CN117511752A - Aspergillus flavus APqn-2 without toxin production, preparation thereof and application thereof in inhibiting toxin production fungi - Google Patents
Aspergillus flavus APqn-2 without toxin production, preparation thereof and application thereof in inhibiting toxin production fungi Download PDFInfo
- Publication number
- CN117511752A CN117511752A CN202311478848.0A CN202311478848A CN117511752A CN 117511752 A CN117511752 A CN 117511752A CN 202311478848 A CN202311478848 A CN 202311478848A CN 117511752 A CN117511752 A CN 117511752A
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- CN
- China
- Prior art keywords
- apqn
- aspergillus flavus
- inhibitor
- strain
- soil
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01B—SOIL WORKING IN AGRICULTURE OR FORESTRY; PARTS, DETAILS, OR ACCESSORIES OF AGRICULTURAL MACHINES OR IMPLEMENTS, IN GENERAL
- A01B79/00—Methods for working soil
- A01B79/02—Methods for working soil combined with other agricultural processing, e.g. fertilising, planting
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B9/00—Preservation of edible seeds, e.g. cereals
- A23B9/16—Preserving with chemicals
- A23B9/24—Preserving with chemicals in the form of liquids or solids
- A23B9/26—Organic compounds; Microorganisms; Enzymes
- A23B9/28—Microorganisms; Enzymes; Antibiotics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention discloses a strain of non-toxic aspergillus flavus APqn-2, a preparation thereof and application thereof in inhibiting toxic fungi, and belongs to the technical field of microorganisms. The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.40536. The non-toxic aspergillus flavus APqn-2 provided by the invention has remarkable inhibition effect on the growth and the toxicity of the toxic aspergillus flavus, the ochromogenes, the fusarium graminearum and the fusarium moniliforme, is favorable for planting and storing crops such as peanuts, corns and the like, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to non-toxic aspergillus flavus APqn-2, a preparation thereof and application thereof in inhibiting toxic fungi.
Background
Grain crops (such as peanuts and corns) are very easy to be polluted by mould in the links of growth, harvesting, storage, transportation, processing and the like, and seriously threaten the health of people and livestock. The main toxigenic fungi in peanuts are aspergillus flavus, aspergillus ochraceus, fusarium graminearum and fusarium moniliforme, and the main toxigenic fungi in corns are aspergillus flavus, aspergillus ochraceus, fusarium graminearum and fusarium moniliforme, and can produce various toxic secondary metabolites such as aflatoxin, zearalenone, ochratoxin, fumonisins and the like. These eubacteria toxins have teratogenic, carcinogenic and nephrotoxic effects. Diseases are also easily generated in the growth process of crops under natural conditions, and the diseases often cause huge losses to agricultural economy. The common chemical prevention and treatment is widely used for many years due to the characteristics of quick response, good effect and the like. However, a series of drawbacks such as environmental pollution, ecological balance destruction, agricultural product quality influence, human and animal health harm, etc. caused by the long-time massive use of chemical medicines have also been presented.
The biological disease control method can not only avoid the defects caused by chemical drug control, but also maintain ecological balance, save energy, reduce cost, and has more stable and durable control effect, thereby gradually attracting attention. Biological control refers to a method of controlling plant diseases by using organisms themselves or their metabolites in nature. Microbial control is the use of microorganisms and various organisms to achieve the goal of one type of microorganism or its products to inhibit another type of pest through certain biological characteristics and ecological phenomena.
The non-toxic aspergillus flavus cannot produce toxin due to the fact that a certain or a certain key genes are deleted in the aflatoxin synthesis gene cluster of the non-toxic aspergillus flavus, so that the non-toxic aspergillus flavus becomes a precondition for competing with toxigenic fungi and inhibiting toxigenic fungi. The non-toxic aspergillus flavus is applied in the field to regulate microbial flora in the soil, and the original toxigenic strain in the soil is gradually replaced in the growth process of crops, so that the possibility that agricultural products are infected by the aspergillus flavus is reduced, and the method is very effective for preventing and controlling mycotoxin pollution of crops before production. Most of researches on inhibiting the growth of the aspergillus flavus in laboratory are carried out at present, the field adaptability is weak, the field has no growth advantage, the effect of inhibiting the aspergillus flavus is not achieved, and meanwhile, the effect of inhibiting the other aspergillus flavus is not good. Therefore, the aspergillus flavus which does not secrete aflatoxin originally can inhibit the generation of various mycotoxins in crops, and the aspergillus flavus can be prepared into biological preparations suitable for complex environments, and is very important for biological prevention and control of the aspergillus flavus and prevention of agricultural products from being polluted by aflatoxin.
Disclosure of Invention
The invention provides a strain of non-toxic aspergillus flavus (Aspergillus flavus) APqn-2, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 40536.
The non-toxic aspergillus flavus APqn-2 has remarkable inhibition effect on the growth and the toxicity of the toxic aspergillus flavus (Aspergillus flavus), aspergillus ochraceus (Aspergillus ochraceus), fusarium graminearum (Fusarium graminearum) and fusarium moniliforme (Fusarium moniliforme).
The invention provides application of the non-toxic aspergillus flavus APqn-2 in preventing and controlling crop disease fungi.
The invention provides application of the non-toxic aspergillus flavus APqn-2 in inhibiting crop disease fungi toxigenic.
The invention provides application of the non-toxic aspergillus flavus APqn-2 in preparing a preparation for preventing and treating crop disease fungi or inhibiting crop disease fungi from producing toxins.
The invention provides a crop disease fungi inhibitor, which contains the non-toxic aspergillus flavus APqn-2.
In one embodiment, the crop disease fungal inhibitor of the present invention is prepared by a method selected from the group consisting of:
inoculating the strain APqn-2 on MEA culture medium, and culturing at 30deg.C for 3-5 days until yellow-green spores are produced; collecting field soil, removing impurities such as stone withered grass and the like, mixing according to the mass ratio of the soil to the wheat to distilled water of 1-2:0.02-0.05:1, and sterilizing at 121 ℃ for 20min to be used as a culture medium; inoculating the activated strain APqn-2 to the soil culture medium, culturing at 30 ℃ for 5-8 days, and shaking once a day to enable the strain APqn-2 to uniformly grow on the culture medium; and adding water with the mass of 5 times of the culture medium into the soil culture medium after the culture is finished, standing and culturing for 3-5 days at 30 ℃, and drying the soil culture medium in a baking oven at 45 ℃ after the culture is finished to prepare the crop disease fungi inhibitor.
In the soil culture medium, the mass ratio of soil to wheat to distilled water is selected from 1.5:0.03:1.
The invention provides a method for preventing and controlling crop disease fungi, which comprises the following steps: the crop disease fungi inhibitor is uniformly spread in a crop field, and the inhibitor is uniformly dispersed in soil through plowing and spinning, so that the purpose of preventing and controlling crop disease fungi is achieved.
In the control method, the usage amount of the crop disease fungi inhibitor is selected from 10-50 kg/mu; preferably 30 kg/mu.
In the present invention, the crops include, but are not limited to, peanut and corn; the disease fungi include, but are not limited to, aspergillus toxigenic, aspergillus ochraceus, fusarium graminearum, and fusarium moniliforme.
The beneficial effects of the invention are as follows:
the invention provides a non-toxic aspergillus flavus APqn-2 which can remarkably inhibit the growth and the toxicity of toxic aspergillus flavus, aspergillus ochraceus, fusarium graminearum and fusarium moniliforme, is favorable for planting and storing crops such as peanuts, corns and the like, and has wide application prospect.
Drawings
FIG. 1 shows the growth morphology of an agrobacterium suspension on a modified Bengalred medium;
FIG. 2 shows the morphology of strain APqn-2 on modified Bengalhong medium;
FIG. 3 shows the morphology of strain APqn-2 in malt extract agar medium (MEA medium);
FIG. 4 shows the morphology of strain APqn-2 in chloronitrosamine 18% glycerol medium (DG 18 medium);
FIG. 5 shows the morphology of strain APqn-2 in an aspergillin agar basal medium (AFPA medium);
FIG. 6 shows the determination of aflatoxin content in the fermentation broth of strain APqn-2.
Detailed Description
The culture medium adopted by the invention is as follows:
(1) Improved Bengalia culture medium
The basic formula of the culture medium comprises the following components:
3g of sucrose, 3g of sodium nitrate, 0.3g of monopotassium phosphate, 0.7g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium chloride, 10g of sodium chloride, 20g of agar powder, 50mg of chloramphenicol, micronutrients and antibiotics.
Wherein, micronutrient (/ L): 0.7mg of borax, 0.5mg of ammonium molybdate, 10mg of ferric sulfate, 0.3mg of copper sulfate pentahydrate, 0.11mg of manganese sulfate monohydrate and 17.5mg of zinc sulfate heptahydrate. The micronutrients were dissolved with a pH of 2.0 (adjusted with HCl). 1mL of the micronutrient solution was added to 1L of medium prior to sterilization.
The preparation method of the culture medium comprises the following steps: sucrose, inorganic salt, trace nutrients and 5mL of Bengalum were dissolved in a volume of deionized water, the pH was adjusted to 6.5, agar was added, heated and stirred until dissolved, chloramphenicol was added, sterilized at 121℃for 15min, the medium was cooled to 50-60℃and then chloronitrosamine (10 mg/L) and streptomycin (50 mg/L) were added, 5-10min later to plates (15-20 mL/L).
Malt extract agar (MEA medium): 30g of malt extract, 3g of soybean peptone, 15g of agar and 1L of distilled water are added.
Chlornitramine 18% glycerol medium (DG 18 medium): 5g of casein peptone, 10g of anhydrous glucose, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 0.002g of chloronitrosamine, 15g of agar and 0.1g of chloramphenicol are dissolved in 1000mL of distilled water, 200g of glycerol is added, and the mixture is uniformly mixed and sterilized at 121 ℃.
AFPA medium: 10g of peptone, 20g of yeast extract powder, 0.5g of ferric ammonium citrate, 0.002g of ammonium chloride, 0.1g of chloramphenicol, 15g of agar and pH of 6.3.
In the invention, the toxic aspergillus flavus, aspergillus ochraceus, fusarium graminearum and fusarium moniliforme are all obtained by self-separation of the applicant.
1. Bacterial separation
The sampling site is the peanut planting soil in Zhang river in Guangdong, and the sampling time is 5 months in 2018. Within a range of 10X 10m, 5 subsamples (soil 2cm wide, 5cm deep) were taken according to diagonal method, 100g each, and mixed as one sample. The collected samples are put into a plastic bag, and the pinholes are pricked to facilitate gas exchange, transported to a laboratory, and stored at 4 ℃ for aspergillus flavus screening. Taking 10g of soil sample, adding 90mL of 0.1% peptone sterile water (w/v), and shaking at room temperature for 30min to obtain 10 -1 A bacterial suspension; taking 0.5mL 10 again -1 Adding 4.5mL of 0.1% peptone sterile water into the bacterial suspension to prepare 10 -2 A diluted bacterial suspension; preparation 10 according to the above method -3 A diluted bacterial suspension. Each dilution was plated onto modified Bengalia red medium, incubated in the dark at 30℃for 5d, and each dilution was repeated 3 times, strain growth was as shown in FIG. 1. Selecting strain growing with yellow-green spores, performing secondary streaking separation on the modified Bengalhong culture medium,until a single colony is obtained, as shown in FIG. 2. The strains of the individual colonies were picked up on malt extract agar medium (MEA medium), cultured at 30℃for 3 days, and then stored at 4℃as shown in FIG. 3. By the method, a single strain of mould is isolated and the strain is numbered APqn-2.
2. Identification of species
The strain APqn-2 was characterized in morphology and in physiological and biochemical by the method described in the "burjie bacteria identification handbook" (eighth edition), with the following specific results:
(1) Morphological features
The strain produces yellowish green spores, opaque colonies and white hyphae on a modified Bengalia red culture medium. Yellow spores were generated on DG18 medium as shown in fig. 4. A color reaction of bright orange was produced on AFPA medium as shown in fig. 5.
(2) Genetic identification
And carrying out molecular identification on the strain APqn-2 through the ITS gene sequence.
Primers used for ITS amplification of the genome of Aspergillus flavus are:
ITS1：5'-TCCGTAGGTGAACCTGCGG-3'(SEQ ID NO:1)；
ITS4：5'-TCCTCCGCTTATTGATATGC-3'(SEQ ID NO:2)。
the PCR amplification conditions were: the PCR amplification reaction procedure was: pre-denaturation at 94℃for 5min,1 cycle; denaturation at 94℃for 30s, annealing at 54℃for 30s, elongation at 72℃for 90s for 30 cycles; finally, the extension is carried out for 7min at 72 ℃. After amplification, the product was stored at 4 ℃. The products were sent to Shanghai Bioengineering Co.Ltd for sequencing and the sequencing results were aligned on BLAST research (http:// www.ncbi.nlm.nih.gov /).
As can be seen from the sequencing, the ITS sequence of strain APqn-2 is as follows:
5'-GAAGGATCATTACCGAGTGTAGGGTTCCTAGCGAGCCCAACCTCCCACCCGTGT TTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATA-3'(SEQ ID NO:3)
the ITS sequence comparison shows that the similarity of the ITS gene sequence of the strain APqn-2 and the small subunit ribosomal RNA gene sequence of the Aspergillus flavus strain GrM is 100%. By adopting a universal primer to detect the expression condition of the strain APqn-2 toxigenic gene, the result shows that 11 toxigenic key genes, namely norB-cypA, aflT, pksA, nor-1, aflR, adhA, norA, ver-1, hypC, hypB and moxY, in genes on the strain APqn-2 toxigenic gene cluster are not expressed, so that the strain does not produce toxigenic genes. Based on morphological identification in combination with molecular biological identification, strain APqn-2 was finally identified as aflatoxin-free aspergillus flavus (Aspergillus flavus).
The strain is preserved in China general microbiological culture collection center (CGMCC) for 24 days in 2023, wherein the preservation number is CGMCC NO.40536, namely, the North Chen West Lu NO. 1, 3 of the Korean region of Beijing, and the postal code 100101 of the microbiological institute of China academy of sciences).
Other materials used in the present invention, such as those not specifically stated, are available through commercial sources. Other terms used herein, unless otherwise indicated, generally have meanings commonly understood by those of ordinary skill in the art. The invention will be described in further detail below in connection with specific embodiments and with reference to the data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
And (3) strain APqn-2 toxigenic verification:
(1) Toxigenic culture
Inoculating the strain APqn-2 on an MEA slant test tube culture medium, and culturing at 28 ℃ for 3d to activate the strain APqn-2; 4mL of sterile water was added to the slant tube medium and rinsed to prepare strain APqn-2 suspension. The number of spores was recorded under a microscope with a hemocytometer. 10mL of toxigenic culture solution is added into a 50mL centrifuge tubeAdding a certain amount of Aspergillus APqn-2 bacterial suspension to make the final spore concentration 10 5 /mL, at 30 degrees C, 200rpm under conditions of 7 days.
Wherein, the composition of the toxigenic culture solution is as follows: 150g sucrose, 20g yeast extract, 10g soytone, 1000mL distilled water, pH 5.9.
(2) Aflatoxin assay
And detecting aflatoxin in the fermentation liquor by adopting an immunoaffinity chromatography purification, liquid chromatography separation and fluorescence detector detection method. The specific operation is as follows: passing 2mL of fermentation liquor through an immunoaffinity chromatography column, eluting with water at a flow rate of 3mL per minute for 2 times, discarding the eluent, allowing air to enter the column, extruding water out of the column, eluting with 1.5mL of methanol for several times, collecting the eluent, concentrating to 0.7mL, diluting to 1mL with water, shaking, loading, separating by high performance liquid chromatography, and detecting by a fluorescence detector.
Chromatographic conditions: the column was Venusil MP C18 (5 μm,4.6 mm. Times.150 mm); column temperature is 40 ℃; the mobile phase is methanol to water (V: v=45:55); the flow rate is 1.3mL/min; the post-column photochemical derivatization method is adopted: a photochemical derivatizer 254nm; the sample is detected by a fluorescence detector, the excitation wavelength is 360nm, the emission wavelength is 450nm, and the sample injection amount is 20 mu L.
The test results are shown in fig. 6.
No aflatoxin was detected in the strain APqn-2 toxigenic fermentation broth, thereby further confirming that the strain APqn-2 is a non-toxigenic Aspergillus flavus strain.
Example 2
And (3) toxin production inhibition test:
(1) Preparation of the culture Medium
The whole unbroken corn and peanut particles were selected, then 10g of uniformly sized peanut and corn were weighed, respectively, and sterilized at 121 ℃ for 15 minutes.
(2) Preparation of bacterial suspension
Inoculating toxigenic Aspergillus flavus, aspergillus ochraceus, fusarium graminearum, and Fusarium moniliforme respectively on MEA slant tube culture medium, culturing at 20deg.C for 5 days, dipping spores on the culture medium in sterile water, shaking uniformly with vortex oscillator, andthe spore concentration was then adjusted to 2X 10 with a hemocytometer 4 spores/mL for use.
Spore concentration of strain APqn-2 was adjusted to 2X 10 with a hemocytometer 4 spore/mL, the strain APqn-2 spore fungus suspension is obtained for standby.
(3) Inhibition effect test
1mL of the strain APqn-2 spore fungus suspension and the spore fungus suspension of the toxigenic fungus (10) were added to the flask respectively 4 :10 4 ) As experimental groups. 1mL of the toxigenic bacteria (10) was then added to the flask 4 ) The spore bacterial suspension mixed with sterile water in equal volume was used as positive control group, and the bottle was gently shaken to cover the spores on peanut and corn. Each group was incubated at 30℃in triplicate for 14 days in the dark.
(4) Mycotoxin assay
Placing the cultured corn and peanut samples into an autoclave, and sterilizing (inactivating mould) at 121 ℃ for 30 min; the sterilized sample is put into a high-speed universal crusher to be crushed, 50mL of 80% methanol is added into a triangular flask, the mixture is vibrated at a high speed for 30min by using a vibrator, the mixture is filtered by using sterilized filter paper, and the extract is measured by using HPLC.
The test results are shown in table 1:
TABLE 1
As shown in Table 1, the strain APqn-2 can inhibit the toxicity of Aspergillus flavus in peanuts and four toxic fungi in corns; the efficiency of inhibiting the aspergillus flavus from producing aflatoxin B1 in peanuts is 92.96 +/-0.94%; in corn, the efficiency of inhibiting fusarium graminearum from producing zearalenone is 82.17 +/-0.94%, the efficiency of inhibiting aspergillus ochraceus from producing ochratoxin A is 74.36+/-0.63%, and the efficiency of inhibiting fusarium moniliforme from producing fumonisin b1 is 77.31 +/-0.54%. The toxicity production inhibition effect is very obvious.
Example 3
And (3) field application:
1. preparation of toxigenic fungus inhibitor
(1) Inhibitor A
Strain APqn-2 was inoculated on MEA medium and cultured at 30 ℃ for 3-5 days until yellow-green spores were produced. Collecting field soil, removing impurities such as stone withered grass, mixing according to the mass ratio of soil, wheat and distilled water of 1.5:0.03:1, and sterilizing at 121deg.C for 20min to obtain culture medium. The activated strain APqn-2 is inoculated on the culture medium, and is cultured for 8 days at 30 ℃ and is shaken once a day, so that the strain APqn-2 grows uniformly on the culture medium. Adding water with the mass of 5 times of the culture medium into the culture medium after the culture is finished, standing at 30 ℃ for 5 days, drying the culture medium in a baking oven at 45 ℃ after the culture is finished, and obtaining the toxigenic fungi inhibitor A, and preserving at normal temperature.
(2) Inhibitor B
Strain APqn-2 was inoculated on MEA medium and cultured at 30 ℃ for 5 days until yellow-green spores were produced. Drying MEA culture medium full of spores in a baking oven at 45 ℃, and crushing to obtain the toxigenic fungi inhibitor B.
(3) Inhibitor C
Strain APqn-2 was inoculated on MEA medium and cultured at 30 ℃ for 5 days until yellow-green spores were produced. Collecting field soil, removing impurities such as stone withered grass, mixing according to the mass ratio of soil, wheat and distilled water of 1.5:0.03:1, and sterilizing at 121deg.C for 20min to obtain culture medium. The activated strain APqn-2 is inoculated on the culture medium, and is cultured for 8 days at 30 ℃ and is shaken once a day, so that the strain APqn-2 grows uniformly on the culture medium. And after the culture is finished, drying the culture medium in a baking oven at 45 ℃ to obtain the toxigenic fungi inhibitor C.
2. Field application method
The inhibitor and the organic fertilizer are uniformly spread in peanut fields and corn fields which are not sowed in spring, the using amount of the inhibitor is 30 kg/mu, and ploughing and rotating are carried out, so that the inhibitor and the organic fertilizer are uniformly dispersed in soil to be used as a test group. Taking the group without inhibitor as a blank group; the other daily management was the same for both the test and control groups. And taking a soil sample before and after the inhibitor is applied and during harvesting, detecting the number of thalli in the soil sample, separating and identifying Aspergillus flavus, and comparing the number change condition of toxigenic fungi in the soil sample before and after the inhibitor is applied.
The test results are shown in table 2:
TABLE 2 inhibition of soil fungi by antagonistic bacteria
As can be seen from Table 2, after the application of strain APqn-2, the number of toxigenic fungi in the soil of each group was significantly suppressed. Meanwhile, the bacteriostatic effect of the inhibitor A is most remarkable. Thus, the different preparation methods of the inhibitor have important influence on the inhibition of toxigenic fungi. The inhibitor (inhibitor B) prepared by taking a microorganism conventional agar Medium (MEA) as a medium has obviously lower inhibition effect on the aspergillus flavus which produces poison in peanut planting fields and the aspergillus flavus which produces poison, fusarium graminearum, aspergillus ochraceus and fusarium moniliforme in corn planting fields than other two inhibitors prepared by taking soil as a medium, and the reason is probably that the inhibitor has increased viability in the soil through liquid-solid combined culture, so that the inhibition capability on the fungi which produce poison is increased. This demonstrates that soil is used as a culture medium to prepare inhibitors which are more suitable for field application and to improve the survival ability of the strain APqn-2 in the field. From the comparison of the inhibitor A and the inhibitor C, in the preparation process of the inhibitor, the solid-liquid combined culture can better improve the inhibition capability of the microbial inoculum to the toxigenic fungi than the solid culture only; the reason for this is probably that the non-toxic bacteria can better adapt to various environments in the soil and enhance stress resistance by taking low-nutrition soil as a culture medium, carrying out solid culture and drying, and then carrying out liquid culture and drying.
3. Corn peanut storage and toxin assay
And (3) airing and weighing each seed sample of the corn and the peanut harvested in the test, respectively filling the seed samples into seed bags, and placing the seed bags in a dry and cool place for storage. The mycotoxin content in corn peanuts stored for 0, 1, 2, 3, 4, 5, 6, 7 and 8 months was measured, and the ability of inhibitor A to inhibit mycotoxin production was calculated as compared with the control group.
The test results are shown in tables 3 and 4:
TABLE 3 variation of aflatoxin content in peanuts during storage
As is clear from Table 3, the peanuts planted in the control group (without inhibitor) were stored for a prolonged period of time, and after 1 month of storage, aflatoxin B was detected 1 Aflatoxin B with prolonged storage time 1 The content is always increased, and when the rice is stored for 7 months, the content of the aflatoxin is 20.3+/-0.6 mug/kg, exceeds the national limit standard of 20 mug/kg, and the rice cannot be eaten. After the inhibitor is applied, aflatoxin is not detected within 7 months of storage time, which indicates that the risk of peanut infection of aspergillus flavus can be obviously reduced when the inhibitor is applied to peanut planting places. The reason for this may be that after the non-toxic aspergillus flavus is applied to the peanut planting field, dominant strains can be formed in the peanut planting field, and the growth and propagation of the toxic aspergillus flavus in the peanut field are inhibited, so that the infection of the toxic bacteria in the peanut is reduced.
TABLE 4 variation of mycotoxin levels in corn during storage
As can be seen from Table 4, the corn (without inhibitor) planted in the control group was stored for 1 month, and aflatoxin B was detected 1 And zearalenone, which was detected after 2 months of storageOchratoxin A and fumonisin B are produced 1 . The mycotoxin content of each has increased with the storage time. Aflatoxin B at 8 months of storage 1 Zearalenone and fumonisin B 1 The content is 25.3+/-0.4 mug/kg, 75.4+/-0.3 mug/kg and 4005.4 +/-7.5 mug/kg respectively, and the content exceeds the respective national limit standards of 20 mug/kg, 60 mug/kg and 4000 mug/kg; when stored for 6 months, the content of ochratoxin A is 6.3+/-0.2 mug/kg, which exceeds the national limit standard of 5 mug/kg. The absence of detection of these toxins within 7 months of storage time after the inhibitor application indicates that the application of the inhibitor to corn plantation significantly reduces the risk of corn infection with these various molds. The reason for this may be that after the non-toxic aspergillus flavus is applied to the corn planting field, dominant strains can be formed in the corn planting field, and the growth and propagation of various toxic fungi in the corn field are inhibited, so that the infection of the toxic fungi in the corn is reduced.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (10)
1. The strain does not produce toxic aspergillus flavus (Aspergillus flavus) APqn-2 and is characterized in that the strain is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 40536.
2. Use of non-toxic aspergillus flavus APqn-2 according to claim 1 for controlling crop disease fungi.
3. Use of non-toxigenic aspergillus flavus APqn-2 as claimed in claim 1 for inhibiting crop disease fungal toxigenesis.
4. Use of non-toxic aspergillus flavus APqn-2 according to claim 1 for preparing a preparation for controlling crop disease fungi or inhibiting crop disease fungi toxigenic.
5. A fungal inhibitor of crop disease comprising non-toxic aspergillus flavus APqn-2 according to claim 1.
6. The crop disease fungal inhibitor of claim 5, said crop disease fungal inhibitor prepared by a process comprising:
inoculating the strain APqn-2 on MEA culture medium, and culturing at 30deg.C for 3-5 days until yellow-green spores are produced; collecting field soil, removing impurities such as stone withered grass and the like, mixing according to the mass ratio of the soil to the wheat to distilled water of 1-2:0.02-0.05:1, and sterilizing at 121 ℃ for 20min to be used as a culture medium; inoculating the activated strain APqn-2 to the soil culture medium, culturing at 30 ℃ for 5-8 days, and shaking once a day to enable the strain APqn-2 to uniformly grow on the culture medium; and adding water with the mass of 5 times of the culture medium into the soil culture medium after the culture is finished, standing and culturing for 3-5 days at 30 ℃, and drying the soil culture medium in a baking oven at 45 ℃ after the culture is finished to prepare the crop disease fungi inhibitor.
7. The crop disease fungal inhibitor of claim 6, wherein the mass ratio of soil, wheat and distilled water in said soil medium is selected from 1.5:0.03:1.
8. A method for controlling crop disease fungi, characterized by the steps of: the crop disease fungi inhibitor according to claim 5 is uniformly spread in a crop field, and the inhibitor is uniformly dispersed in soil by ploughing and rotary land so as to achieve the purpose of preventing and controlling crop disease fungi.
9. The method of claim 8, wherein the crop disease fungal inhibitor is used in an amount selected from the group consisting of 10 to 50 kg/mu; preferably 30 kg/mu.
10. The use according to any one of claims 2 to 4, or the inhibitor according to any one of claims 5 to 7, or the method according to any one of claims 8 to 9, wherein the disease fungus is selected from aspergillus toxigenic, aspergillus ochraceus, fusarium graminearum or fusarium moniliforme.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311478848.0A CN117511752A (en) | 2023-11-08 | 2023-11-08 | Aspergillus flavus APqn-2 without toxin production, preparation thereof and application thereof in inhibiting toxin production fungi |
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