory organs, wherein the shortwave monad is preserved in China general microbiological culture collection center (CGMCC) No.19122 at 12-10 of 2019.
Further, the fermentation broth of Brevundimonas is prepared by the following method:
streaking a single bacterial colony of Brevundimonas on an NB solid plate, and culturing at 28 ℃ for 24 hours;
picking single colony from the NB solid flat plate into NB liquid culture medium, culturing at 25-35 ℃ and 180rpm for 12-24 h, inoculating into a container filled with NB liquid culture medium according to 1-5% of inoculum size, and culturing in a shaking table at constant temperature of 25-35 ℃ for 3-6 days to obtain fermentation mixed liquor;
and centrifuging the fermentation mixed solution at 10000-12000rpm for 10min, and sucking the supernatant to obtain the fermentation solution of Brevundimonas.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
the application of the shortwave monad in inducing the curvularia shapessary to produce predatory organs is characterized in that the shortwave monad fermentation liquor and the curvularia shapesii spore suspension are mixed and cultured, so that the curvularia shapesii can be induced to produce a large number of predatory organs, or the shortwave monad fermentation liquor volatile matter is introduced into a culture container for culturing the curvularia shapesii spore suspension, and the curvularia shapesii can be induced to produce a large number of predatory organs.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a predatory organ of Brevibacterium sp.1 induced production of Arthrospira oligosporum;
FIG. 2 is a predatory organ of Brevibacterium sp.2 induced production of Arthrospira oligosporum;
FIG. 3 is a predatory organ of Brevibacterium sp.3 induced production of Arthrospira oligosporum;
FIG. 4 shows the mycelium of Arthropoda shaeformis which did not produce predatory organs in the control group of example 1.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The whole method route of the invention is as follows:
1. preparation of Brevundimonas (Brevundimonas sp.) fermentation broths
Culture medium: NB medium was conventional. The composition and preparation method of the culture medium are as follows:
NB medium: 10g of peptone, 3g of beef extract, 5g of sodium chloride, adding water for dissolution, and fixing the volume to 1L, and adding 18g/L of agar into NB solid medium for sterilization at 121 ℃ for 20 minutes.
Single colony of strain was streaked on NB solid plate and cultured in incubator at 28℃for 24 hours. Selecting single bacterial colony, culturing at 25-35 deg.c and 180rpm for 12-24 hr, inoculating to triangular flask with NB liquid culture medium in 1-5% amount, and culturing at 25-35 deg.c in constant temperature shaking table (150-200 rpm) for 3-6 days. Separating bacterial liquid and bacterial cells by using a high-speed centrifuge at 10000-12000rpm for 10min, sucking supernatant, and placing in a refrigerator at-4 ℃ for standby to obtain the active fermentation liquid used by the invention.
2. Culture of the nematode-trapping fungus Arthrospira oligospora (Arthrobotrys oligospora) and preparation of spore suspension
PDA medium: 200g of potato slices are boiled for 30min and filtered, 20g of glucose and 18g of agar are added, water is added to a volume of 1L, and the mixture is autoclaved for 20min.
CMA medium: corn kernels 30g/L (boiled for more than 30 min); yeast extract 0.6-1%; agar: 18g/L, and autoclaved for 20min.
Preparation of a spore suspension of aschersonia (Arthrobotrys oligospora): inoculating the strain on PDA or CMA solid plate, culturing in a 28 deg.C incubator for 5-8 days to activate the strain, transferring the activated strain into a triangular flask containing CMA solid culture medium, culturing at 28 deg.C for 8-12 days, adding appropriate amount of sterile water and glass beads into the triangular flask, shaking to wash all mycelia, and filtering with 6 layers of mirror paper to obtain spore suspension.
Induction experiment of 3 Conyza shaeformis (Arthrobotrys oligospora) predatory organs
WA medium: aqueous agar medium of 1.5% agar.
The culture of Brevundimonas sp is taken, the supernatant of the fermentation broth and the suspension of the aschersonia sp are evenly mixed according to the ratio of 1:1-1:10 (v/v) and coated on a WA plate with the length of 6-9 cm. A WA plate of 6-9cm WAs plated with NB medium and a suspension of Conyza shaeformis spores at a ratio of 1:1-1:10 (v/v) as negative control. The flat plate is placed in a constant temperature incubator at 25-28 ℃ for culture. Three replicates were made for each group. Two days later, the formation of predatory organs (three-dimensional bacterial networks) was observed with an optical microscope.
Or: a9 cm double-baffle plate WAs poured on one side with WA solid medium, 0.5-1mL of a spore suspension of Arthropoda shaeformis (Arthrobotrys oligospora) WAs spread on the WA medium, 1-2mL of Brevundimonas sp WAs added to the side of the double-baffle plate without WA medium, and the culture WAs performed at constant temperature at 28 ℃. Three replicates were made for each group. After 3 days of culture, the presence or absence of predatory organ production was observed under a microscope.
The application of Brevibacterium in inducing the production of predatory organs by using Arthrospira oligospora will be described in detail below with reference to examples and experimental data.
Example 1
(1) Preparation of Brevundimonas (Brevundimonas sp.) fermentation broths
Brevundimonas sp. On NB solid medium in 28℃incubator for 24h, and transferred to 250mL Erlenmeyer flasks sterilized with 100mL NB liquid medium at 28℃and 180rpm shaking for 24h as seed solution. Inoculating into 250mL triangular flask sterilized with 100mL NB liquid medium at 1%, culturing at 28deg.C with shaking table at 180rpm for 4 days, centrifuging (10000 rpm,10 min) the fermentation broth to remove thallus, sucking supernatant with a pipetting gun, and collecting sterile triangular flask for use.
(2) Preparation of Conyza shapesii (Arthrobotrys oligospora) spore suspension
The method comprises inoculating Arthrospora shapesii (Arthrobotrys oligospora) on PDA solid plate, culturing for 6 days in a 28 deg.C incubator upside down, transferring the activated strain into a triangular flask containing CMA solid culture medium, culturing for 10 days at 28 deg.C, adding appropriate amount of sterile water and glass beads into the triangular flask, shaking to wash all mycelia, and filtering with 6 layers of mirror paper to obtain spore suspension.
(3) Induction experiment
Mixing the bacterial fermentation supernatant obtained in the step (1) with the spore suspension obtained in the step (2) according to the volume ratio of 1:5, uniformly coating on a WA culture medium of 9cm, and culturing at a constant temperature of 28 ℃. NB liquid medium and spore suspension (1:5, v/v) were added to WA solid medium, mixed well, spread and incubated in a constant temperature incubator at 28℃to serve as a control. Three replicates were made for each group. After 2 days of incubation, the test group had developed a three-dimensional network of predatory organs with aschersonia (Arthrobotrys oligospora) and the control group had substantially no three-dimensional network of predatory organs formed.
Example 2
(1) Preparation of Brevundimonas (Brevundimonas sp.) fermentation broths
Brevundimonas sp. On NB solid medium in 28℃incubator for 24h, and transferred to 250mL Erlenmeyer flasks sterilized with 120mL NB liquid medium at 30℃and 160rpm shaking for 20h as seed solution. Inoculating into 250mL triangular flask sterilized with 120mL NB liquid medium according to 3% inoculum size, culturing at 30deg.C for 3 days with 160rpm shaking table, centrifuging (12000 rpm,10 min) the fermentation broth to remove thallus, sucking supernatant with pipetting gun, and collecting in sterile triangular flask for use.
(2) Preparation of Conyza shapesii (Arthrobotrys oligospora) spore suspension
The method comprises inoculating Arthrospora shapesii (Arthrobotrys oligospora) on PDA solid plate, culturing for 8 days in a 28 deg.C incubator upside down, transferring the activated strain into a triangular flask containing CMA solid culture medium, culturing for 8 days at 28 deg.C, adding appropriate amount of sterile water and glass beads into the triangular flask, shaking to wash all mycelia, and filtering with 6 layers of mirror paper to obtain spore suspension.
(3) Induction experiment
Mixing the bacterial fermentation supernatant obtained in the step (1) with the spore suspension obtained in the step (2) according to the volume ratio of 1:8, uniformly coating the mixture on a 6cm WA plate, and culturing at the constant temperature of 28 ℃. NB broth and spore suspension (1:8, v/v) were added to WA medium, mixed well and spread in a 28℃incubator for incubation as a control. Three replicates were made for each group. After 2 days of incubation, the test group had developed a three-dimensional network of predatory organs with aschersonia (Arthrobotrys oligospora) and the control group had substantially no three-dimensional network of predatory organs formed.
Example 3
(1) Preparation of Brevundimonas (Brevundimonas sp.) fermentation broths
Brevundimonas sp. On NB solid medium in 28℃incubator for 24h, and transferred to 250mL Erlenmeyer flasks sterilized with 100mL NB liquid medium at 35℃and 180rpm shaking for 24h as seed solution. Inoculating into 250mL triangular flask sterilized with 100mL NB liquid medium at 5%, culturing at 35deg.C with shaking table at 180rpm for 5 days, centrifuging (10000 rpm,10 min) the fermentation broth to remove thallus, sucking supernatant with pipetting gun, and collecting in sterile triangular flask.
(2) Preparation of Conyza shapesii (Arthrobotrys oligospora) spore suspension
Inoculating Arthrospora shapesii (Arthrobotrys oligospora) on a PDA solid flat plate, culturing for 8 days in a 28 ℃ incubator in an inversion mode, transferring the activated strain slices into a triangular flask filled with CMA solid culture medium, culturing for 12 days at the 28 ℃, adding a proper amount of sterile water and glass beads into the triangular flask, shaking to wash all mycelia, filtering with 6 layers of mirror-cleaning paper, and preparing spore suspension.
(3) Induction experiment
A WA solid medium WAs poured onto one side of a 9cm double-septum plate, 1mL of a conidium of Arthropoda shaeformis (Arthrobotrys oligospora) WAs spread on the WA medium, and 1mL of Brevundimonas sp. Mycorrhizal fluid WAs added to the side of the double-septum plate where the WA medium WAs not present, and the culture WAs carried out at a constant temperature of 28 ℃. Three replicates were made for each group. After 3 days of culture, the test group had aschersonia (Arthrobotrys oligospora) produced a three-dimensional network of predatory organs, whereas the control group (side of double-baffle plate without WA medium added with NB liquid medium) had substantially no three-dimensional network of predatory organs. Demonstrating that volatile materials produced by Brevundimonas sp can induce Arthrospora shapessary Arthrobotrys oligospora to produce a three-dimensional network of predatory organs.
In fig. 1-4, the right plot is a larger magnification plot than the left plot.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (7)
1. Brevundimonas spBrevundimonassp. inducing the aschersonia oligosporisArthrobotrys oligospora) The application of the Brevundimonas in producing predatory organs is characterized in that the Brevundimonas is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 19122 in 12-10 2019.
2. The use according to claim 1, characterized in that it comprises:
mixing the fermentation liquor of Brevundimonas with the suspension of the aschersonia with the volume ratio of 1 (1-10), and culturing to obtain the aschersonia with predatory organs.
3. Use according to claim 2, characterized in that the fermentation broth of shortwave uniconas is prepared by the following method:
streaking a single bacterial colony of Brevundimonas on an NB solid plate, and culturing at 28 ℃ for 24 hours;
picking single colony from the NB solid flat plate into NB liquid culture medium, culturing at 25-35 ℃ and 180rpm for 12-24 h, inoculating into a container filled with NB liquid culture medium according to 1-5% of inoculum size, and culturing in a shaking table at constant temperature of 25-35 ℃ for 3-6 days to obtain fermentation mixed liquor;
and centrifuging the fermentation mixed solution at 10000-12000rpm for 10min, and sucking the supernatant to obtain the fermentation solution of Brevundimonas.
4. The use according to claim 2, characterized in that the suspension of aschersonia paucispora is prepared by the following method:
inoculating the Arthrospora shapessary strain on a PDA or CMA solid plate, and culturing the activated strain in an incubator at 28 ℃ for 5-8 days in an inverted manner;
transferring the activated strain into a container filled with CMA solid culture medium, culturing at 28 ℃ for 8-12 days, adding a proper amount of sterile water and glass beads into the container, shaking and washing all mycelia, and filtering with 6 layers of mirror-cleaning paper to obtain the Conyza shapewear spore suspension.
5. The use according to claim 2, wherein the fermentation broth of shortwave monad and the suspension of the aschersonia parvula spores are mixed according to the volume ratio of 1 (1-10) and then cultured, and the method specifically comprises the following steps:
uniformly mixing fermentation liquor of Brevundimonas and the aschersonia paucispora spore suspension according to the volume ratio of 1 (1-10), coating the mixture on a WA plate, and standing the WA plate for 2 days at the temperature of 25-28 ℃.
6. The application of the fermentation liquor of the shortwave monad in inducing the aschersonia aleyrodis to produce predatory organs is characterized in that the shortwave monad is preserved in China general microbiological culture Collection center (CGMCC) No.19122 in 12-10-2019.
7. The use according to claim 6, wherein the fermentation broth of shortwave monad is prepared by the following method:
streaking a single bacterial colony of Brevundimonas on an NB solid plate, and culturing at 28 ℃ for 24 hours;
picking single colony from the NB solid flat plate into NB liquid culture medium, culturing at 25-35 ℃ and 180rpm for 12-24 h, inoculating into a container filled with NB liquid culture medium according to 1-5% of inoculum size, and culturing in a shaking table at constant temperature of 25-35 ℃ for 3-6 days to obtain fermentation mixed liquor;
and centrifuging the fermentation mixed solution at 10000-12000rpm for 10min, and sucking the supernatant to obtain the fermentation solution of Brevundimonas.
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Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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