CN110122508B

CN110122508B - Antagonistic antibacterial agent for producing toxic aspergillus flavus, preparation method and application thereof

Info

Publication number: CN110122508B
Application number: CN201910451854.4A
Authority: CN
Inventors: 张建成; 张初署; 孙杰
Original assignee: Shandong Peanut Research Institute
Current assignee: Shandong Peanut Research Institute
Priority date: 2019-05-28
Filing date: 2019-05-28
Publication date: 2021-01-12
Anticipated expiration: 2039-05-28
Also published as: CN110122508A

Abstract

The invention discloses an antagonistic bacterium agent for producing aspergillus flavus, a preparation method and application thereof, belonging to the technical field of antagonistic bacterium agents for harmful microorganisms. The active ingredients of the antagonistic bacteria agent for producing the aspergillus flavus are aspergillus flavus PEAS-10 which does not produce the aspergillus flavus toxin and aspergillus flavus PEASH-12 which does not produce the aspergillus flavus toxin; the aspergillus flavus strain in the antagonistic antibacterial agent can grow and propagate rapidly in the field, can efficiently inhibit the growth, propagation and toxin production of toxin-producing aspergillus flavus, can reduce peanut diseases, remarkably improve peanut yield, and has the advantages of low aflatoxin content in the harvested peanuts, long peanut storage period and excellent effect compared with single microbial agent treatment.

Description

Antagonistic antibacterial agent for producing toxic aspergillus flavus, preparation method and application thereof

Technical Field

The invention belongs to the technical field of antagonistic bacteria agents for harmful microorganisms, and particularly relates to an antagonistic bacteria agent for producing aspergillus flavus, a preparation method and application thereof.

Background

Aflatoxins are metabolites of aspergillus flavus and aspergillus parasiticus. A large amount of experimental data show that the aflatoxin can cause experimental liver cancer to be induced by human beings and various animals, is the strongest chemical carcinogen found at present, and has 75 times greater capability than that of the dimethyl nitrosamine to induce liver cancer. Aflatoxin is also a virulent agent, with 68 times of arsenic and 10 times of potassium cyanide, and can cause severe liver damage and death in a short period of time.

Peanuts are the crop most susceptible to Aspergillus flavus infection. The peanuts are likely to be infected with aspergillus flavus in the whole growth process, and particularly in the later growth period, the pollution of the aspergillus flavus is aggravated after the seed coats of the peanuts are damaged due to the change of temperature and humidity and the harm of sick rats and the like. After the peanuts are harvested, the influence of air temperature, air humidity and storage conditions can cause aspergillus flavus infection more easily. In the process of reproduction and metabolism of the aspergillus flavus, a large amount of toxins (mainly aflatoxin B1) are generated to pollute peanuts and products thereof. It has been found that this toxin is likely to be present in improperly preserved peanuts and peanut oil, peanut beverages, and peanut butter. Due to the pollution of aspergillus flavus, the growth of peanuts can be inhibited, so that the yield of the peanuts is reduced, and the yield is reduced by about 10 percent.

The aflatoxin pollution of the peanuts mainly comprises pollution before the peanuts are harvested and pollution after the peanuts are harvested. The peanuts are easy to be infected by aspergillus flavus before being harvested, and researches show that the soil is a main source of the aspergillus flavus of the peanuts, and the aspergillus flavus in the peanut pods is directly related to the aspergillus flavus in the soil, so that the researches on the field biological prevention and control of the peanut aspergillus flavus pollution are of great significance in order to effectively prevent and reduce the pollution of the peanut aflatoxin.

Biocontrol aflatoxin is a substance that utilizes beneficial (or at least harmless) organisms and metabolites thereof to change the layout of microorganisms, inhibit the growth of toxigenic strains or inhibit the synthesis of toxins thereof, thereby achieving the level of reducing aflatoxin of agricultural products; or the aflatoxin is adsorbed and degraded by the actions of biological adhesion, degradation and the like, so that the aim of removing the aflatoxin is fulfilled. Compared with other treatment methods, the biological prevention and control method has the advantages of simple operation, no damage to the original quality of agricultural products, safety, high efficiency, environmental friendliness and the like, and represents a new direction for green control of aflatoxin.

The field control of the peanut aspergillus flavus pollution is mainly characterized in that water supply is ensured during the peanut pod development period in the later growth period of the peanuts, the situation that the infection chances of the aspergillus flavus are increased due to the breakage of seed coats caused by drought before harvesting is avoided, the occurrence of other diseases, insect pests and mouse pests is avoided, and the pod damage caused by plowing is avoided in the pod bearing period and the pod development period. Sun-drying the legume in time after harvesting to make the water content less than 5%, and screening new peanut varieties with resistance. However, the aspergillus flavus has strong viability, and the produced spores can resist various severe natural conditions, so that the infection of the aspergillus flavus cannot be completely avoided.

At present, the aspergillus flavus which does not produce the toxin is separated from the soil, only the research of inhibiting the growth of the aspergillus flavus which produces the toxin in a laboratory is carried out, and the field experimental research is not carried out. At present, some antagonistic bacteria are not suitable for field growth, have no growth advantage in the field and cannot play a role in inhibiting the production of toxic aspergillus flavus. Moreover, the single microbial inoculum has the problems of poor adaptability and poor prevention and treatment effect.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a compound antagonistic antibacterial agent which can rapidly grow and propagate in the field, can efficiently inhibit the growth, propagation and toxin production of toxin-producing aspergillus flavus and has excellent effect compared with single microbial agent treatment.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an antagonistic bacterium agent for producing toxic aspergillus flavus, the effective components of which are aspergillus flavus PEAS-10 which does not produce aflatoxin and aspergillus flavus PEASH-12 which does not produce aflatoxin;

the aspergillus flavus PEAS-10 which does not produce toxin is preserved in the year 2018, on the 08 th month and on the 01 th day: the China general microbiological culture Collection center (CGMCC) has a collection number of CGMCC NO:15997 and the address of the CGMCC is as follows: west road No. 1, north west of chaoyang district, beijing, the requested preservation unit is peanut institute of shandong province;

the aspergillus flavus PEASH-12 which does not produce toxin is preserved in the following period of 2018, 08 and 01 months: china general microbiological culture Collection center (CGMCC) with the collection number of CGMCC NO:15998 and the address of: west road No. 1, north zhou yang ward, beijing, the requested depository is peanut institute of shandong province.

On the basis of the scheme, the number of spores of Aspergillus flavus PEAS-10 which does not produce aflatoxin in the antagonistic antibacterial agent is more than or equal to 10⁸Per gram; the number of spores of Aspergillus flavus PEASH-12 which does not produce aflatoxin is more than or equal to 10⁸Per gram.

On the basis of the scheme, the preparation method of the antagonistic antibacterial agent for producing the toxic aspergillus flavus comprises the following steps:

(1) respectively inoculating the strains on an MEA culture medium, and culturing for 3-5 days at 30 ℃ until yellow-green spores are generated;

(2) respectively inoculating the activated non-toxigenic aspergillus strains to a sterilized microbial inoculum culture medium, culturing for 5-8 days at 30 ℃, and shaking once every day to ensure that the aspergillus flavus grows uniformly on the culture medium; after culturing, the number of aspergillus flavus spores is more than or equal to 10⁸Per gram of medium;

(3) mixing the culture medium containing Aspergillus flavus PEAS-10 and Aspergillus flavus PEASH-12 cultured in (2) according to a certain proportion, and finally obtaining the antagonistic bacteria agent for producing the toxic Aspergillus flavus by the spore number proportion of the two strains being PEAS-10: PEASH-12 ═ 1: 1-1: 3, and preserving at normal temperature

On the basis of the scheme, the mixing ratio of spores of aspergillus flavus PEAS-10 and aspergillus flavus PEASH-12 in the toxin-producing aspergillus flavus antagonistic bacteria agent is 2: 3.

On the basis of the scheme, the microbial inoculum culture medium is prepared by the following method:

crushing peanut shell into 0.5 multiplied by 0.5cm, mixing according to the mass ratio of 1: 1-2: 3 of the peanut shell and distilled water, and simultaneously adding 1-1.5 mass percent of CaCl₂And 0.5% KCl, sterilized at 121 ℃ for 20 min.

The application of the antagonistic antibacterial agent for producing the toxic aspergillus flavus prepared by the method is used for inhibiting the growth and the toxicity production of the aspergillus flavus, reducing crop diseases, improving the utilization rate of organic fertilizers, improving the yield of crops, reducing the content of aflatoxin in agricultural products during harvesting and prolonging the storage period of the agricultural products.

On the basis of the scheme, the crops are peanuts or corns.

A method for inhibiting growth and toxin production of aspergillus flavus comprises the step of spraying the toxin-producing aspergillus flavus antagonistic antibacterial agent prepared by the method at the rhizosphere of crops by 30 kg/mu 1 month before the crops are harvested.

A method for reducing crop diseases, improving utilization rate of organic fertilizer or increasing crop yield comprises the steps of spraying the toxin-producing aspergillus flavus antagonistic antibacterial agent prepared by the method to the rhizosphere of crops at a rate of 30 kg/mu 1 month before the crops are harvested, harvesting in due time, airing and storing in dry and cool places.

A method for reducing aflatoxin content in agricultural products during harvesting or prolonging storage period of agricultural products comprises spraying the obtained antagonistic antibacterial agent for producing aflatoxin at 30 kg/mu in the rhizosphere of crops 1 month before harvesting, harvesting at proper time, air drying, and storing in dry and cool place.

The technical scheme of the invention has the advantages that:

the aspergillus flavus strain which does not produce aflatoxin in the composite antagonistic antibacterial agent can rapidly grow and propagate in the field, can efficiently inhibit the growth, propagation and toxin production of toxin-producing aspergillus flavus, has obvious effects of field prevention and control of aflatoxin pollution, and has obvious prevention effect on root rot and stem rot of flowers when the antagonistic antibacterial agent is used for treating peanuts; the peanut yield is obviously increased; after the treated peanuts are harvested, the content of aflatoxin is low, and the storage period of the peanuts can be prolonged. Compared with single microbial inoculum treatment, the effect is excellent.

Drawings

FIG. 1 measurement of aflatoxin content in the fermentation broth of the strain PEASH-12;

FIG. 2 measurement of aflatoxin content in the fermentation broth of strain PEAS-10.

Detailed Description

Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.

The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

The Aspergillus flavus which does not produce toxin is Aspergillus flavus (PEAS-10) which is preserved in 2018 in 08 and 01 months: the China general microbiological culture Collection center (CGMCC) has a collection number of CGMCC NO:15997 and the address of the CGMCC is as follows: west road No. 1, north zhou yang ward, beijing, the requested depository is peanut institute of shandong province.

The Aspergillus flavus which does not produce toxin is Aspergillus flavus (PEASH-12) which is preserved in 2018 in 08 and 01 months: china general microbiological culture Collection center (CGMCC) with the collection number of CGMCC NO:15998 and the address of: west road No. 1, north zhou yang ward, beijing, the requested depository is peanut institute of shandong province.

EXAMPLE 1 acquisition of strains PEAS-10 and PEASH-12

Separation, purification and identification of bacterial strain

1. Collecting samples: samples were collected from peanut growing lands (2018.05, sampling sites were red flag fields of the Onychongcun and the Gangxi improved variety breeding farm in the Jialin region of the southern city, Sichuan province, respectively), and 5 subsamples (2cm wide and 5cm deep soil) were taken from each sample (100g) within a range of 10 × 10m by the diagonal method and mixed to obtain one sample. Putting the collected sample into a plastic bag, pricking pin holes to facilitate gas exchange, transporting to a laboratory, storing at 4 ℃ for screening aspergillus flavus.

2. And (5) separating and purifying the strain.

(1) Preparation of soil sample bacterial suspension

10g of soil sample was added with 90mL of 0.1% peptone sterile water (w/v), and shaken at room temperature for 30min to prepare 10^-1Bacterial suspension; then 0.5mL of 10^-1The bacterial suspension was added with 4.5mL of 0.1% peptone sterile water to prepare 10^-2Diluting the bacterial suspension; preparation of 10 by the above method^-3A diluted bacterial suspension.

(2) Isolation and purification of the strains

0.1mL of the inoculum was taken for each dilution, spread on modified Bengal red medium, incubated at 30 ℃ in the dark for 5 days, and repeated 3 times for each dilution. And (3) selecting the aspergillus flavus with yellow-green spores to perform secondary streak separation on the improved Bangla red culture medium until a single colony is obtained. Aspergillus flavus of a single colony is picked up on an MEA slant test tube culture medium, cultured for 3d at 30 ℃ and stored at 4 ℃.

By the method, the strain PEAS-10 is separated from a soil sample of the village of the country of the Tumen in the Jialin city of the southern China, Sichuan province, and the strain PEASH-12 is separated from a soil sample of the red flag branch field of the fine breed breeding farm of the West China.

(3) Identification

Identification of the Strain PEAS-10

Morphological identification

The isolated strains of the invention are on a modified Bengal Red Medium: producing white hypha and yellow-green spores by aspergillus flavus; yellow spores were produced on DG18 medium and a bright orange color reaction on AFPA medium; and the strain is cultured in a toxin-producing culture solution, and aflatoxin is not produced.

Molecular identification

The strain PEAS-10 was molecularly identified by ITS gene sequence.

Primers used for amplification of Aspergillus flavus genome ITS were:

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 program is as follows: pre-denaturation at 94 ℃ for 5min for 1 cycle; denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 90s for 30 cycles; final extension at 72 ℃ for 7 min. After amplification, the product was stored at 4 ℃. The product was sent to Shanghai Bioengineering, Inc. for sequencing, and the sequencing results were aligned on BLAST research (http:// www.ncbi.nlm.nih.gov /).

Sequencing shows that the ITS sequence of the bacterial strain PEAS-10 is as shown in SEQ ID No. 3:

ACCTGCGGAAGGATCATTACCGAGTGTAGGGTTCCTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAT

the ITS sequence comparison shows that the similarity of the ITS gene sequence of the strain PEAS-10 and the small subunit ribosomal RNA gene sequence of the aspergillus flavus strain LWU-31 is 100%.

The expression condition of the toxic gene of the strain PEAS-10 is detected by adopting a universal primer, and the result shows that seven toxic key genes of nor-1, afiR, omtA, ordA, ver-1, verA and verB in the genes on the toxic gene cluster of the strain PEAS-10 are not expressed, so that the strain does not produce toxicity.

The morphological identification and the molecular biology identification result show that the strain PEAS-10 is aspergillus flavus which does not produce aflatoxin; it was stored in 2018 on 08/01: the China general microbiological culture Collection center (CGMCC) has a collection number of CGMCC NO:15997 and the address of the CGMCC is as follows: west road No. 1, north zhou yang ward, beijing, the requested depository is peanut institute of shandong province.

Identification of the Strain PEASH-12

Morphological identification

Molecular identification

The strain PEASH-12 was molecularly characterized by ITS gene sequence.

Primers used for amplification of Aspergillus flavus genome ITS were:

ITS1：5’-TCCGTAGGTGAACCTGCGG-3’(SEQ ID No.1)；

ITS4：5’-TCCTCCGCTTATTGATATGC-3’(SEQ ID No.2)。

Sequencing shows that the ITS sequence of the bacterial strain PEASH-12 is as follows SEQ ID No. 4:

GACCTGCGGAAGGATCATTACCGAGTGTAGGGTTCCTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAT

the ITS sequence comparison shows that the coverage rate of the ITS gene sequence of the strain PEASH-12 and the Aspergillus flavus strain S2599 small subunit ribosomal RNA gene sequence is 99 percent, and the similarity is 100 percent.

The general primers are adopted to detect the expression condition of the toxic gene of the strain PEASH-12, and the result shows that eight toxic key genes of afiT, nor-1, afiR, omtA, ordA, ver-1, verA and verB in the genes on the toxic gene cluster of the strain PEASH-12 are not expressed, so that the strain does not produce toxicity.

The morphological identification and the molecular biology identification result show that the bacterial strain PEASH-12 is aspergillus flavus which does not produce aflatoxin; it was stored in 2018 on 08/01: china general microbiological culture Collection center (CGMCC) with the collection number of CGMCC NO:15998 and the address of: west road No. 1, north zhou yang ward, beijing, the requested depository is peanut institute of shandong province.

Example 2 analysis of the toxicity of Aspergillus flavus PEAS-10 and PEASH-12

(1) Culture for producing toxin

Respectively inoculating Aspergillus flavus PEAS-10 and PEASH-12 strains on an MEA slant test tube culture medium, and culturing at 28 ℃ for 3d to activate the strains; 4mL of sterile water is added to a slant tube culture medium and washed to respectively prepare a Aspergillus flavus PEAS-10 suspension and an Aspergillus flavus PEASH-12 suspension. The number of spores was recorded under a microscope using a hemocytometer.

Adding 10mL of toxigenic culture solution into 50mL of centrifuge tube, adding a certain amount of Aspergillus flavus PEAS-10 or PEASH-12 bacterial suspension, and making the final concentration of spores be 10⁵mL, 30 ℃, 200rpm, cultured for 7 days.

(2) Aflatoxins B in toxigenic culture solutions₁Measurement of (2)

Respectively detecting by adopting the methods of immunoaffinity chromatography purification, liquid chromatography separation and fluorescence detector detectionAFB in the fermentation liquor₁. The specific operation is as follows: passing 2mL of fermentation liquid through an immunoaffinity chromatography column, eluting with 20mL of water for 2 times at a flow rate of 3mL per minute, discarding eluent, allowing air to enter the column, extruding water out of the column, eluting with 1.5mL of methanol for several times, collecting eluent, concentrating to 0.7mL, diluting with water to 1mL, shaking up, loading, separating by high performance liquid chromatography, and detecting by a fluorescence detector.

Chromatographic conditions are as follows: the chromatographic column is Venusil MP C18(5 μm, 4.6 mm. times.150 mm); the column temperature was 40 ℃; the mobile phase is methanol and water (V: V ═ 45: 55); the flow rate is 1.3 mL/min; post-column photochemical derivatization: 254nm for photochemical derivitizer; the fluorescence detector is used for detecting the fluorescence, the excitation wavelength is 360nm, the emission wavelength is 450nm, and the sample injection amount is 20 mu L. The results are shown in FIG. 1.

Aflatoxin is not detected in the aflatoxin-producing fermentation broth of both the aspergillus flavus strain PEAS-10 and the aspergillus flavus strain PEASH-12, and the aspergillus flavus strain PEAS-10 and the aspergillus flavus strain PEASH-12 are further proved to be non-toxigenic strains.

Example 3

Antagonistic bacterium agent for preventing and controlling aflatoxin pollution and preparation method thereof

(1) The microorganisms used were: aspergillus flavus PEAS-10 and Aspergillus flavus PEASH-12

(2) Activating strains: the strains are respectively inoculated on an MEA culture medium and cultured for 3-5 days at the temperature of 30 ℃ until yellow green spores are generated.

(3) Preparation of a microbial inoculum culture medium: crushing peanut shell to about 0.5 multiplied by 0.5cm, mixing the peanut shell and distilled water according to the mass ratio of 1: 1-2: 3, and simultaneously adding 1-1.5 mass percent of CaCl₂And 0.5% KCl, sterilized at 121 ℃ for 20 min.

(3) Respectively inoculating the activated non-toxigenic aspergillus strains to a sterilized microbial inoculum culture medium, culturing for 5-8 days at 30 ℃, and shaking once every day to ensure that the aspergillus flavus grows uniformly on the culture medium; after culturing for 5-8 days, the number of aspergillus flavus spores reaches 10⁸More than one strain per gram of culture medium.

(4) Mixing the cultured culture medium containing Aspergillus flavus PEAS-10 and Aspergillus flavus PEASH-12 according to a certain proportion, and finally, controlling the spore number ratio (PEAS-10: PEASH-12) of the two strains to be 2: 3 to prepare the microbial inoculum for preventing and controlling the aflatoxin pollution. Preserving at normal temperature.

The antagonistic bacteria agent of the invention has the effect of inhibiting the aspergillus flavus from producing toxin

1. Inhibition test in laboratory

1) Test method

(1) Preparation of the culture Medium

Intact corn and peanut granules are selected, 10g of even size peanuts and corn are weighed separately and sterilized at 121 ℃ for 15 minutes.

(2) Preparation of the bacterial suspension

Inoculating Aspergillus flavus (Aspergillus flavus NRRL 3357 standard strain (Aspergillus flavus NRRL 3357 provided by professor Homoba Hayata of Zhongshan university) on MEA slant tube culture medium, culturing at 20 deg.C for 5 days, dipping spores on the culture medium with cotton swab in sterile water, shaking with vortex oscillator, and adjusting spore concentration to 2 × 10 with blood counting cell plate⁴Spores/ml for use.

Weighing 0.1g of microbial inoculum for preventing and controlling aflatoxin pollution in sterile water, uniformly oscillating by using a vortex oscillator, and then adjusting the spore concentration to 2 x 10 by using a blood counting chamber⁴Spores/ml for use.

(3) Test for inhibitory Effect

Adding the diluted 1ml of antagonistic bacteria and the toxin-producing Aspergillus flavus (10) into the triangular flask respectively⁴:10⁴) Spore suspension was used as experimental group. Then 1ml of toxigenic bacteria (10) was added to the flask⁴) An isovolumetrically mixed spore suspension with sterile water was used as a positive control and the bottles were gently shaken to cover the spores onto peanuts and corn. Each was cultured in triplicate at 30 ℃ for 14 days in the dark.

(4) Determination of aflatoxin content

Placing the cultured corn and peanut samples into an autoclave, and sterilizing at 121 deg.C for 30min (to inactivate Aspergillus flavus); the sterilized sample is put into a high-speed universal pulverizer to be smashed, then 50ml of 80% methanol is added into a triangular flask, the mixture is vibrated for 30min at a high speed by an oscillator, and then the filtered extract is measured by HPLC by using sterilized filter paper.

2) Test results

TABLE 1 Effect of antagonistic bacteria on inhibiting toxigenic bacteria

As can be seen from table 1, the antagonistic antibacterial agent has an inhibition toxin production rate of 86.45% for the toxigenic bacteria in the peanuts and an inhibition toxin production rate of 88.21% for the toxigenic bacteria in the corns, and the antagonistic antibacterial agent can well inhibit the toxigenic bacteria. While PEAS-10 alone inhibits the toxigenicity rate in peanuts by 74.02% and in corn by 81.19%; the single PEASH-12 has the poison production inhibition rate of 83.81% in peanuts and 80.33% in corns, so that the composite antagonistic bacteria agent has the advantage of obviously improving the poison production inhibition rate compared with single bacteria.

2. In the field

1) Test method

And (3) after 1 month before the peanuts are harvested, scattering the aspergillus flavus toxigenic bacteria antagonistic antibacterial agent (the aspergillus flavus toxigenic bacteria antagonistic antibacterial agent prepared in the example 3) at the rhizosphere of the peanuts by 30 kg/mu, taking a group without the application of the antagonistic antibacterial agent as a blank control group, and keeping the other daily management test groups as the same as the blank control group.

And taking

soil samples

10 and 20 days after the antagonist is applied until the soil samples are harvested once respectively, detecting the quantity of thalli in the soil samples, separating and identifying aspergillus flavus, and comparing the quantity of the aspergillus flavus in the soil samples after the antagonist is applied and the proportion change condition of the toxin-producing aspergillus flavus.

2) Analysis of reproductive capacity of non-toxigenic aspergillus flavus in soil

TABLE 2 changes in the amount and ratio of Aspergillus flavus in soil after administration of the antagonist

As can be seen from Table 2, the number of Aspergillus flavus colonies in the soil of the control group (no application of the antagonistic antibacterial agent) was 213.45cfu/g, and the proportion of the toxigenic Aspergillus flavus was 70.23%, whereas the number of Aspergillus flavus colonies in the soil was rapidly increased from 6874.10cfu/g after the application of the antagonistic antibacterial agent for 10 days, and the soil Aspergillus flavus was rapidly increased, and the proportion of the toxigenic bacteria was rapidly decreased to 1.11%; after 20 days of applying the strain, the colony number of the aspergillus flavus in the soil reaches 8857.21cfu/g, and the proportion of the toxin-producing aspergillus flavus is reduced to 0.92 percent; when the strain is harvested, the colony number of the aspergillus flavus in the soil reaches 9087.45cfu/g, and the proportion of the aspergillus flavus producing the toxin is reduced to 0.78%;

from the results, the bacteria which do not produce poison grow and reproduce in the soil rapidly after the antagonistic antibacterial agent is applied, the colony number of the aspergillus flavus in the soil increases rapidly after 20 days of the application of the bacteria, and then the growth tends to be slow, which indicates that the effect of applying the bacteria which do not produce poison is the best 20 days before the peanut is harvested after the application of the bacteria; meanwhile, after the non-toxigenic bacteria are applied, the non-toxigenic bacteria can rapidly grow and propagate in the peanut soil, the growth and propagation of the non-toxigenic aspergillus flavus can be competitively inhibited, the proportion of the toxigenic bacteria is reduced, experiments show that after the non-toxigenic bacteria are applied, the proportion of the toxigenic bacteria is reduced from 70.23% of a control group to 0.78% before harvesting, and the proportion of the toxigenic bacteria is rapidly reduced, so that the proportion of the toxigenic bacteria infecting peanuts is reduced, and the pollution risk of the peanut aflatoxin is reduced.

3) Prevention and control of peanut root rot

When the peanuts are harvested, the morbidity of the peanut root rot of the control group and the peanuts applied with the antagonistic bacteria agent is counted, the incidence rate of the root rot is determined by taking the root rot pathogenic strain/total peanut strain, and the result is shown in table 3.

TABLE 3 peanut root rot onset after administration of antagonistic bacterial agents

Group of	Incidence of root rot (%)
		Control group	18.21
Antagonistic bacteria agent group	2.03

As can be seen from Table 3, the incidence of peanut root rot after the application of the antagonistic antibacterial agent is reduced to 2.03% from 18.21% of the control group, and the analysis reason is that the antagonistic antibacterial agent can inhibit the peanut root rot while inhibiting the aspergillus flavus producing the toxin.

4) Prevention and control of peanut stem rot

When the peanuts are harvested, the morbidity of the stem rot of the peanuts in the control group and the peanuts after the antagonistic bacteria agent is applied is counted, the incidence rate of the stem rot is determined by taking the stem rot pathogenic strain/total peanut strain as the incidence rate, and the result is shown in a table 4.

TABLE 4 peanut root rot onset after administration of antagonistic bacterial agents

Group of	Incidence of Stem rot (%)
		Control group	9.78
Antagonistic bacteria agent group	1.03

As can be seen from Table 4, the incidence of peanut root rot after administration of the antagonistic bacteria was reduced from 9.78% to 1.03% in the control group.

5) Peanut storage and toxin determination

After the peanuts are harvested, each seed sample is independently aired and weighed, and the seeds are respectively put into seed bags and stored in a dry and cool place. And (3) measuring the aflatoxin content of the peanuts stored for 0, 1, 2, 3, 4, 5, 6, 7 and 8 months, and calculating the capability of the aflatoxin which does not produce the toxin to inhibit the production of the aflatoxin in the peanuts compared with a control group.

TABLE 5 changes in aflatoxin content in peanuts during storage

As can be seen from Table 5, the control group was peanuts in a peanut field without the antagonist of the present invention, aflatoxins could be detected at the time of harvest, and as the storage time was extended, the aflatoxins content was 20.45. mu.g/kg at five months of storage, which exceeded the national limit standard of 20. mu.g/kg, and the aflatoxins were out of the range of standard and could not be eaten. The content of aflatoxin in the peanut of the control group increases rapidly with the prolonging of the storage period, and reaches 100.45 mug/kg by the eighth month.

The test group can not detect the aflatoxin within 7 months of storage time, which shows that the risk of aflatoxin infection in the peanut storage process can be obviously reduced by applying the antagonistic antibacterial agent to the peanut planting field. The aflatoxin can be detected in the 6 th month of storage of peanuts treated by a single PEAS-10 microbial inoculum or a single PEASH-12 microbial inoculum; the compound antagonistic antibacterial agent can effectively reduce the content of aflatoxin in the peanuts and prolong the storage period of the peanuts.

6) Effect of peanut yield

The peanut yield of the control group and the peanuts after the antagonistic bacteria agent is applied are counted at the time of peanut harvest, and the results are shown in a table 6.

TABLE 6 Effect of peanut yield following administration of antagonists

Group of	Peanut yield (kg/666.67 m)²)
		Control group	259.32
Antagonistic bacteria agent group	324.15

As can be seen from Table 6, the peanut yield after administration of the antagonist was from 259.32kg/666.67m of the control²The temperature is increased to 324.15kg/666.67m²The yield is increased by 25 percent, because the pollution of the aspergillus flavus is reduced by the application of the antagonistic antibacterial agent, and the quantity of bad fruits is reduced; in addition, the antagonistic antibacterial agent contains peanut shells, and the peanut shells are scattered in the field, so that the organic matters in the soil can be increased, and the peanut yield is improved.

Secondly, the influence of the culture method on the aspergillus flavus which does not produce toxin

1. Influence of microbial inoculum culture medium on growth and reproduction of aspergillus flavus not producing toxin

Test groups:

(3) Preparation of a microbial inoculum culture medium: crushing peanut shell to about 0.5 multiplied by 0.5cm, mixing the peanut shell and distilled water according to the mass ratio of 2: 3, and simultaneously adding 1 percent of CaCl by mass₂And 0.5% KCl, sterilized at 121 ℃ for 20 min.

(3) Respectively inoculating the activated non-toxigenic aspergillus strains to a sterilized microbial inoculum culture medium, culturing at 30 ℃, shaking once every day to enable the aspergillus flavus to be culturedThe growth is uniform on the substrate; after culturing for 5 days, detecting that the number of aspergillus flavus spores is more than or equal to 10⁸One/g medium.

Control group 1:

the microorganisms used were: aspergillus flavus PEAS-10 and Aspergillus flavus PEASH-12;

activating strains: the strains are respectively inoculated on an MEA culture medium and cultured for 3-5 days at the temperature of 30 ℃ until yellow green spores are generated.

Preparation of a microbial inoculum culture medium: pulverizing peanut shell into 0.5 × 0.5cm, mixing with distilled water at a mass ratio of 2: 3, and sterilizing at 121 deg.C for 20 min.

Respectively inoculating the activated non-toxigenic aspergillus strains to a sterilized microbial inoculum culture medium, culturing at 30 ℃, shaking once every day to ensure that the aspergillus flavus grows uniformly on the culture medium; after culturing for 7 days, detecting the number of aspergillus flavus spores to be more than or equal to 10⁸One/g medium.

Mixing the cultured culture medium containing Aspergillus flavus PEAS-10 and Aspergillus flavus PEASH-12 according to a certain proportion, and finally, controlling the spore number ratio (PEAS-10: PEASH-12) of the two strains to be 2: 3 to prepare the microbial inoculum for preventing and controlling the aflatoxin pollution. Preserving at normal temperature.

Control group 2:

Preparation of a microbial inoculum culture medium: mixing the wheat and the distilled water according to the mass ratio of 2: 3, and simultaneously adding 1 percent of CaCl by mass₂And 0.5% KCl, sterilized at 121 ℃ for 20 min.

Respectively inoculating the activated non-toxigenic aspergillus strains after sterilizationCulturing the aspergillus flavus on the microbial inoculum culture medium at 30 ℃ and shaking once a day to ensure that the aspergillus flavus grows uniformly on the culture medium; after 8 days of culture, the number of aspergillus flavus spores is detected to be more than or equal to 10⁸One/g medium.

The results show that the microbial inoculum culture medium used by the invention is beneficial to the growth and the propagation of the aspergillus flavus without producing the toxin, and the culture time for reaching the aspergillus flavus spores with effective concentration is short.

2. Influence of microbial inoculum culture medium on field viability of non-toxigenic aspergillus flavus

(1) Test group

And (3) after the peanuts are harvested for 1 month, scattering the aspergillus flavus toxigenic bacteria antagonistic bacteria prepared by the test group at the rhizosphere of the peanuts by 30 kg/mu, and taking a group without applying the antagonistic bacteria as a blank control group, wherein other daily management test groups are the same as the blank control group.

And taking a soil sample 30 days after the antagonist is applied, detecting the quantity of thalli in the soil sample, separating and identifying the aspergillus flavus, and comparing the quantity of the aspergillus flavus in the soil sample after the antagonist is applied and the proportion change condition of the toxin-producing aspergillus flavus.

(2) Control group 1

And (3) after the peanuts are harvested for 1 month, scattering the aspergillus flavus toxigenic bacteria antagonistic antibacterial agent prepared by the control group 1 at the rhizosphere of the peanuts with the dosage of 30 kg/mu, and performing other daily management in the same way as the step (1).

(3) Control group 2

And (3) after the peanuts are harvested for 1 month, scattering the aspergillus flavus toxigenic bacteria antagonistic antibacterial agent prepared by the control group 2 at the rhizosphere of the peanuts with the dosage of 30 kg/mu, and performing other daily management in the same way as the step (1).

And taking a soil sample once 30 days after the antagonistic bacteria agent is applied, detecting the quantity of thalli in the soil sample, separating and identifying the aspergillus flavus, and comparing the quantity of the aspergillus flavus in the soil sample after the antagonistic bacteria agent is applied and the proportion change condition of the toxin-producing aspergillus flavus.

The results are shown in Table 7.

TABLE 7 Aspergillus flavus amount and ratio change after applying antagonistic bacteria agent cultured by different culture methods

The results in Table 7 show that the aspergillus flavus cultured by the method does not produce toxic aspergillus flavus, has strong field viability and good inhibition effect on the toxic aspergillus flavus.

Influence of the antagonist prepared by the invention on the utilization rate of the organic fertilizer

Test groups:

(3) Respectively inoculating the activated non-toxigenic aspergillus strains to a sterilized microbial inoculum culture medium, culturing at 30 ℃, shaking once every day to ensure that the aspergillus flavus grows uniformly on the culture medium; after culturing for 5 days, detecting that the number of aspergillus flavus spores is more than or equal to 10⁸One/g medium.

Blank control group: will be provided withCrushing peanut shell to about 0.5 multiplied by 0.5cm, mixing the peanut shell and distilled water according to the mass ratio of 2: 3, and simultaneously adding 1 percent of CaCl by mass₂And 0.5% KCl, sterilized at 121 deg.C for 20min, cultured at 30 deg.C for 5 days, and shaken once a day.

And (3) scattering the antagonistic agent of the experimental group at the peanut rhizosphere position by 30 kg/mu 1 month before the peanut is harvested, meanwhile, scattering the culture medium of the control group at the peanut rhizosphere position by 30 kg/mu to serve as a blank control group, and keeping the other daily management test groups and the control group to be the same. Immediately collecting the soil of the test group and the control group after applying the microbial inoculum, and measuring the content of organic matters.

The soil of the test group and the control group was collected at the time of harvesting the peanuts, the organic matter content in the soil was measured, and the organic matter utilization ratio of the test group and the control group was calculated (organic matter utilization ratio/% (organic matter content in soil at the time of harvesting/organic matter content in soil immediately after applying the microbes) × 100), and the results are shown in table 8.

TABLE 8 utilization of organic matter in soil after administration of antagonistic agents

Group of	Organic matter utilization (%)
		Control group	58
Antagonistic bacteria agent group	84

As can be seen from table 8, the utilization rate of organic matters in the soil after the application of the antagonistic bacteria is significantly increased compared with that of the control group, which indicates that the antagonistic bacteria prepared by the invention not only can reduce the occurrence of peanut diseases, but also can increase the utilization rate of organic matters in the soil and improve the yield of peanuts.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Sequence listing

<110> institute for peanut research in Shandong province

<120> antagonistic bacterium agent for producing aspergillus flavus, preparation method and application thereof

<130> 2019

<141> 2019-05-28

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> Artificial sequence (Aspergillus flavus)

<400> 1

tccgtaggtg aacctgcgg 19

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Aspergillus flavus)

<400> 2

tcctccgctt attgatatgc 20

<210> 3

<211> 574

<212> DNA

<213> Aspergillus flavus PEAS-10

<400> 3

acctgcggaa ggatcattac cgagtgtagg gttcctagcg agcccaacct cccacccgtg 60

tttactgtac cttagttgct tcggcgggcc cgccattcat ggccgccggg ggctctcagc 120

cccgggcccg cgcccgccgg agacaccacg aactctgtct gatctagtga agtctgagtt 180

gattgtatcg caatcagtta aaactttcaa caatggatct cttggttccg gcatcgatga 240

agaacgcagc gaaatgcgat aactagtgtg aattgcagaa ttccgtgaat catcgagtct 300

ttgaacgcac attgcgcccc ctggtattcc ggggggcatg cctgtccgag cgtcattgct 360

gcccatcaag cacggcttgt gtgttgggtc gtcgtcccct ctccgggggg gacgggcccc 420

aaaggcagcg gcggcaccgc gtccgatcct cgagcgtatg gggctttgtc acccgctctg 480

taggcccggc cggcgcttgc cgaacgcaaa tcaatctttt tccaggttga cctcggatca 540

ggtagggata cccgctgaac ttaagcatat caat 574

<210> 4

<211> 574

<212> DNA

<213> Aspergillus flavus PEASH-12

<400> 4

gacctgcgga aggatcatta ccgagtgtag ggttcctagc gagcccaacc tcccacccgt 60

gtttactgta ccttagttgc ttcggcgggc ccgccattca tggccgccgg gggctctcag 120

ccccgggccc gcgcccgccg gagacaccac gaactctgtc tgatctagtg aagtctgagt 180

tgattgtatc gcaatcagtt aaaactttca acaatggatc tcttggttcc ggcatcgatg 240

aagaacgcag cgaaatgcga taactagtgt gaattgcaga attccgtgaa tcatcgagtc 300

tttgaacgca cattgcgccc cctggtattc cggggggcat gcctgtccga gcgtcattgc 360

tgcccatcaa gcacggcttg tgtgttgggt cgtcgtcccc tctccggggg ggacgggccc 420

caaaggcagc ggcggcaccg cgtccgatcc tcgagcgtat ggggctttgt cacccgctct 480

gtaggcccgg ccggcgcttg ccgaacgcaa atcaatcttt ttccaggttg acctcggatc 540

aggtagggat acccgctgaa cttaagcata tcat 574

Claims

1. An antagonistic bacterium agent for producing aspergillus flavus, which is characterized in that: the effective components of the aspergillus flavus strain are aspergillus flavus PEAS-10 which does not produce aflatoxin and aspergillus flavus PEASH-12 which does not produce aflatoxin;

the aspergillus flavus PEASH-12 which does not produce toxin is preserved in the following period of 2018, 08 and 01 months: china general microbiological culture Collection center (CGMCC) with the collection number of CGMCC NO:15998 and the address of: west road No. 1, north west of chaoyang district, beijing, the requested preservation unit is peanut institute of shandong province;

the preparation method of the toxin-producing aspergillus flavus antagonistic antibacterial agent comprises the following steps:

(3) mixing the culture medium containing Aspergillus flavus PEAS-10 and Aspergillus flavus PEASH-12 cultured in the step (2) according to a certain proportion, and finally obtaining the antagonistic bacteria agent for producing the toxic Aspergillus flavus by the spore number proportion of the two strains being PEAS-10 to PEASH-12 which is 1: 1-1: 3;

the microbial inoculum culture medium is prepared by the following method:

crushing peanut shell to 0.5 multiplied by 0.5cm, mixing according to the mass ratio of 1: 1-2: 3 of the peanut shell and distilled water, and simultaneously adding 1-1.5 percent of CaCl by mass percent₂And 0.5% KCl, sterilized at 121 ℃ for 20 min.

2. The antagonistic antibacterial agent against aspergillus flavus producing a toxin according to claim 1, characterized in that: the number of spores of Aspergillus flavus PEAS-10 which does not produce aflatoxin in the antagonistic antibacterial agent is more than or equal to 10⁸Per gram; the number of spores of Aspergillus flavus PEASH-12 which does not produce aflatoxin is more than or equal to 10⁸Per gram.

3. The toxin producing aspergillus flavus antagonistic antibacterial agent as claimed in claim 1, characterized in that: the mixing ratio of spores of aspergillus flavus PEAS-10 and aspergillus flavus PEASH-12 in the toxin-producing aspergillus flavus antagonistic bacteria agent is 2: 3.

4. The application of the antagonistic antibacterial agent for producing the toxic aspergillus flavus of any one of claims 1 to 3 is characterized in that the antagonistic antibacterial agent is used for inhibiting the growth and the toxicity production of the aspergillus flavus, reducing the diseases of crops, improving the utilization rate of organic fertilizers, improving the yield of the crops, reducing the content of the aflatoxin in agricultural products during harvesting and prolonging the storage period of the agricultural products.

5. Use according to claim 4, characterized in that: the crops are peanuts or corns.

6. A method for inhibiting growth and toxin production of aspergillus flavus is characterized by comprising the following steps: spraying the toxic aspergillus flavus antagonistic bacterium producing agent as claimed in any one of claims 1 to 3 to the rhizosphere of crops at 30 kg/mu 1 month before the crops are harvested.

7. A method for reducing crop diseases, improving the utilization rate of organic fertilizers or improving the crop yield is characterized in that:

spraying the toxicity-producing aspergillus flavus antagonistic antibacterial agent as claimed in any one of claims 1 to 3 to the rhizosphere of crops at 30 kg/mu 1 month before the crops are harvested, harvesting in due time, airing and storing in dry and cool places.

8. A method of reducing aflatoxin content or extending the shelf life of agricultural products at harvest, comprising: spraying the toxicity-producing aspergillus flavus antagonistic antibacterial agent as claimed in any one of claims 1 to 3 to the rhizosphere of crops at 30 kg/mu 1 month before the crops are harvested, harvesting in due time, airing and storing in dry and cool places.