CN114561297A - Aspergillus flavus and application thereof - Google Patents
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Abstract
The invention relates to aspergillus flavus and application thereof, belonging to the technical field of microorganisms. The Aspergillus flavus XL-0048 has been deposited in China general microbiological culture Collection center (CGMCC) at 27 months 9 and 2021 with a deposition number of CGMCC No. 23268. The strain has obvious inhibition effect on 3 plant pathogenic fungi, namely sugarcane alternaria alternata, corn northern leaf blight and orange brown spot germ, has wide application prospect, and is easy to popularize and apply.
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 endophytic fungi of plants, and belongs to the family of Neurospora of the order Neurospora of the class Deuteromycetes.
Sugarcane black spot is a worldwide disease caused by Ustilago scitaminea Syd, and is also the most main fungal disease which is harmful to sugarcane production in China. The black spot disease generally occurs in sugarcane planting provinces in China, is more serious especially on dry land sugarcane and perennial root sugarcane, the incidence rate of some sugarcane fields is as high as 80% -90%, and the sustainable development of the sugarcane industry is seriously influenced.
The corn leaf spot is a corn leaf disease caused by corn leaf spot (Setosphaeria turcica), and after the corn leaf spot is infected by a disease bacterium, water stain-like spots appear on plant leaves firstly, and then the plant leaves expand to two ends along a leaf vein to form typical spindle-shaped disease spots, so that the photosynthesis of the leaves is seriously influenced. The method occurs under the conditions of high humidity and medium temperature, and can cause the crop to reduce the yield by more than 50 percent when infected seriously by infected varieties, even the crop is not harvested.
The citrus brown spot is a defoliating disease caused by the fungus Alternaria alternate, and germs mainly damage tender leaves, new shoots and young fruits (the fruits can be damaged in the whole growth period of high-susceptibility varieties), cause defoliation, fruit drop and dead shoots, and cause the non-shedding fruits to cause the lesion spots to be not sold on the market. The fruit of the affected plant may be infected and cause severe fruit drop from the moment the fruit is set up until the moment the fruit is harvested.
Phytopathogenic fungi refer to those fungi which can parasitize plants and cause diseases. More than 8000 species of plant pathogenic fungi have been described. The fungus can cause more than 3 million plant diseases, accounts for 80 percent of the total plant diseases, and belongs to a first pathogen. In recent years, a great deal of research shows that biological control not only has an inhibiting 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 strain which has the characteristics of easy culture, high yield, stable culture character and the like, and the culture of the aspergillus flavus strain has higher bacteriostatic activity on the plant pathogenic fungi of sugarcane alternaria alternata, corn leaf blight and orange brown spot germ.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides Aspergillus flavus (Aspergillus flavus) XL-0048 which is preserved in China general microbiological culture Collection center (CGMCC) at 27 months 9 and 2021 with the preservation number of CGMCC No. 23268.
In a second aspect, the invention provides the use of Aspergillus flavus XL-0048 in the preparation of a bacteriostatic agent.
Further, preferably, the fungi inhibited by the bacteriostatic agent comprise alternaria alternata, alternaria zeae and alternaria citri.
Further, it is preferable that the active ingredient in the bacteriostatic agent comprises the culture of Aspergillus flavus (Aspergillus flavus) XL-0048 or an extract of the culture of Aspergillus flavus (Aspergillus flavus) XL-0048.
Further, it is preferable 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 slant into PDB liquid culture medium, culturing in shaking table at 28 deg.C and 180rpm for 15-20 days, and taking out to obtain liquid culture;
the PDA slant culture medium contains: 3g/L of potato extract powder, 20g/L of glucose, 14g/L of agar and 5.6 +/-0.2 of pH value;
the PDB liquid culture medium contains: 5g/L of potato extract powder, 15g/L of glucose, 10g/L of peptone and 5g/L of sodium chloride.
Further, preferably, the extract is obtained by extracting an Aspergillus flavus (Aspergillus flavus) XL-0048 culture serving as a raw material with an organic solvent.
Further, it is preferable that the organic solvent is ethyl acetate.
In a third aspect the present invention provides a culture of Aspergillus flavus (XL-0048) as described above.
In a fourth aspect, the present invention provides an extract of a culture of Aspergillus flavus (Aspergillus flavus) XL-0048 as described above.
The Aspergillus flavus XL-0048 culture can also be obtained by culturing Aspergillus flavus XL-0048 serving as a raw material in a PDA culture medium, a PDB culture medium and the like which are conventional in the field.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides aspergillus flavus and application thereof, and the aspergillus flavus is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 27 months 9 and 2021 with the preservation number of CGMCC No. 23268. The culture and the extract of the culture have obvious inhibiting effect on 3 strains of sugarcane Alternaria alternata syd (NB-01), corn northern leaf spot bacteria (NB-06) and orange brown spot bacteria (Alternaria alternata, YB-05), and the Minimum Inhibitory Concentration (MIC) of a crude extract of the liquid culture on the corn northern leaf spot bacteria can reach 12.5ug/ml, so that the liquid culture 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 the plate; FIG. 1-B is a back view of the plate;
aspergillus flavus XL-0048 which has been deposited in China general microbiological culture Collection center (CGMCC No. 23268) at 27.9.2021 with the preservation number of CGMCC No. 3, the institute of microbiology, China academy of sciences, North Kogyo-south China.
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 invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
Example 1 Source and identification of Aspergillus flavus XL-0048
(ii) A source of Aspergillus flavus XL-0048
A fungus is separated from Salvia miltiorrhiza which is picked from Shenyang pharmaceutical university botanical garden in Shenyang of Liaoning province in 2017 in 4 months, and is named as XL-0048.
Identification of XL-0048
The ITS sequencing Sequence of XL-0048 is shown in a Sequence table (SEQ ID NO. 1), and has the highest Sequence similarity with Sequence ID MW723884.1, wherein the similarity is 98.59%.
Based on the above identification results, XL-0048 belongs to Aspergillus flavus.
Example 2 major morphological features of Aspergillus flavus XL-0048
Inoculating Aspergillus flavus XL-0048 on PDA culture medium, culturing at 28 deg.C for 3 days until the diameter of colony reaches 6cm, and changing the color of colony front from white to yellow and yellow-green with its growth to be semi-villous. The color of the mature spores is changed into brown, the surfaces of the spores are flat or have radial grooves, the reverse surfaces of the spores are colorless or brown, and the conidial heads are loose and radial and then loose and columnar. The conidiophores are very coarse when observed by a slide microscope. The top capsule is in a flask shape or a nearly spherical shape. Conidia are in a chain shape on the peduncle, and the periphery of the conidia is provided with small bulges, spheres and roughness.
The PDA culture medium formula comprises: 3g/L of potato extract powder, 20g/L of glucose, 14g/L of agar and 5.6 +/-0.2 of pH value.
The single colony morphology of the strain on a PDA plate is shown in figure 1, and figure 1-A is a front view of the 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
A filter paper method is adopted to determine the bacteriostatic activity of the fungus (XL-0048) to be screened on the test fungus (3 pathogenic fungi), and the specific method is as follows:
culture of bacterial liquid: respectively streaking and inoculating the stored fungi to be screened and tested fungi (3 strains of pathogenic fungi) on a PDA slant culture medium, placing the slant culture medium in an incubator at 28 ℃ for culturing for 72h, directly inoculating the grown slant culture medium into a spare PDB liquid culture medium, placing the culture medium in a shaking table at 28 ℃ and 180rpm for culturing for 48h, and taking out the slant culture medium for later use to obtain the test bacterial liquid.
Secondly, under the aseptic condition, using the fungi to be tested as indicator bacteria, respectively taking a proper amount of bacteria liquid and a PDA flat plate, uniformly coating, clamping three aseptic filter paper sheets by using aseptic tweezers, and respectively dropwise adding a proper amount of bacteria liquid of the strains to be screened on each paper sheet. Culturing in 28 deg.C constant temperature incubator for 3-5 days, and observing whether there is antibacterial effect.
The PDA slant culture medium comprises the following components in parts by weight: 3g/L of potato soaking powder, 20g/L of glucose, 14g/L of agar and 5.6 +/-0.2 of pH value.
Experimental results show that XL-0048 has obvious inhibiting effect on 3 plant pathogenic fungi, namely sugarcane alternaria alternata, corn leaf spot and orange brown spot.
Example 4 liquid culture of Aspergillus flavus (Aspergillus flavus) XL-0048
(1) Activation of the strain: inoculating the preserved aspergillus flavus XL-0048 on a PDA slant culture medium by streaking, and culturing for 3 days in an incubator at 28 ℃;
liquid culture of the strain: preparing 200 ml/bottle PDB liquid culture medium, directly inoculating the grown slant into a liquid shake flask, culturing in a shaking table at 28 deg.C and 180rpm for 20 days, and taking out to obtain liquid culture (i.e. fermentation liquid).
(2) Activation of the strain: inoculating the preserved aspergillus flavus XL-0048 on a PDA slant culture medium by streaking, and culturing for 5 days in an incubator at 28 ℃;
liquid culture of the strain: preparing 200 ml/bottle PDB liquid culture medium, directly inoculating the grown slant into a liquid shake flask, culturing in a shaking table at 28 deg.C and 180rpm for 15 days, and taking out to obtain liquid culture (i.e. fermentation liquid).
(3) Activation of the strain: inoculating the preserved aspergillus flavus XL-0048 on a PDA slant culture medium by streaking, and culturing for 4 days in an incubator at 28 ℃;
liquid culture of the strain: preparing 200 ml/bottle PDB liquid culture medium, directly inoculating the grown slant into a liquid shake flask, culturing in a shaking table at 28 deg.C and 180rpm for 17 days, and taking out to obtain liquid culture (i.e. fermentation liquid).
The PDA slant culture medium comprises the following components: 3g/L of potato extract powder, 20g/L of glucose, 14g/L of agar and 5.6 +/-0.2 of pH value.
The PDB liquid culture medium formula comprises: 5g/L of potato extract powder, 15g/L of glucose, 10g/L of peptone and 5g/L of sodium chloride, and the pH value is natural.
EXAMPLE 5 preparation of crude extract of Aspergillus flavus (Aspergillus flavus) XL-0048 Ethyl acetate
The fermentation liquor of example 4 was extracted with an equal volume of ethyl acetate by the following method: oscillating for 30min to fully mix the fermentation liquor with ethyl acetate, standing for 24h for liquid separation, taking out the ethyl acetate phase, namely the upper phase, performing reduced pressure 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 a crude extract of the bacterial liquid ethyl acetate, and preserving in a refrigerator at 4 ℃ for later use.
EXAMPLE 6 determination of the MIC value of an Aspergillus flavus (Aspergillus flavus) XL-0048 Ethyl acetate crude extract
The Minimum Inhibitory Concentration (MIC) of the crude extract of the bacterial liquid ethyl acetate in the embodiment 5 is further determined by a 96-well plate method, which comprises the following steps:
culture of test bacterial liquid: and (3) streaking and inoculating the preserved test fungi (3 pathogenic fungi) on a PDA slant culture medium, culturing in an incubator at 28 ℃ for 72h, directly inoculating the grown slant into a standby PDB liquid culture medium, culturing in a shaking table at 28 ℃ and 180rpm for 48h, and taking out for standby to obtain the test bacteria liquid.
Preparing a sample solution: 1mg of the crude ethyl acetate extract of the bacterial liquid obtained in example 5 was added with 100uL of DMSO to dissolve the crude ethyl acetate extract sufficiently, and then the volume of the mixture was determined by using DMSO to obtain a 100ug/mL sample solution.
Measuring MIC by a 96-well plate method: taking a sterile 96-well plate, respectively adding 100 mu L of PDB liquid culture medium into 1-7 wells, taking 100 mu L of sample solution to add into No.1 well, uniformly mixing (the concentration is 50 ug/mL), taking 100 mu L of sample solution out of No.1, adding into No.2 well, uniformly mixing, sequentially diluting to No. 7 well, taking 100 mu L of sample solution out of No. 7 well, and discarding. The sample concentration in the No. 1-7 holes is 50, 25, 12.5, 6.25, 3.125, 1.563 and 0.7815ug/mL in sequence, and then 100 mu L of test bacterium liquid is respectively added. On the same 96-well plate, a hole added with 100 μ L DMSO is used as a blank control, a hole added with only 100 μ L test bacteria solution without sample solution is used as a positive control, and a hole added with only 100 μ L PDB liquid culture medium without test bacteria solution is used as a negative control. The minimum clear concentration of culture in the observation well is the MIC.
The PDA culture medium formula comprises: 3g/L of potato extract powder, 20g/L of glucose, 14g/L of agar and 5.6 +/-0.2 of pH value.
The PDB liquid culture medium formula comprises: 5g/L of potato extract powder, 15g/L of glucose, 10g/L of peptone and 5g/L of sodium chloride, and the pH value is natural.
The Minimal Inhibitory Concentrations (MIC) of Aspergillus flavus (Aspergillus flavus) XL-0048 against different pathogenic fungi are 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 ethyl acetate crude extract of XL-0048 bacterial liquid has significant inhibitory effect on 3 plant pathogenic fungi, namely, sugarcane alternaria alternata, corn northern leaf blight and orange brown blotch, and the 3 fungi.
The foregoing shows and describes the general principles, essential 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, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. 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> Yangling future Zhongke environmental protection science and technology Limited
<120> Aspergillus flavus and application 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 (9)
1. Aspergillus flavus (Aspergillus flavus) XL-0048 which has been deposited in China general microbiological culture Collection center (CGMCC) at 27 th month 9 in 2021 with the preservation number of CGMCC No. 23268.
2. Use of the Aspergillus flavus (Aspergillus flavus) XL-0048 according to claim 1 for the preparation of a bacteriostatic agent.
3. The use of Aspergillus flavus (XL-0048) according to claim 2 in the preparation of a bacteriostatic agent for inhibiting fungi including Saccharum nigricans, northern leaf blight, and Monascus aurantiacus.
4. Use of Aspergillus flavus (Aspergillus flavus) XL-0048 in the preparation of a bacteriostatic agent according to claim 2, wherein the active ingredient in said bacteriostatic agent comprises an extract of said Aspergillus flavus (Aspergillus flavus) XL-0048 culture or Aspergillus flavus (Aspergillus flavus) XL-0048 culture.
5. Use of the Aspergillus flavus (Aspergillus flavus) XL-0048 according to claim 4 for the preparation of a bacteriostatic agent, characterized in 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 slant into PDB liquid culture medium, culturing in shaking table at 28 deg.C and 180rpm for 15-20 days, and taking out to obtain liquid culture;
the PDA slant culture medium contains: 3g/L of potato extract powder, 20g/L of glucose, 14g/L of agar and 5.6 +/-0.2 of pH value;
the PDB liquid culture medium contains: 5g/L of potato extract powder, 15g/L of glucose, 10g/L of peptone and 5g/L of sodium chloride.
6. The use of Aspergillus flavus XL-0048 according to claim 4 for the preparation of a bacteriostatic agent, characterized in that said extract is obtained by extraction of an organic solvent from a culture of Aspergillus flavus XL-0048.
7. Use of Aspergillus flavus (XL-0048) according to claim 4 for the preparation of a bacteriostatic agent, characterized in that the organic solvent is ethyl acetate.
8. A culture of Aspergillus flavus (Aspergillus flavus) XL-0048 according to claim 4 or 5.
9. An extract of a culture of Aspergillus flavus (Aspergillus flavus) XL-0048 according to claim 4, 6 or 7.
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