CN113846020B - Aspergillus flavus XZCY1805 without toxicity production and application thereof - Google Patents
Aspergillus flavus XZCY1805 without toxicity production and application thereof Download PDFInfo
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Abstract
The invention relates to a non-toxic aspergillus flavus strain and application thereof. The non-toxic aspergillus flavus XZCY1805 does not produce various toxins such as Aflatoxin (AFT), cyclopia anitic acid (CPA), aflatoxin (Aflatrem), and versicolor aflatoxin (ST), and is a biocontrol strain with high safety and strong competitive advantage. The non-toxic aspergillus flavus XZCY1805 has strong toxicity inhibition effect on standard toxic aspergillus flavus strain 3.4408 and toxic aspergillus flavus separated from different peanut producing places, and can remarkably reduce the contents of aflatoxin, cyclopinic acid, aflatoxin and versicolor aflatoxin in agricultural products such as peanuts.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to non-toxic aspergillus flavus XZCY1805 and application thereof.
Background
Aflatoxin (AFT) is a secondary metabolite generated after host plants are infected by fungi such as aspergillus flavus (Aspergillus flavus) and aspergillus parasiticus (Aspergillus parasiticus), is the most toxic mycotoxin for polluting agricultural products, has acute and chronic toxicity, and can be used for cancerogenic, teratogenic and mutagenic. The common species of aflatoxins mainly comprise aflatoxin B 1 (AFB 1 ) Aflatoxin B 2 (AFB 2 ) Aflatoxin G 1 (AFG 1 ) Aflatoxin G 2 (AFG 2 ). The aflatoxin B has the greatest toxicity and the strongest carcinogenicity 1 The toxicity is 10 times of potassium cyanide and 68 times of arsenic, the carcinogenicity is 10000 times of six and six, and the cancer is classified as a class I carcinogen by the International organization for cancer research (International Agency for Research on Cancer, IARC) of the world health organization (World Health Organization, WHO). In addition to aflatoxin, aspergillus flavus can also produce toxic secondary metabolites such as cyclopia anitic acid (Cyclopiazonic acid, CPA), aflatoxin (afatrem), variegated aflatoxin (ST), and the like, which threatens the safety of consumption and the life and health of people. CPA is an indole derivative and has toxic effects on humans and various animals, including liver degeneration and necrosis, myocardial injury, kidney injury, and the like. Aspergillus flavus tremorine is an indole diterpenoid mycotoxin, has tumorigenic properties and acute neurotoxic effects, and can cause tremor of animals. The aflatoxin is an intermediate product in the later stage of the process of synthesizing the aflatoxin by the aspergillus flavus, and the toxicity and carcinogenicity of the aflatoxin are highly valued in various countries in the world because the aflatoxin has a similar structure. In the areas with serious aflatoxin pollution in China, agricultural products such as peanuts, corns, rice, nuts and the like and foods are reported to be detected, and mixed pollution phenomena of the agricultural products, such as cyclopia anioic acid, aflatoxin and the like exist, so that the agricultural product consumption safety in China is threatened, and the industrial development and export trade are limited. Wherein the peanut and the corn are most easily infected by fungi such as aspergillus flavus and aflatoxinToxin contamination. Therefore, researching field prevention and control of aspergillus flavus pollution of agricultural products reduces aspergillus flavus infection from the source and mycotoxin pollution generated by the aspergillus flavus infection, and has important significance for guaranteeing quality safety of agricultural products in China and improving international competitiveness of agricultural products in China.
The biological prevention and control method has the advantages of no damage to the original quality of agricultural products, safety, high efficiency, environmental protection and the like, and is an effective way for green control of aspergillus flavus and aflatoxin. The non-toxic aspergillus flavus is utilized to competitively inhibit the growth and colonization of field toxic aspergillus flavus, so that the number of toxic bacteria is reduced, the pollution level of the aflatoxin is reduced, and the method is a very effective biological control method for controlling the aflatoxin pollution in agricultural products such as peanuts, corns and the like at the source. The research of the non-toxic aspergillus flavus biocontrol bacteria in China is in the beginning stage, and no cases of popularization and application in fields exist. The prior disclosure is a biological control report with high safety for non-toxic aspergillus flavus used as biological control and the application thereof (such as CN 110157626A, CN 110305796A, CN 110129212A, CN 107177516 and CN 104789480A), but aiming at the non-toxic metabolites such as non-aflatoxin, non-cyclic apigenin acid, non-aflatoxin, heterochromatic aflatoxin and the like, which can bring about the pollution risk of mycotoxins such as cyclic apigenin acid, aflatoxin and the like after the bacterial strains are applied in fields. It has been found that after non-toxic Aspergillus flavus AF36 (Afla-/CPA+) is applied as a biocontrol agent, the content of cyclopia animate acid in corn and peanut is increased, which brings new risk potential. Therefore, the aspergillus flavus with high safety, which does not produce various toxins such as aflatoxin, cyclopia anitic acid, aflatoxin and the like, is screened, developed and utilized, toxin pollution prevention and control are carried out before crop harvest, and the method has wide application prospect in the aspects of effectively reducing the risk of the aflatoxin, the cyclopia anitic acid, the aflatoxin and the like entering a food chain and improving the quality and safety level of agricultural products. Through a large number of experimental work, 1 strain of non-toxic aspergillus flavus XZCY1805 which does not produce secondary metabolites such as aflatoxin, cyclopia anipulic acid, aflatoxin, and versicolor aflatoxin is screened from 97 strains which do not produce aflatoxin, and the non-toxic aspergillus flavus XZCY1805 has strong competitive advantage, and can obviously reduce the pollution risk of aflatoxin, cyclopia anipulic acid, aflatoxin, versicolor aflatoxin and other mycotoxins in agricultural products such as peanuts.
Disclosure of Invention
The invention aims to solve the technical problem of providing a non-toxic aspergillus flavus strain which does not produce various toxins such as aflatoxin, cyclopia animic acid, aflatoxin, versicolor aflatoxin and the like and application thereof.
The non-toxic aspergillus flavus strain XZCY1805 (Aspergillus flavus) is preserved in China center for type culture collection (abbreviated CCTCC) in 9 months and 18 days in 2020, and the preservation number is CCTCC NO: M2020521, and the preservation address is: chinese, university of martial arts, martial arts.
The non-toxic aspergillus flavus strain XZCY1805 is separated from a Tibetan-style peanut planting field.
Screening: the aspergillus flavus XZCY1805 is screened from 1600 aspergillus flavus strains through strain toxigenic capability evaluation and biocontrol inhibition effect test, and the process is as follows: collecting more than 2000 parts of peanut and soil samples from the northeast, northern, yangtze river basin and southern peanut main producing areas of China, and obtaining 1600 strains of aspergillus flavus through separation, purification and identification; after the toxin production capacity of the strain is measured, 97 strains of non-toxin-producing aspergillus flavus (mainly separated from 15 peanut planting places such as Henan, shandong, hubei, jiangxi, tibet and the like) are selected to be respectively co-cultured with a toxin-producing standard strain 3.4408 on peanut powder and evaluated for toxin production inhibition effect, and 11 strains with obvious inhibition effect on the toxin-producing aspergillus flavus are initially screened out; further co-culturing 11 strains of non-toxic aspergillus flavus with the toxic aspergillus flavus bacteria separated from different areas (Liaoning Fuxin, hebei Tang mountain, sichuan peng 'an, tibet's corner, hubei Hongan, hubei yang patrol and Jiangxi camphor tree) on peanut powder and peanut kernels, evaluating the toxic inhibition effect, and screening out 3 strains with obvious competitive inhibition effects on the toxic strains from different geographical sources; the 3 strains of non-toxic aspergillus flavus are further subjected to determination of toxic secondary metabolites such as aflatoxin, cyclopia anioni acid, aflatoxin, versicolor aflatoxin and the like, and 1 strain XZCY1805 which does not produce aflatoxin, cyclopia anioni acid, aflatoxin and versicolor aflatoxin is screened out, so that the growth and reproductive capacity of the non-toxic aspergillus flavus can be obviously competitive inhibited, and the content of aflatoxin, cyclopia anioni acid, aflatoxin and versicolor aflatoxin in agricultural products such as peanuts can be reduced.
The non-toxic aspergillus flavus strain XZCY1805 has the characteristics of rapid growth and reproduction, has strong competitive advantage, can obviously compete and inhibit the growth and reproduction of toxic aspergillus flavus, and reduces the content of mycotoxins such as aflatoxin, cyclopinic acid, aflatoxin, and versicolor aflatoxin in agricultural products such as peanuts. AFB of non-toxigenic bacteria XZCY1805 against toxigenic bacteria 3.4408 in peanut powder 1 The toxigenic inhibition rate of (2) is 99.86%, and the toxigenic inhibition rate is 99.86% for AFT and AFB isolated from toxigenic strains of different producing areas 1 The toxin production inhibition rate ranges of 81.5 to 94.4 percent and 84.5 to 98.2 percent respectively; the total amount of aflatoxin in peanut kernels is reduced by 51.5% -97.8%, and aflatoxin B 1 The content is reduced by 51.2 to 100.0 percent; potting test of peanut field, AFT and AFB 1 The toxicity production inhibition effect of the composition is remarkable, and the inhibition rates are 76.5% and 68.0% respectively. The non-toxigenic bacteria XZCY1805 has the capability of effectively competing to inhibit the growth and reproduction of toxigenic bacteria and reducing the probability of the toxigenic bacteria infecting crops, thereby reducing the content of mycotoxins such as aflatoxin, cyclopia anioic acid, aflatoxin, versicolor aflatoxin and the like in agricultural products. Compared with other Aspergillus flavus biocontrol bacteria which do not produce aflatoxin, the biological control bacteria do not produce multiple toxins such as aflatoxin, cyclopia animic acid, aflatoxin, versicolor aflatoxin and the like, and have higher safety.
The colony morphology of the non-toxic aspergillus flavus strain XZCY1805 is as follows: the strain produced white hyphae and yellowish green spores on PDA medium, yellowish green spores on DG-18 medium, and bright orange characteristic colonies on the back of AFPA medium. After culturing in DG-18 medium at 28+ -1deg.C for 5 days, the colony diameter is 7.73cm, and the spore yield is 5.5X10 8 In particular, the strain was compared with the standard strain 3.4408 (colony diameter 7.0cm, spore yield 4.25X10) of Aspergillus flavus under the same conditions 8 And so on) is stronger in growth and reproductive ability.
The method for inhibiting the generation of aflatoxin by using the non-toxic aspergillus flavus strain XZCY1805 is provided, and the non-toxic aspergillus flavus strain XZCY1805 is inoculated on a biological sample to inhibit the generation of mycotoxin.
The biological sample can be peanut powder or peanut kernels.
According to the scheme, the mycotoxins comprise aflatoxin, cyclopia anioic acid, aflatoxin, omutzfeldin and the like, and the aflatoxin comprises aflatoxin B 1 Aflatoxin B 2 Aflatoxin G 1 And aflatoxin G 2 。
Specifically, the non-toxic aspergillus flavus strain XZCY1805 is activated in DG-18 solid culture medium, cultured for 5 days under the dark condition of 28+/-1 ℃, and then the conidium on the surface of the culture medium is washed by sterilized 0.1% Tween 80, and the conidium suspension is obtained by dilution with sterile water for use.
According to the above scheme, the concentration of the spore suspension can be 1×10 6 And each ml.
A method for preventing and reducing the risk of aflatoxin, cyclopia anioic acid, aflatoxin and variegated qutoxin contamination in agricultural products in the field is provided. The aspergillus flavus XZCY1805 conidium suspension is utilized to be spread on the rhizosphere of crops.
According to the scheme, the spore concentration of the aspergillus flavus XZCY1805 suspension is preferably 1 multiplied by 10 6 The dosage is preferably 80L/mu.
According to the scheme, the agricultural product is peanut, and the application time is preferably the peanut flowering and needle-setting period.
The invention has the beneficial effects that:
(1) The non-toxic aspergillus flavus XZCY1805 has strong toxin production inhibition effect on standard toxic aspergillus flavus strain 3.4408 and the toxic aspergillus flavus separated from different flower production places, is a biocontrol strain with high safety and strong competitive advantage, can obviously inhibit the toxic aspergillus flavus from infecting crops, and reduces the pollution of aflatoxin, cyclopia anioic acid, aflatoxin, versicolor aflatoxin and other mycotoxins in agricultural products such as peanuts. Has remarkable toxicity-producing inhibition effect on peanut powder, peanut kernels and peanut seedlings.
(2) The non-toxic aspergillus flavus XZCY1805 does not produce Aflatoxin (AFT), does not produce various toxins such as cyclopia anib (CPA), aflatoxin (ST), aflatoxin (Aflatrem) and the like, and is a biocontrol strain with high safety. The method has the characteristics of rapid growth and reproduction, has strong competitive inhibition capability, can obviously compete and inhibit the growth and reproduction of the toxin-producing aspergillus flavus, and reduces the content of mycotoxins such as aflatoxin, cyclopia anioic acid, aflatoxin, versicolor aflatoxin and the like.
Drawings
Table 1: non-toxic aspergillus flavus XZCY1805 has the toxicity producing property;
table 2: the aspergillus flavus XZCY1805 without toxin production has the effect of inhibiting the toxin production of aspergillus flavus on peanut powder;
table 3: non-toxic aspergillus flavus XZCY1805 for producing AFT and AFB on peanut kernels 1 An inhibition effect;
table 4: non-toxic aspergillus flavus XZCY1805 for producing AFG (alpha-fetoprotein) on peanut kernels 1 、AFG 2 Inhibition effect
Table 5: the XZCY1805 with different concentrations has the effect of inhibiting the toxicity of the aspergillus flavus;
table 6: the non-toxic aspergillus flavus XZCY1805 has an effect of inhibiting the aflatoxin content of the potted peanuts;
fig. 1: the strain XZCY1805 grows yellow-green spores on the DG-18 medium and generates bright orange color reaction on the AFPA medium;
fig. 2: non-toxic aspergillus flavus XZCY1805 with different spore concentrations is used for producing toxic aspergillus flavus AFT and AFB 1 Inhibition rate.
Detailed Description
Example 1: isolation, purification and identification of strain XZCY1805
Collecting more than 2000 parts of peanut and soil samples from the northeast, northern, yangtze river basin and southern peanut main producing areas of China, and obtaining 1600 strains of aspergillus flavus through separation, purification and identification; after the aflatoxin production capacity of the strain is measured, 97 strains of non-toxic aspergillus flavus (mainly separated from 15 peanut planting places such as Henan, shandong, hubei, jiangxi and Tibet) are selected for carrying out competition inhibition effect research and toxicity production capacity evaluation: co-culturing peanut powder with a toxigenic standard strain 3.4408 and evaluating toxigenic inhibition effect, and primarily screening 11 strains with obvious inhibition effect on toxigenic aspergillus flavus; further co-culturing 11 strains of non-toxic aspergillus flavus with the toxic aspergillus flavus bacteria separated from different areas (Liaoning Fuxin, hebei Tang mountain, sichuan peng 'an, tibet's corner, hubei Hongan, hubei yang patrol and Jiangxi camphor tree) on peanut powder and peanut kernels, evaluating the toxic inhibition effect, and screening out 3 strains with obvious competitive inhibition effects on the toxic strains from different geographical sources; the 3 strains of non-toxic aspergillus flavus are further subjected to determination of toxic secondary metabolites such as aflatoxin, cyclopia anioni acid, aflatoxin, versicolor aflatoxin and the like, and 1 strain XZCY1805 which does not produce aflatoxin, cyclopia anioni acid, aflatoxin and versicolor aflatoxin is screened out, so that the growth and reproductive capacity of the non-toxic aspergillus flavus can be obviously competitive inhibited, and the content of the aflatoxin, cyclopia anioni acid, aflatoxin and versicolor aflatoxin in agricultural products such as peanuts can be reduced. The strain XZCY1805 which does not produce aflatoxin, cyclopia acid, aflatoxin and versicolor is separated from Tibet tsuki peanut planting land.
The above sample for screening, for a soil sample, the separation process is: and (3) carrying out treatments such as plant residue removal, large-particle stone removal, soil grinding, uniform mixing and the like on the soil sample. 10.0g of ground soil sample is weighed and added into 90mL of sterilized water, and the mixture is placed into a shaking table to shake for 2 hours to prepare 100mL of sample suspension. mu.L of the suspension was added to DG-18 medium plates and the mixture was spread with a spreading bar. Then the coated flat plate is put into a constant temperature incubator, and cultivated under the conditions of 28+/-1 ℃ and 90% relative humidity and darkness. After 5 days, colonies with yellow-green spores are picked up, transferred to a new DG-18 culture medium plate, and subjected to secondary streak purification culture until single colonies are obtained for later use.
For peanut samples, the separation process was: crushing peanuts to obtain peanut powder, preparing a sample suspension, and separating and purifying by the method to obtain single bacterial colonies for later use. And (3) identification:
morphological identification
The strain XZCY1805 grows yellow-green spores on DG-18 culture medium, the colony diameter after 5 days of culture at 28+/-1 ℃ is 7.73cm, and the spore yield is 5.5X10 8 Accordingly, the colony diameter of Aspergillus flavus standard strain 3.4408 under the same conditions was 7.0cm, and the spore yield was 4.25X10 8 And each. Therefore, the aspergillus flavus XZCY1805 provided by the invention has the advantage of rapid growth and propagation.
Colonies growing with yellow-green spores were picked from DG-18 medium and transferred to selective medium AFPA (aflatoxin agar, aspergillus flavus and Aspergillus parasiticus Agar) for cultivation at 28.+ -. 1 ℃ and relative humidity of 90% under dark conditions. After 5 days the colour of the back of the colonies was observed, producing a bright orange colour reaction on AFPA medium (fig. 1), identified initially as aspergillus flavus or aspergillus parasiticus.
Molecular biological identification
The strain XZCY1805 is subjected to molecular identification by a fungus ITS gene sequence and a calcium regulatory gene sequence.
Primers used for ITS amplification of the genome of Aspergillus flavus are:
ITS1：5′-TCCGTAGGTGAACCTGCGG-3′
ITS4：5′-TCCTCCGCTTATTGATATGC-3′
the primers used for amplifying the gene encoding the calmodulin of the aspergillus flavus genome are as follows:
CF1:5′-AGGCCGAYTCTYTGACYGA-3′
CF4:5′-TTTYTGCATCATRAGYTGGAC-3′
the PCR amplification conditions of the ITS gene sequence are as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s for 35 cycles; finally, the extension is carried out for 10min at 72 ℃. After amplification, the product was stored at 4 ℃. Sequencing was carried out on NCBI website (https:// blast. NCBI. Nlm. Nih. Gov/blast. Cgi) by the Wuhan division of the biological sciences Co., beijing.
The calmodulin encoding gene sequence PCR amplification conditions are: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 57℃for 30s, extension at 72℃for 1min for 35 cycles; finally, the extension is carried out for 10min at 72 ℃. After amplification, the product was stored at 4 ℃. Sequencing to the Wuhan division of Beijing Qinke biotechnology Co., ltd, the sequencing result being at NCBI website
The comparison was carried out on (https:// blast. Ncbi. Nlm. Nih. Gov/blast. Cgi).
Sequencing shows that the ITS sequence of the strain XZCY1805 is shown in SEQ ID No. 1; the calmodulin sequence is shown in SEQ ID No. 2.
The comparison of ITS amplified sequences in NCBI website through BLASTN database shows that the similarity of ITS gene sequence of the strain XZCY1805 and the RNA gene sequence of the ribosomal small subunit of Aspergillus flavus (Aspergillus flavus) is 99.67%. The comparison of the calmodulin coding gene amplified sequence in NCBI website by BLASTN database shows that the calmodulin coding gene sequence of the strain XZCY1805 has 98.86 percent of similarity with the calmodulin A gene sequence of aspergillus flavus (Aspergillus flavus).
The strain XZCY1805 is identified as aspergillus flavus (Aspergillus flavus) by combining morphological and molecular biological identification results.
Example 2: toxigenic analysis of strain XZCY1805
1. Strain toxigenic culture
Inoculating the non-toxic aspergillus flavus strain XZCY1805 into DG-18 solid culture medium for activation. After 5 days of cultivation in dark at 28+ -1deg.C, the Aspergillus flavus conidia on the plate were obtained by washing with sterilized 0.1% Tween 80 to obtain spore suspension. Spore suspension concentrations were determined using a hemocytometer. Pouring a certain amount of spore liquid into a triangular flask containing sterilized 30ml liquid sand culture medium to give final concentration of 4X10 5 And each ml. The flask was placed on a shaker at 28.+ -. 1 ℃ and 200rpm for dark culture.
2. Determination of the toxin content of the Strain
After 5 days, the mycelium pellet culture solution is filtered by using sterile gauze, mycelium pellets are discarded, the toxin-producing culture solution is collected and stored in a centrifuge tube, and the mixture is kept stand for 1h, and 500 mu l of toxin-producing culture solution is removed and centrifuged in 2mlAdding 500 μl of methanol into the tube, uniformly swirling, centrifuging for 10min at 20000rmp, carefully sucking out the supernatant in the tube (preventing the needle from contacting the bottom of the tube) with a 1ml syringe, passing the toxin extract in the syringe through a 0.22 μm organic system filter membrane, and filling into a sample bottle, and measuring the content of toxic metabolites such as aflatoxin, cyclopeanic acid, aflatoxin, and versicolor aflatoxin by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS), wherein the detection results are shown in Table 1. Chromatographic conditions: the chromatographic column is C 18 (100 mm. Times.2.1 mm,3 μm); the column temperature is 40 ℃, the sample injection volume is 2 mu L, and the temperature of a sample tray is 10 ℃; mobile phase a was methanol to water (V: v=5:95), mobile phase B was methanol to water (V: v=5:95), both containing 0.1% formic acid and 10mM ammonium formate, at a flow rate of 0.3ml/min; gradient elution procedure: 0-1min:85A,1-3min:85-50% of A,3-5min: 50-30% of A,5-10min:30-0% of A,10-13min:0% A,13-15min:0-85% of A,15-20min:85% A.
Mass spectrometry conditions: ESI (+) mode: the air curtain air is 20mL/min; the atomizer temperature was 450 ℃; 20mL/min of atomizer; assisted by heating air pressure 40mL/min. The quantification mode is selected ion detection (SIM); analysis software analysis 1.14. Chromatographic column: waters Symmetry C18 column (2.1X105 mm,3.5 μm); mobile phase a:10mM acetic acid in water; mobile phase B: 100% methanol; column temperature 40 ℃; the flow rate is 0.3mL/min; gradient procedure: b increases to 90% at 0-10 min and decreases from 90% to 50% at 10-15 min.
As can be seen from Table 1, strain XZCY1805 does not produce aflatoxin, cyclopia animate acid, versicolor aflatoxin, and aflatoxin.
TABLE 1 toxigenic Properties of non-toxigenic Aspergillus flavus XZCY1805
a AFT is an abbreviation for aflatoxin (aflatoxin B) 1 Aflatoxin B 2 Aflatoxin G 1 And aflatoxin G 2 Is a total amount of (b);
b CPA is cyclopia anib acid; c ST is aflatoxin of versicolorThe method comprises the steps of carrying out a first treatment on the surface of the dA flavem is Aspergillus flavus tremorine;
e ND indicates undetected.
Example 3: inhibition effect of strain XZCY1805 on peanut powder on aspergillus flavus producing toxin
1. Spore suspension preparation
The non-toxic Aspergillus flavus strain XZCY1805, the standard strain 3.4408 and the non-toxic Aspergillus flavus strain Aspergillus flavus separated from different regions are respectively named SCPA-32-12 (Sichuan tarragon), XZCY-24-6 (Tibet Santa Clara), LNFX-25-1 (Liaoning Fuxin), HBHA-129-1 (Hubei Hongan), HBYL-12-7 (Hubei Yang Luo), HBTS-94-2 (Hebei Tangshan) and JXZS-118-9 (Jiangxi camphor tree) which are respectively inoculated into a DG-18 solid culture medium for activation. After 5 days of incubation at 28.+ -. 1 ℃ in the dark, the conidia of A.flavus on the plates were washed with sterilized 0.1% Tween 80 in a 10ml centrifuge tube to give a spore suspension. Determining spore suspension concentration under microscope with a blood cell counting plate, diluting spore concentration to 1×10 with sterile water 6 And (5) each ml of the solution is ready for use.
2. Competition inhibition culture
10.0g of peanut powder was weighed into a sterile petri dish (9 cm), and a control group and a treatment group were set: preparation of Aspergillus toxigenic (spore concentration 1×10) 6 Equal volume of spore suspension (control) mixed with sterile water (250. Mu.l each) and non-toxic Aspergillus flavus XZCY1805 (spore concentration 1X 10) 6 Individual/ml) and Aspergillus flavus standard strain 3.4408 or Aspergillus flavus (spore concentration 1×10) which are isolated from different peanut producing regions 6 Equal volumes of spore suspension (treatment group) were mixed (250 μl each), the mixed spore suspension was inoculated uniformly onto peanut powder, 3 biological replicates were made for each treatment, and the samples were placed in a constant temperature incubator and cultured in continuous darkness at 28 ℃ ±1 ℃ with relative humidity of 90% for 14 days.
3. Aflatoxin content determination
After the culture, transferring peanut powder in a culture dish into a 50ml centrifuge tube, adding 15ml of 70% methanol solution (containing 4% NaCl), mixing uniformly by vortex, placing on a shaker for shaking for 2h, centrifuging at 4500r/min,taking 1ml supernatant, passing through organic filter membrane, and detecting aflatoxin B by High Performance Liquid Chromatography (HPLC) 1 、B 2 、G 1 、G 2 Is contained in the composition. The total Aflatoxin (AFT) is the sum of the above 4 aflatoxins.
HPLC conditions C18 column (4.6 mm. Times.150 mm,5 μm); the column temperature is 35 ℃; mobile phase: methanol: water (V: v=45:55); the flow rate is 0.8mL/min; post-column photochemical derivatization with a photochemical derivatizer of 254nm; fluorescence detector (excitation wavelength 360nm, emission wavelength 440 nm), sample injection amount 10 μl, and measurement time 22min.
The calculation formula of the toxigenic inhibition rate comprises the following steps: inhibition ratio (%) = [1- (treatment group aflatoxin content)/(control group aflatoxin content) ]x100, formula I.
As can be seen from Table 2, the peanut powder is used as the matrix, and the non-toxigenic bacteria XZCY1805 is used for AFB of toxigenic bacteria 3.4408 1 The toxigenic inhibition rate of (2) is 99.86%, and the toxigenic inhibition rate is 99.86% for AFT and AFB isolated from toxigenic strains of different producing areas 1 、AFB 2 The toxin production inhibition rate ranges of 81.5% -94.4%, 84.5% -98.2% and 39.8% -93.7% respectively, which shows that the non-toxin producing bacteria XZCY1805 has broad-spectrum and high-efficiency competition inhibition of the toxin production capacity of the toxin producing aspergillus flavus, thereby reducing the content of aflatoxin in peanuts.
TABLE 2 inhibition of Aspergillus flavus toxigenic on peanut powder by Aspergillus flavus XZCY1805
Wherein AFT is total aflatoxin (from aflatoxin B) 1 Aflatoxin B 2 Aflatoxin G 1 And aflatoxin G 2 Composition of the components
Example 4: inhibition effect of bacterial strain XZCY1805 on peanut kernels on aspergillus flavus producing toxins
1. Spore suspension preparation
Same as in example 3.
2. Competition inhibition culture
Selecting mature, full, healthy, pest-free, disease-spot-free, complete and uniform-sized peanut seeds with normal color. Soaking peanut kernels in 70% ethanol solution for 2min for surface sterilization, and then washing with sterile distilled water for 3 times to wash off residual ethanol on the peanut surfaces and ensure that the water content of the peanut seeds is about 20% and is finished within 13.0 min.
A control group and a treatment group were set, and a strain of Aspergillus toxigenic (spore concentration 11X 10) was prepared, respectively 6 Spore suspension (control group) mixed with sterile water volumes (12.5 ml each) and non-toxic Aspergillus flavus XZCY1805 (spore concentration 1X 10) 6 Individual/ml) and a standard strain of Aspergillus flavus 3.4408 and isolated, different regions of virulent Aspergillus flavus (spore concentration 1X 10) 6 Equal volumes of spore suspension (treatment group) mixed (12.5 ml each) per ml, 10 peanut kernels were placed into them for 5min, and then placed in sterile petri dishes with sterile forceps. Each treatment was repeated for 3 organisms and samples were placed in a constant temperature incubator and incubated in continuous darkness at 28.+ -. 1 ℃ and 90% relative humidity for 7 days.
3. Aflatoxin content determination
Sterilizing the cultured peanut kernels at high temperature and high pressure (121deg.C for 30 min), drying at constant temperature in a drying oven (110deg.C for 1 h), cooling, grinding into peanut powder with a coffee machine, weighing 1.0g of peanut powder sample, loading into a centrifuge tube, adding 5ml of 70% methanol solution (containing 4% NaCl), mixing, shaking on a shaker for 2h, centrifuging at 4500r/min, passing through an immunoaffinity column and an organic filter membrane, and detecting aflatoxin B with High Performance Liquid Chromatography (HPLC) 1 、B 2 、G 1 、 G 2 Is contained in the composition. The total Aflatoxin (AFT) is the sum of the above 4 aflatoxins.
Chromatographic conditions are described in example 3.
The formula for calculating the toxigenic inhibition rate is the same as that described in example 3, namely formula I. As can be seen from tables 3 and 4, the peanut kernels are used as culture medium, and the non-toxigenic bacteria XZCY1805 are used for AFT and AFB of toxigenic bacteria 3.4408 1 、AFB 2 The toxigenic inhibition rates of (a) are respectively 97.8%, 100.0% and 80.7%, and the toxigenic inhibition rates are respectively used for AFT and AFB of toxigenic strains separated from different peanut producing areas 1 、AFB 2 、AFG 1 、 AFG 2 The toxin production inhibition rate ranges of 51.5% -88.8%, 51.2% -100.0%, 17.4% -89.6%, 72.8% -100.0% and 40.9% -100.0% respectively, which shows that the non-toxin producing bacteria XZCY1805 can effectively compete and inhibit the toxin production capacity of the toxin producing aspergillus, and remarkably reduce the aflatoxin pollution level in peanuts.
TABLE 3 inhibition of AFT and AFB production by non-toxic Aspergillus flavus XZCY1805 on peanut kernels
AFT is the total aflatoxin (from aflatoxin B) 1 Aflatoxin B 2 Aflatoxin G 1 And aflatoxin G 2 Composition of the components
TABLE 4 non-toxic Aspergillus flavus XZCY1805 AFG production on peanut kernels from toxic Aspergillus flavus 1 AFG 2 Inhibition effect
Example 5: inhibition experiments with different concentration ratios
Preparation of spores with a concentration of 1X 10 3 、1×10 4 、1×10 5 、1×10 6 Non-toxic Aspergillus flavus XZCY1805 and 1×10 in a volume of one/ml 5 The preparation method of each/ml spore suspension of Aspergillus flavus 3.4408 is the same as in example 3.
Control and treatment groups were set:
preparation of A.flavus Standard Strain 3.4408 (1×10) 5 ) Mixing with sterile water (12.5 ml each) to obtain spore suspension with spore number of 5×10 4 A.flavus standard strain 3.4408 (control) per ml.
Mixing Aspergillus flavus XZCY1805 with Aspergillus flavus standard strain 3.4408 at volume ratio of 1:1 to obtain spores with concentration ratio of 5×10 2 :5×10 4 (1:100)、5×10 3 :5×10 4 (1:10)、5×10 4 :5×10 4 (1:1)、 5×10 5 :5×10 4 A mixture of (10:1) A.flavus SX0104 and A.flavus standard strain 3.4408.
The sterilized and washed peanut kernels are respectively soaked in a control group and a treatment group for 5min, then are clamped by sterile forceps and placed in a sterile culture dish, and are placed in a constant temperature incubator for continuous dark culture for 7 days under the condition of 28 ℃ and 90% relative humidity.
Sterilizing the cultured peanut kernels at high temperature and high pressure (121 ℃ for 30 min), drying the peanut kernels in a constant temperature drying oven (110 ℃ for 1 h), grinding the peanut kernels into peanut powder by using a coffee machine after cooling, weighing 1g of peanut powder sample, loading the peanut powder sample into a centrifuge tube, adding 5ml of 70% methanol solution (containing 4% NaCl), mixing uniformly by vortex, placing the peanut powder sample on an oscillator for shaking for 2h, centrifuging the peanut kernels at 4500r/min, passing through an immunoaffinity column and an organic filter membrane, and detecting aflatoxin B by using high performance liquid chromatography 1 、B 2 、G 1 、G 2 The aflatoxin inhibition was calculated according to example 3, and the results are shown in table 5 and fig. 2, under the chromatographic conditions described in example 3.
As can be seen from Table 5 and FIG. 2, the non-toxic Aspergillus flavus with different concentration ratios has the inhibition effect on the mildew production of the mildew, the inhibition rate of the non-toxic Aspergillus flavus on the mildew production of the mildew is increased along with the increase of the concentration of spores, and the number ratio of spores of the non-toxic Aspergillus flavus to spores of the mildew production 3.4408 is 1:100 to 10: in the case of 1, the AFT toxigenic inhibition rate of the aspergillus flavus XZCY1805 on the toxigenic strain 3.4408 in peanut kernels is 45.4-96.3%. Lower concentration ratio of 5×10 2 :5×10 4 At (1:100), the inhibition rate of the total aflatoxin AFT is 45.4%. When the spore concentration ratio of the aspergillus flavus XZCY1805 to the aspergillus flavus standard strain 3.4408 is 5 multiplied by 10 3 :5×10 4 At (1:10), the inhibition rate of the total aflatoxin amount is 66.8%; concentration ratio of 5X 10 4 :5×10 4 At (1:1), the inhibition rate was 76.1%; at a concentration ratio of 5X 10 5 :5×10 4 At (10:1), the inhibition rate reached a maximum of 96.3%.
TABLE 5 inhibition of Aspergillus flavus toxigenic by XZCY1805 at different concentrations
Example 6 inhibition effect of Strain XZCY1805 on potted peanut aflatoxin
1. Non-toxic aspergillus flavus XZCY1805 potting application method
The peanut seedlings are moved into a flowerpot (flowerpot area is 0.3 m) 2 4 peanut plants per pot, no damage to peanut seedlings and soil at root). 9mL of a conidium suspension of non-toxic Aspergillus flavus XZCY1805 (concentration: 1X 10) 6 Each mL) was spread on the rhizosphere of each peanut plant (test group), the blank group was spread with an equal volume of sterile water, and other daily management test groups were identical to the blank group. The test was set up with 3 biological replicates.
2. Harvesting peanut sample collection
And (3) collecting peanut samples in the peanut harvesting period, airing and drying, and after the samples are subjected to shrinkage division by a quartering method, detecting the aflatoxin content.
3. Peanut pod toxigenic culture
In order to reduce the risk of aflatoxin contamination caused by improper storage environment, an aflatoxin contamination experiment under extreme environmental conditions (toxigenic cultivation conditions) was performed. The harvested dried peanut pods were placed in petri dishes, 5 pods were placed in each dish, and 3 replicates were set per sample. Placing into an incubator, culturing at 28+ -1deg.C and relative humidity of 90%, and detecting aflatoxin content after 20 days.
4. Detection of aflatoxin content and toxigenic inhibition effect in peanuts
Drying peanut pod in constant temperature oven (110deg.C, 1 h), cooling, grinding into peanut powder with coffee machine, weighing 1.0g peanut powder sample, loading into centrifuge tube, adding 5ml 70% methanol solution (containing 4% NaCl), mixing, shaking on vibrator for 2h, centrifuging at 4500r/min, passing through immunoaffinity column and organic filter membrane, and detecting aflatoxin B with High Performance Liquid Chromatography (HPLC) 1 、B 2 、G 1 、G 2 Is contained in the composition. The total Aflatoxin (AFT) is the sum of the above 4 aflatoxins.
Chromatographic conditions were as described in example 3.
The formula for calculating the toxigenic inhibition rate is the same as that described in example 3, namely formula I. The results are shown in Table 6
Toxin detection results show that aflatoxin is not detected in both the peanut control group and the experimental group after harvesting and drying.
As can be seen from Table 6, under the condition of toxigenic culture, the aflatoxin content in peanut pod of the blank group was 69.3. Mu.g/kg, and aflatoxin B 1 The content of (3) is 40.3 mug/kg; the aflatoxin content in the peanut pods of the test group is 16.3 mug/kg, and aflatoxin B 1 The content was 12.9. Mu.g/kg. The invention shows that the aspergillus flavus XZCY1805 without producing toxins has the capability of effectively competing and inhibiting the growth and propagation of the toxigenic bacteria and reducing the proportion of the toxigenic bacteria to the infected crops, thereby reducing the mycotoxin pollution content in agricultural products and ensuring the AFT and AFB in peanuts 1 The inhibition ratios of (a) were 76.5% and 68.0%, respectively.
Table 6 inhibition effect of non-toxic Aspergillus flavus XZCY1805 on aflatoxin content of potted peanut
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Claims (8)
1. Aspergillus flavus strain XZCY1805 which does not produce toxicityAspergillus flavus) The method is characterized in that: the non-toxic aspergillus flavus strain is preserved in China Center for Type Culture Collection (CCTCC) at 9 months and 18 days in 2020, and the preservation number is M2020521.
2. The aspergillus flavus strain XZCY1805 according to claim 1, wherein the non-toxigenic aspergillus flavus strain XZCY1805 does not produce aflatoxin, pipaninic acid, aflatoxin and versicolor aflatoxin.
3. A method for inhibiting aflatoxin production by using the non-toxigenic aspergillus flavus strain XZCY1805 as claimed in claim 1, characterized in that: inoculating the non-toxic aspergillus flavus strain XZCY1805 on a biological sample to inhibit the generation of aflatoxin.
4. A method according to claim 3, characterized in that: the biological sample is peanut powder or peanut kernels.
5. A method according to claim 3, characterized in that: the aflatoxin comprises aflatoxin B 1 Aflatoxin B 2 Aflatoxin G 1 And aflatoxin G 2 。
6. A method according to claim 3, characterized in that: the non-toxic aspergillus flavus strain XZCY1805 is prepared into a component conidium suspension for use.
7. A method for preventing and reducing the risk of aflatoxin contamination in agricultural products in a field, comprising: the aspergillus flavus XZCY1805 conidium suspension is utilized to be spread at the rhizosphere of peanuts.
8. The method according to claim 7, wherein: the agricultural product is peanut, and the application time is the peanut flowering and needle-setting period.
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