CN114561297B - Aspergillus flavus and application thereof - Google Patents
Aspergillus flavus and application thereof Download PDFInfo
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- CN114561297B CN114561297B CN202111501827.7A CN202111501827A CN114561297B CN 114561297 B CN114561297 B CN 114561297B CN 202111501827 A CN202111501827 A CN 202111501827A CN 114561297 B CN114561297 B CN 114561297B
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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
- 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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/34—Aspergillus
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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
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to aspergillus flavus and application thereof, and belongs to the technical field of microorganisms. The aspergillus flavus (Aspergillus flavus) XL-0048 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 23268 in the year 2021, month 9 and day 27. The strain has remarkable inhibition effect on 3 plant pathogenic fungi, namely sugarcane black spot pathogen, corn big spot pathogen and orange brown spot pathogen, and has wide application prospect and easy popularization and application.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to aspergillus flavus and application thereof.
Background
Aspergillus flavus (Aspergillus flavus) is one of important plant endophytic fungi, belonging to the genus Alternaria of the family Alternariaceae, the order Alternaria, the class Alternaria, and the class Alternaria.
Sugarcane black spot is a worldwide disease caused by black powder fungus (Ustilago scitaminea Syd.) and is also the most dominant fungal disease that jeopardizes sugarcane production in China. Black spot disease commonly occurs in various sugarcane planting provinces in China, is more serious especially on dry-land sugarcane and perennial root sugarcane, has the incidence rate of up to 80% -90% in certain sugarcane fields, and seriously affects the sustainable development of sugarcane industry.
The corn leaf spot disease is a corn leaf disease caused by corn leaf spot bacteria (Setosphaeria turcica), water spots appear on plant leaves after the plant leaves are infected by the bacteria, and then typical clostridia spots are formed by expanding along the leaf veins to two ends, so that leaf photosynthesis is seriously affected. When the plant infection happens under the conditions of high humidity and medium temperature, the crop yield can be reduced by more than 50% even when the infected variety is seriously infected, and the plant infection is even dead.
Citrus brown spot is a defoliation disease caused by fungi Alternaria alternate, and bacteria mainly damage tender leaves, new shoots and young fruits (damage to high-sensitivity varieties and the whole growing period of the fruits), cause defoliation, fallen fruits and dead shoots, and cause the disease spots on the surface of the non-fallen fruits to be unable to be marketed and sold. The fruits of the infected plants may become infected from just fruit setting until just before fruit picking and cause severe fruit drop.
Phytopathogenic fungi refer to those fungi that can be parasitic to plants and cause diseases. There are over 8000 plant pathogenic fungi already described. The fungus can cause more than 3 kinds of plant diseases, accounting for 80% of the total plant diseases, and belongs to the first major pathogen. In recent years, a great deal of research shows that biological control has not only an inhibitory effect on pathogenic bacteria but also is environment-friendly and pollution-free, so that research on biological control of plant diseases is receiving more and more attention from researchers.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide the aspergillus flavus which has the characteristics of easy culture, high yield, stable culture property and the like, and the culture of the aspergillus flavus has higher antibacterial activity on plant pathogenic fungi such as sugarcane black spot pathogen, corn big spot pathogen and orange brown spot pathogen.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the present invention provides Aspergillus flavus (Aspergillus flavus) XL-0048, which has been deposited in China general microbiological culture Collection center, with the accession number CGMCC No.23268, on the 9 th month 27 of 2021.
The second aspect of the invention provides the use of aspergillus flavus (Aspergillus flavus) XL-0048 as described above in the preparation of a bacteriostatic agent.
Further, preferably, the fungi inhibited by the bacteriostatic agent comprise sugarcane black spot pathogen, corn big spot pathogen and orange brown spot pathogen.
Further, it is preferable that the active ingredient in the bacteriostatic agent comprises an extract of the culture of Aspergillus flavus (Aspergillus flavus) XL-0048 or Aspergillus flavus (Aspergillus flavus) XL-0048.
Further, it is preferred that the culture of Aspergillus flavus (Aspergillus flavus) XL-0048 is a liquid culture; the culture method comprises the following steps: inoculating Aspergillus flavus (Aspergillus flavus) XL-0048 on PDA slant culture medium, and culturing in 28 deg.C incubator for 3-5 days;
directly inoculating the grown inclined plane into PDB liquid culture medium, culturing in shaking table at 28deg.C and 180rpm for 15-20 days, and taking out to obtain liquid culture;
the PDA slant culture medium comprises: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2;
the PDB liquid medium contains: potato extract powder 5g/L, glucose 15g/L, peptone 10g/L and sodium chloride 5g/L.
Further, it is preferable that the extract is obtained by extracting a culture of Aspergillus flavus (Aspergillus flavus) XL-0048 as a raw material with an organic solvent.
Further, it is preferable that the organic solvent is ethyl acetate.
A third aspect of the invention provides a culture of Aspergillus flavus (Aspergillus flavus) XL-0048 as described above.
A fourth aspect of the invention provides an extract of a culture of Aspergillus flavus (Aspergillus flavus) XL-0048 as described above.
The Aspergillus flavus (Aspergillus flavus) XL-0048 culture can also be obtained by taking Aspergillus flavus (Aspergillus flavus) XL-0048 as a raw material and culturing in a PDA culture medium, a PDB culture medium and the like which are conventional in the art.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides aspergillus flavus and application thereof, which are preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.23268 in 2021, 9 and 27. The culture and the extract of the culture have remarkable inhibition effect on 3 fungi of sugarcane black spot pathogen (Ustilago scitaminea Syd, NB-01), corn big spot pathogen (Setosphaeria turcica, NB-06) and orange brown spot pathogen (Alternaria alternata, YB-05), and the Minimum Inhibitory Concentration (MIC) of the crude extract of the liquid culture on the corn big spot pathogen can reach 12.5ug/ml, so that the preparation method has good application prospect.
Drawings
FIG. 1 is a single colony morphology (PDA plate) of Aspergillus flavus XL-0048; wherein, FIG. 1-A is a front view of a flat panel; FIG. 1-B is a back view of a flat panel;
aspergillus flavus (Aspergillus flavus) XL-0048 was deposited in China general microbiological culture Collection center (CGMCC) at 9/27 of 2021, with the deposit number of CGMCC No.23268, and the deposit address of North Chen Xiyu No. 1/3, china academy of sciences microbiological study.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.
Example 1 Source and identification of Aspergillus flavus (Aspergillus flavus) XL-0048
(1) Source of Aspergillus flavus (Aspergillus flavus) XL-0048
A fungus was isolated from Salvia Miltiorrhiza, picked from a plant garden at Shenyang university of medical science, liaoning, 9.4.2017, and named XL-0048.
(2) Identification of XL-0048
The ITS sequencing Sequence of XL-0048 is shown in the Sequence table (SEQ ID NO. 1), and has the highest Sequence similarity with Sequence ID MW723884.1, and the similarity is 98.59%.
XL-0048 belongs to Aspergillus flavus (Aspergillus flavus) according to the above identification.
EXAMPLE 2 main morphological characteristics of Aspergillus flavus (Aspergillus flavus) XL-0048
Aspergillus flavus XL-0048 is inoculated on PDA culture medium, cultured at 28 deg.c for 3 days to reach colony diameter of 6cm, and the colony has front color changed from white to yellow and yellow green to semi-villus. The spore becomes brown after maturation, the surface of the spore is flat or has radial grooves, the back surface of the spore is colorless or brown, and the conidium head is loose and radial, and then is loose and columnar. And the conidiophores are rough when observed by a film-making microscopic examination. The top bag is flask-shaped or nearly spherical. Conidiophores are chain-shaped on the small peduncles, and small protrusions, spheres and roughness are arranged around the conidiophores.
The PDA culture medium formula comprises the following components: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2.
The single colony morphology diagram of the strain on a PDA flat plate is shown in figure 1, and figure 1-A is a front view of the flat plate in figure 1-A; FIG. 1-B is a back view of the plate.
EXAMPLE 3 Activity screening of Aspergillus flavus (Aspergillus flavus) XL-0048
The antibacterial activity of fungi to be screened (XL-0048) on the fungi to be tested (3 pathogenic fungi) is measured by a filter paper sheet method, and the specific method is as follows:
(1) culturing bacterial liquid: respectively streaking and inoculating preserved fungi to be screened and fungi to be tested (3 pathogenic fungi) on a PDA slant culture medium, placing the PDA slant culture medium in a 28 ℃ incubator for culturing for 72 hours, directly inoculating the grown slant into a standby PDB liquid culture medium, placing the PDB liquid culture medium in a shaking table at 28 ℃ and 180rpm for culturing for 48 hours, and taking out the liquid culture medium for standby, thus obtaining the fungus testing liquid.
(2) Under the aseptic condition, using the fungus to be tested as an indicator fungus, respectively taking a proper amount of fungus liquid and a PDA flat plate, uniformly coating, clamping three aseptic filter paper sheets by using aseptic tweezers, and respectively dripping a proper amount of fungus liquid of a strain to be screened on each paper sheet. Culturing in a constant temperature incubator at 28deg.C for 3-5 days, and observing antibacterial effect.
The PDA slant culture medium comprises the following formula: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2.
The experimental result shows that XL-0048 has remarkable inhibition effect on 3 plant pathogenic fungi, namely sugarcane black spot pathogen, corn big spot pathogen and orange brown spot pathogen.
EXAMPLE 4 liquid culture of Aspergillus flavus (Aspergillus flavus) XL-0048
(1) Activation of the strain: inoculating the preserved Aspergillus flavus XL-0048 on PDA slant culture medium, and culturing in a 28 deg.C incubator for 3 days;
liquid culture of strains: preparing 200 ml/bottle PDB liquid culture medium, directly inoculating the grown inclined plane into liquid shake flask, culturing in shake flask at 28deg.C and 180rpm for 20 days, and taking out to obtain liquid culture (i.e. fermentation broth).
(2) Activation of the strain: inoculating the preserved Aspergillus flavus XL-0048 on PDA slant culture medium, and culturing in a 28 deg.C incubator for 5 days;
liquid culture of strains: preparing 200 ml/bottle PDB liquid culture medium, directly inoculating the grown inclined plane into liquid shake flask, culturing in shake flask at 28deg.C and 180rpm for 15 days, and taking out to obtain liquid culture (i.e. fermentation broth).
(3) Activation of the strain: inoculating the preserved Aspergillus flavus XL-0048 on PDA slant culture medium, and culturing in a 28 deg.C incubator for 4 days;
liquid culture of strains: preparing 200 ml/bottle PDB liquid culture medium, directly inoculating the grown inclined plane into liquid shake flask, culturing in shake flask at 28deg.C and 180rpm for 17 days, and taking out to obtain liquid culture (i.e. fermentation broth).
The PDA slant culture medium comprises the following formula: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2.
The formula of the PDB liquid culture medium comprises the following components: potato soaked powder 5g/L, glucose 15g/L, peptone 10g/L, sodium chloride 5g/L, and pH value is natural.
EXAMPLE 5 preparation of a crude extract of Aspergillus flavus (Aspergillus flavus) XL-0048 ethyl acetate
The fermentation broth of example 4 was extracted with an equal volume of ethyl acetate by: oscillating for 30min, fully mixing the fermentation liquor with ethyl acetate, standing for 24h to separate liquid, taking out ethyl acetate phase, namely an upper phase, performing reduced pressure rotary evaporation at 42 ℃ (the vacuum degree is generally-0.08 Mpa to-0.01 Mpa), completely evaporating the solvent by using a rotary evaporator to obtain bacterial liquid ethyl acetate crude extract, and preserving in a refrigerator at 4 ℃ for later use.
EXAMPLE 6 determination of MIC value of Aspergillus flavus (Aspergillus flavus) XL-0048 Ethyl acetate crude extract
The bacterial liquid ethyl acetate crude extract of example 5 was further measured for Minimum Inhibitory Concentration (MIC) by a 96-well plate method, and the specific method is as follows:
(1) culturing a test bacterial liquid: and (3) streaking and inoculating the preserved test fungi (3 pathogenic fungi) on a PDA slant culture medium, placing the PDA slant culture medium in a 28 ℃ incubator for culturing for 72 hours, directly inoculating the grown slant into a standby PDB liquid culture medium, placing the PDB liquid culture medium in a shaking table at 28 ℃ and 180rpm for culturing for 48 hours, and taking out the liquid culture medium for standby, thus obtaining the test fungus liquid.
(2) Sample solution preparation: 1mg of the crude ethyl acetate extract of the bacterial liquid of example 5 was taken, and 100uL of DMSO was added to dissolve it sufficiently, and the volume was determined by DMSO to obtain a sample solution of 100 ug/mL.
(3) MIC determination by 96-well plate method: taking a sterile 96-well plate, adding 100 mu L of PDB liquid culture medium into 1-7 wells, adding 100 mu L of sample solution into 1-well, mixing uniformly (the concentration is 50 ug/mL), taking 100 mu L out of 1-well, adding into 2-well, mixing uniformly, diluting to 7-well in sequence, taking 100 mu L out of 7-well, and discarding. Sample concentrations in wells 1-7 were 50, 25, 12.5, 6.25, 3.125, 1.563 and 0.7815ug/mL, respectively, with 100. Mu.L of the test bacteria solution. On the same 96-well plate, a well with 100. Mu.L of DMSO was used as a blank control, a well with no sample solution but 100. Mu.L of the test bacterial liquid was used as a positive control, and a well with no test bacterial liquid but 100. Mu.L of PDB liquid medium was used as a negative control. The minimum concentration of clarified culture medium in the observation wells was MIC.
The PDA culture medium formula comprises the following components: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2.
The formula of the PDB liquid culture medium comprises the following components: potato soaked powder 5g/L, glucose 15g/L, peptone 10g/L, sodium chloride 5g/L, and pH value is natural.
The Minimum Inhibitory Concentration (MIC) of Aspergillus flavus (Aspergillus flavus) XL-0048 against various pathogenic fungi is shown in Table 1.
TABLE 1 Minimum Inhibitory Concentration (MIC) of Aspergillus flavus XL-0048 against various pathogenic fungi
| Selecting test fungi | MIC/（ug/mL） |
| NB-01 | 25 |
| NB-06 | 12.5 |
| YB-05 | 25 |
As can be seen from Table 1, the crude extract of ethyl acetate of XL-0048 bacterial liquid has remarkable inhibition effect on 3 plant pathogenic fungi, namely, sugarcane black spot pathogen, corn big spot pathogen and orange brown spot pathogen, and the 3 fungi.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
SEQ ID NO.1
gcttcgagtg acggttctag cgagcccacc tcccacccgt gtttactgta ccttagttgc 60
ttcggcgggc ccgccattca tggccgccgg gggctctcag ccccgggccc gcgcccgccg 120
gagacaccac gaactctgtc tgatctagtg aagtctgagt tgattgtatc gcaatcagtt 180
aaaactttca acaatggatc tcttggttcc ggcatcgatg aagaacgcag cgaaatgcga 240
taactagtgt gaattgcaga attccgtgaa tcatcgagtc tttgaacgca cattgcgccc 300
cctggtattc cggggggcat gcctgtccga gcgtcattgc tgcccatcaa gcacggcttg 360
tgtgttgggt cgtcgtcccc tctccggggg ggacgggccc caaaggcagc ggcggcaccg 420
cgtccgatcc tcgagcgtat ggggctttgt cacccgctct gtaggcccgg ccggcgcttg 480
ccgaacgcaa atcaatcttt tccaggttga cctcggatca ggtagggata cccgttgaac 540
ttaagcaatc aaaaacgggg gaaaaaaaag aaaa 574。
Sequence listing
<110> Yang Ling future Environment protection technology Co., ltd
<120> A. Flavus and use thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 574
<212> DNA
<213> 1 (Artificial sequence)
<400> 1
gcttcgagtg acggttctag cgagcccacc tcccacccgt gtttactgta ccttagttgc 60
ttcggcgggc ccgccattca tggccgccgg gggctctcag ccccgggccc gcgcccgccg 120
gagacaccac gaactctgtc tgatctagtg aagtctgagt tgattgtatc gcaatcagtt 180
aaaactttca acaatggatc tcttggttcc ggcatcgatg aagaacgcag cgaaatgcga 240
taactagtgt gaattgcaga attccgtgaa tcatcgagtc tttgaacgca cattgcgccc 300
cctggtattc cggggggcat gcctgtccga gcgtcattgc tgcccatcaa gcacggcttg 360
tgtgttgggt cgtcgtcccc tctccggggg ggacgggccc caaaggcagc ggcggcaccg 420
cgtccgatcc tcgagcgtat ggggctttgt cacccgctct gtaggcccgg ccggcgcttg 480
ccgaacgcaa atcaatcttt tccaggttga cctcggatca ggtagggata cccgttgaac 540
ttaagcaatc aaaaacgggg gaaaaaaaag aaaa 574
Claims (3)
1. Aspergillus flavus (Aspergillus flavus) XL-0048 was deposited in China general microbiological culture Collection center, with the accession number CGMCC No.23268, on the 9 th month of 2021 and on the 27 th day.
2. The use of aspergillus flavus (Aspergillus flavus) XL-0048 in the preparation of a bacteriostatic agent according to claim 1, wherein the bacteriostatic agent inhibits fungi comprising cercospora saccharalis (Ustilago scitaminea syd.), alternaria corn (Setosphaeria turcica), alternaria alternata (Alternaria alternata);
the active ingredients in the bacteriostatic agent comprise extracts of a culture of aspergillus flavus (Aspergillus flavus) XL-0048;
the extract is obtained by taking an aspergillus flavus (Aspergillus flavus) XL-0048 culture as a raw material and extracting with an organic solvent; the organic solvent is ethyl acetate.
3. The use of aspergillus flavus (Aspergillus flavus) XL-0048 in the preparation of a bacteriostatic agent according to claim 2, wherein the culture of aspergillus flavus (Aspergillus flavus) XL-0048 is a liquid culture; the culture method comprises the following steps: inoculating Aspergillus flavus (Aspergillus flavus) XL-0048 on PDA slant culture medium, and culturing in 28 deg.C incubator for 3-5 days;
directly inoculating the grown inclined plane into PDB liquid culture medium, culturing in shaking table at 28deg.C and 180rpm for 15-20 days, and taking out to obtain liquid culture;
the PDA slant culture medium comprises: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2;
the PDB liquid medium contains: potato extract powder 5g/L, glucose 15g/L, peptone 10g/L and sodium chloride 5g/L.
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