CN111534460B - Preparation method and application of bacillus beiLeisi capable of efficiently antagonizing fusarium graminearum - Google Patents
Preparation method and application of bacillus beiLeisi capable of efficiently antagonizing fusarium graminearum Download PDFInfo
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Abstract
The invention discloses a preparation method and application of Bacillus beiLeisi capable of efficiently antagonizing fusarium graminearum, and belongs to the technical field of microorganisms. The bacillus beiLeisi NF002 of the invention can efficiently antagonize fusarium graminearum, can effectively inhibit the generation of the DON toxin of the fusarium graminearum and can degrade the DON toxin in wheat grains, the bacillus beiLeisi NF002 can be used as a biological control material for wheat scab, and the bacillus beiensis has good application prospect no matter in the aspects of developing new biological pesticides or new biological control microbial inoculum.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a bacillus beiLeisi capable of efficiently antagonizing fusarium graminearum, a preparation method and application thereof.
Background
Wheat scab is one of the major diseases of wheat and is common worldwide. Wheat scab can be infected from wheat seedlings to ear emergence, and mainly causes seedling withering, stalk rot and ear rot, wherein the damage of the ear rot is the most serious. The wheat scab in most areas of China is caused by fusarium graminearum (A)Fusarium graminearum) And (4) causing. Fusarium graminearum caused wheat head blight can not only seriously affect wheat yield, but also affect wheat quality, and deoxynivalenol (fusarium nivale) can be generated in infected grainsdeoxynivalanolDON) and zearalenone (Zearalenone). Wherein the DON toxin has the characteristics of stable chemical property and no decomposition by heating. The DON toxin can cause poisoning of people and animals, and mainly has acute poisoning symptoms of nausea, vomiting, dizziness, fever, headache, numbness of hands and feet, systemic hypodynamia, diarrhea and the like, and severe patients can have fluctuation of breath, pulse, body temperature and blood pressure, and hemorrhage, abortion and even death. Therefore, the quality of food made of wheat as a raw material is seriously affected and the health of human and animals is harmed.
At present, most of chemical agents are used for preventing and treating wheat scab, but the chemical agents cause serious harm to human health and environment. Therefore, the microbial control is a hot spot of current research by virtue of the advantages of no pollution, no public nuisance, no residue, difficult generation of drug resistance and the like. The current research involves many types of microorganisms, among which bacillus has its distinct advantages: firstly, bacillus has broad-spectrum bacteriostasis; secondly, the bacillus has the characteristics of wide distribution, strong stress resistance and the like, is beneficial to development, processing, storage and use, and has wide research prospect and utilization value. A large number of existing research results show that the bacillus can inhibit the growth of pathogenic bacteria through antagonism, competition and antibacterial substance secretion, and can induce plants to obtain systemic resistance so as to achieve the effect of inhibiting plant pathogenic bacteria.
The invention provides a strain of Bacillus beleisi (a strain of natural biocontrol bacteria) derived from wheat kernels in Zhongxiang Hubei rice and wheat rotation areasBacillus velezensis) NF002, the strain can prevent and treat wheat head blight, inhibit fusarium graminearum spore germination, hypha growth and DON toxin synthesis, and degrade DON toxin in wheat grains. The invention also provides a preparation method and an application method of the fermentation culture and spray preparation of the strain.
Disclosure of Invention
The bacillus beiLeisi NF002 can effectively antagonize fusarium graminearum, can effectively inhibit the generation of DON toxin of the fusarium graminearum and can degrade the DON toxin in wheat grains, and the bacillus beiLeisi NF002 is used as a biological control material for wheat scab, so that the bacillus beiLeisi has a good application prospect no matter in the aspects of developing new biological pesticides or new biological control inoculants.
Through a large amount of screening and tests of the inventor, the Bacillus beijerinckii for efficiently antagonizing fusarium graminearum is finally obtained, and the Bacillus beijerinckii is Bacillus beijerinckii (Bacillus subtilis)Bacillus velezensis) NF002, deposited in China center for type culture Collection, with the deposit number: CCTCC NO, M20191000, preservation date: 03/12/2019, Wuhan university, China.
The strain NF002 can inhibit the growth of hyphae and the germination of spores of fusarium graminearum, inhibit the synthesis of DON toxin of fusarium graminearum and degrade the DON toxin in wheat grains, and is used for preventing and treating wheat scab.
The application of the bacillus beijerinckii for efficiently antagonizing fusarium graminearum can be used for spraying the bacillus beijerinckii at the early stage of wheat flowering during wheat scab prevention.
The preparation method of the bacillus beijerinckii for efficiently antagonizing fusarium graminearum comprises the following steps:
(1) selecting NF002 single colony obtained by screening from a flat plate, inoculating into a sterilized LB liquid culture medium, culturing for 24h at 28 ℃ and 180 r/min, and activating;
(2) transferring the activated bacterial liquid obtained in the step (1) into a sterilized fermentation medium according to the inoculation amount of 5%, and culturing for 2 days at the temperature of 28 ℃ and under the condition of 180 r/min to obtain fermentation liquor;
(3) adding 0.1% (v/v) Tween 20 into the fermentation liquor obtained in the step (2) to dilute to a final concentration of 1.0 × 108CFU/mL to obtain the spray preparation.
The preparation method of the bacillus beijerinckii for efficiently antagonizing fusarium graminearum comprises the following steps of (1): 10.0g of sucrose, 3.0 g of yeast powder, 5.0 g of peptone, 5.0 g of NaCl and K2HPO4 0.5 g，MgSO4 0.1 g，MnSO4 0.01 g，FeSO40.001 g, adding deionized water to 1.0L, pH 7.0, sterilizing at 115 deg.C for 30 min.
Compared with the prior art, the invention has the following beneficial effects:
(1) compared with the prior art, the method for screening the strain of the antagonistic fusarium graminearum is more efficient.
(2) According to the preparation method of the bacterial strain fermentation culture and spray preparation, the total number of bacterial colonies can reach 3.7 multiplied by 10 after the Bacillus beilesensis NF002 is fermented for 2 days10CFU/mL, the number of spores can reach 6.15 multiplied by 109CFU/mL, no water insoluble component exists in the formula of the culture medium, and the obtained fermentation liquid can be suitable for spray application to prevent and control wheat scab.
(3) The Bacillus belgii NF002 provided by the invention can obviously inhibit the germination of fusarium graminearum spores, the growth of hyphae and the generation of DON toxin (the inhibition rate reaches 96.41 percent), and can degrade the existing DON in wheat grains (the degradation rate reaches 55.36 percent)
(4) The Bacillus belgii NF002 spray preparation provided by the invention can effectively inhibit the growth of fusarium graminearum in wheat fields, prevent and control the occurrence of scab, reduce the disease index by 25.94%, and also reduce the DON content in wheat grains (the DON toxin content of wheat is reduced by 89.19%), thereby improving the quality of wheat.
(5) The Bacillus belgii provided by the invention is derived from wheat grains, is a wheat symbiotic bacterium, is used as a biological control material for wheat scab, and has good application prospect no matter in the aspect of developing new biological pesticides or new biological control microbial agents.
Description of the drawings:
FIG. 1 is a graph showing the effect of NF002 on the inhibition of Fusarium graminearum on PDA plates.
FIG. 2 is the colony morphology of NF002 on LB plates.
FIG. 3 is a phylogenetic tree of NF 002.
FIG. 4 is a graph of the effect of 20% NF002 fermentation supernatant on Fusarium graminearum inhibition on PDA plates.
FIG. 5 is an inverted microscope observation of the inhibitory effect of NF002 on Fusarium graminearum hyphae.
FIG. 6 shows the NF002 concentration versus 2X 10 for different bacteria concentrations5CFU/mL Fusarium graminearum spore germination inhibiting effect.
FIG. 7 is a graph of the effect of NF002 on DON production by Fusarium graminearum in wheat kernels.
FIG. 8 is the effect of NF002 on the degradation of DON in wheat kernels.
FIG. 9 shows the control effect of NF002 on Fusarium graminearum infected wheat ears.
Detailed Description
The following are specific embodiments of the present invention and further description of the technical solutions of the present invention, but the present invention is not limited to the scope of the embodiments, and all changes or equivalent substitutions that do not depart from the spirit of the present invention are included in the scope of the present invention.
The media and reagent components involved in the examples:
LB culture medium: 10.0g of tryptone, 5.0 g of yeast extract, 10.0g of NaCl and 1.0L of distilled water to constant volume; sterilizing at 121 deg.C for 20 min at pH 7.0.
PDA culture medium: 200.0 g of potato, 20.0 g of glucose, 20.0 g of agar and 1.0L of distilled water with constant volume; sterilizing at 115 deg.C for 30 min under natural pH.
PDB culture medium: 200.0 g of culture medium potato, 20.0 g of glucose and 1.0L of distilled water with constant volume; sterilizing at 115 deg.C for 30 min under natural pH.
CMC culture medium: 20.0 g of CMC-Na; na (Na)2HPO4 2.5 g；KH2PO41.5 g; peptone 2.5 g; distilled water is added to a constant volume of 1.0L; sterilizing at 121 ℃ for 20 min at the pH of 7.0-7.5.
NF002 fermentation medium: 10.0g of sucrose, 3.0 g of yeast powder, 5.0 g of peptone, 5.0 g of NaCl and K2HPO40.5 g，MgSO4 0.1g，MnSO4 0.01 g，FeSO40.001 g, distilled water to constant volume of 1.0L; pH 7.0, sealing with 6 layers of gauze, and sterilizing at 115 deg.C for 30 min.
Test fusarium graminearum strains: the fusarium graminearum strain used for the experiment is purchased from China center for culture collection management of industrial microorganisms, and the collection number is as follows: CICC 2697.
Example 1 screening of Fusarium graminearum antagonistic bacteria in wheat grains
Preparation of fusarium graminearum spores: fusarium graminearum was inoculated into a 250 mL flask containing 100 mL of CMC medium and cultured with shaking at 180 rpm at 25 ℃ for 7 days. Filtering the culture with two layers of sterile gauze to remove mycelium to prepare a suspension of macroconidium, centrifuging at 4 deg.C and 10000 rpm for 20 min, collecting the spores, washing the spores twice with distilled water, and adjusting the concentration of the suspension to 2X 10 with 0.1% (v/v) Tween 20 solution5 CFU/mL。
And (3) wheat grain selection of antagonistic fusarium graminearum: cleaning wheat grains from the rice and wheat rotation area of Zhongxiang phosphorite Hubei with 75% alcohol, soaking in 1% sodium hypochlorite solution for 10min, and cleaning with sterile water for 2 times. With spore liquid concentration of 2 × 105Infecting the wheat grains by CFU/mL fusarium graminearum, and culturing at 28 ℃ for 24 h; infected wheat grains were spotted on sterilized large plates (about 100 grains per plate) spread with moist gauze and cultured at 28 ℃ for 5 days. And selecting the wheat grains without the growth of the fusarium graminearum as the wheat grains for antagonizing the fusarium graminearum to further screen antagonistic bacteria.
Antagonistic fusarium graminearum primary screening: the wheat grains without the growth of the wheat grain fusarium graminearum are transferred to a PDA flat plate with the center connected with a fusarium graminearum block with the diameter of 7mm, the distance between the block and the wheat grains is 3 cm, and the wheat grains are cultured for 3 days at the temperature of 28 ℃. And selecting bacteria with bacteriostatic effects, inoculating the bacteria into an LB liquid culture medium for activation, then carrying out streak separation, and repeating the steps until single colonies of antagonistic fusarium graminearum are separated.
And (3) performing secondary screening on antagonistic fusarium graminearum strains: inoculating fusarium graminearum blocks with the diameter of 7mm to one side of the center of a PDA flat plate, simultaneously symmetrically scribing the separated single colony bacterial liquid to the other side of the center of the flat plate, wherein the distance between the fusarium graminearum blocks and the bacterial liquid is 40 mm. The width of the zone of inhibition (distance of the edge of the colony of antagonistic bacteria from the edge of the colony of fusarium graminearum) was measured after 5 days, with the unconjugated strain as a control. Bacteriostatic rate = (control colony diameter-treated colony diameter)/control colony diameter × 100%. Each treatment was repeated 3 times.
Through the screening, a strain of high-efficiency antagonistic fusarium graminearum with the number of NF002 is obtained, and as shown in figure 1, the inhibition rate of the strain on fusarium graminearum reaches 58.97 +/-1.25%.
Example 2 morphological and molecular biological characterization of NF002 Strain
Morphological identification: inoculating the screened single antagonistic bacterium NF002 strain in LB liquid culture medium overnight for activation, coating the obtained bacterial liquid on an LB flat plate, carrying out inverted culture at 28 ℃ for 2 days, and observing the morphological characteristics of the bacterial colony, wherein the bacterial colony is white, semitransparent, smooth and moist, slightly raised and round and neat when cultured for 24 hours as shown in figure 2.
Molecular biological identification: inoculating the screened antagonistic bacterium single strain into a PA bottle filled with 5 mL of liquid LB culture medium, culturing overnight at 28 ℃ to generate a large amount of bacterium liquid, and carrying out 16S PCR by using the bacterium liquid as a template. The PCR amplification system (50. mu.L) was 27F (5)，-AGAGTTTGATCCTGGCTCAG-3，) And 1492R (5)，-TACGGCTACCTTGTTACGACTT-3，) mu.L of each primer, 1. mu.L of the inoculum, 1. mu.L of Easy Taq DNA Polymerase (5U/. mu.L), 5. mu.L of 10 × Easy Taq buffer, 4. mu.L of dNTP (2.5 mM), ddH2O 37 μL。95℃ 10 min；94℃ 30 cycles of 30 s at 61 ℃ for 45s and 72 ℃ for 1 min for 45 s; 10min at 72 ℃. The PCR amplification product, together with the pClone007 vector, was sent to Wuhan engine Biotech company for sequence analysis, and the sequence was determined to be BLAST online comparison at NCBI, and MEGA 6.0 software was used to construct phylogenetic trees.
As shown in FIG. 3, antagonistic bacterium NF002 was identified as Bacillus belgii (R) ((R))Bacillus velezensis) And has been preserved in China center for type culture Collection with the preservation number: CCTCC NO, M20191000, preservation date: 12 months 03 days 2019.
EXAMPLE 3 preparation of fermentation broth and spray preparation of Bacillus beilesiensis NF002
Inoculating the screened antagonistic bacteria NF002 single strain in LB liquid culture medium for overnight activation, inoculating the antagonistic bacteria NF002 single strain in NF002 fermentation culture medium according to the inoculum size of 5% (5% of the volume of the fermentation culture medium), and performing shake culture at 28 ℃ and 180 rpm for 48 h. Directly diluting the fermentation liquid in a gradient manner, coating on an LB plate to count the total bacteria number, taking the fermentation liquid, carrying out water bath at 80 ℃ for 10min, and then diluting in a gradient manner, coating and counting the spore number. After 48 h of shake culture, the total bacterial count of NF002 fermentation liquor reaches 3.72 multiplied by 1010CFU/mL, number of spores up to 6.15X 109CFU/mL。
Adding 0.1% (v/v) Tween 20 into the fermentation liquid to dilute to a final concentration of 1.0 × 108CFU/mL to obtain the spray preparation. The culture medium has no water insoluble component, and the obtained fermentation liquid is suitable for spray application to prevent and treat wheat scab.
Example 4 experiment of antagonism of Fusarium graminearum in 20% fermentation supernatant of Bacillus beiLeisi 002
Centrifuging the NF002 fermentation liquor, taking supernatant, filtering and sterilizing the supernatant, mixing the supernatant with a PDA culture medium according to a ratio of 20%, pouring the mixture into a flat plate, inoculating fusarium graminearum blocks with the diameter of 7mm into the center of the flat plate, and observing and recording the inhibition effect of the fusarium graminearum blocks by taking a blank experiment as a control. As shown in FIG. 4, the 20% NF002 fermentation supernatant fusarium graminearum has an obvious inhibition effect, and the inhibition rate reaches 70.21%.
Example 5 Effect of Bacillus beilesiensis NF002 on Fusarium graminearum hyphae
Inoculating activated fusarium graminearum on the center of a PDA culture medium flat plate, and punching mycelium blocks at the edges of bacterial colonies by using a puncher with the diameter of 7mm after 3 days; the mycelia mass was inoculated into 50 mL of PDB culture medium and shaken at 180 rpm at 28 ℃ for 24h to prepare a uniform mycelia suspension. 1 mL of mycelium suspension and 1 mL of NF002 fermentation broth are taken to be added into 3 mL of PDB culture solution, sterile water is taken as a control, oscillation is carried out for 24h at 25 ℃ and 180 rpm, and the morphological change of the mycelium is observed under an inverted microscope. As shown in FIG. 5, the surface of the mycelia of the control group was smooth, the protoplasts were uniform, and the top of the fusarium graminearum mycelia after addition of NF002 bacterial solution showed significant apical encapsulation.
Example 6 experiment of inhibition effect of NF002 of different bacterial concentrations on Fusarium graminearum
Preparation of 4.0X 10 as in example 15CFU/mL Fusarium graminearum spore suspension and 1.0X 1010，1.0×109，1.0×108，1.0×107，1.0×106，1.0×105， 1.0×104，1.0×103Mixing CFU/mL NF002 fermentation broth in equal volume, culturing at 28 deg.C and 180 rpm for 12 h, adding 5 μ L mixed solution on PDA plate, culturing at 28 deg.C for 48 h, as shown in FIG. 6, when the bacterial solution concentration is 5.0 × 107When CFU/mL is adopted, NF002 can obviously inhibit the concentration of spore liquid to be 2.0 multiplied by 105Germination of Fusarium graminearum spores CFU/mL.
Example 7 Effect of Bacillus beilesiensis NF002 on DON production by Fusarium graminearum in wheat grains
Fusarium graminearum spore suspension was prepared as described in example 1. Soaking wheat at room temperature for 24h, draining, placing into a conical flask, and sterilizing at 121 deg.C for 1 h. And (2) adding 5 mL of fusarium graminearum spore suspension and 5 mL of NF002 bacterial suspension into 50 g of sterilized wheat, uniformly mixing in a conical flask, replacing the NF002 bacterial suspension with 5 mL of sterile water as a negative control, replacing the NF001 bacterial suspension with 5 mL of carbendazim (1 g/L) as a positive control, and culturing for 20 days at 28 ℃ in the dark. The method for measuring the DON content of the wheat grains refers to GBT23503-2009, namely immunoaffinity chromatography and purification high performance liquid chromatography for measuring deoxynivalenol in food. The experimental result is shown in fig. 7, NF002 can obviously inhibit the growth of fusarium graminearum hyphae on wheat grains, and simultaneously inhibit the production of DON toxin, and the inhibition rate is 96.41%.
Example 8 NF002 degradation experiment on wheat DON toxin
Fusarium graminearum spore suspension was prepared as described in example 1. Drying and crushing the wheat grains which are infected and turned into red by fusarium graminearum spores, sieving the crushed wheat grains with a sieve with the aperture of 1 mm, and sterilizing the crushed wheat grains for 30 min at 115 ℃ for later use. Adding NF002 bacterial liquid and sterile water into 30 g of sterilized and pulverized wheat grains to 100 g of sterilized and pulverized wheat grains, and making the concentration of the NF002 bacterial liquid be 1.0 x 108And (3) detecting the content of the DON toxin after culturing for 5 days at the temperature of 28 ℃ and 180 rpm by CFU/g. The method for measuring the DON content of the wheat grains comprises the following steps: refer to GBT23503-2009, immunoaffinity chromatography for determination of deoxynivalenol in food, and purification by high performance liquid chromatography. The experimental result is shown in fig. 8, and the degradation rate of NF002 to DON in wheat grains reaches 55.36%.
Example 9 experiment of control of wheat scab with NF002 spray preparation
2.0×105Preparation of CFU/mL Fusarium graminearum spore suspension: see example 1.
1.0×108Preparation of CFU/mL NF002 spray formulation: see example 2.
The pesticide spraying method and the spore infection method and time are as follows: at the beginning of wheat flowering (about 10% of wheat ears are flowering), a polyethylene compressed air sprayer is used at 40 mL/m2Mixing 1.0X 108The NF002 bacterial suspension (or carbendazim wettable powder) is uniformly sprayed on the wheat head, covered with a plastic film and moisturized for 24 hours. After 24h, the plastic film was removed and the same polyethylene compressed air sprayer was used at 40 mL/m2The concentration is 2 x 105The fusarium graminearum conidia are uniformly sprayed on the wheat ears, and a plastic film is covered to keep moisture for 24 hours.
Evaluation of NF002 resistance to wheat scab: after 16d (milk stage), the disease severity of the wheat is investigated and counted according to the disease grading standard of wheat scab resistance evaluation technical specification (NY/T1443.4-2007).
TABLE 1 grading of wheat scab
DI (disease index) =
Biocontrol effect (%) = (control group disease severity-treatment group disease severity)/control group disease severity × 100%
The method for measuring the DON content of the wheat grains comprises the following steps: reference is made to GBT 23503-2009' determination of deoxynivalenol in food by immunoaffinity chromatography and purification high performance liquid chromatography
As shown in the result of FIG. 9, the NF002 bacterial liquid is sprayed under the laboratory condition, so that the fusarium graminearum can be completely inhibited from infecting the wheat ears.
As shown in Table 2, the results of field experiments show that after NF002 is sprayed, compared with the group without pesticide spraying, the disease index of wheat is reduced by 25.94%, and the DON toxin content of wheat is reduced by 89.19%. Therefore, after the NF002 bacterial liquid is sprayed, the growth of fusarium graminearum can be effectively inhibited, the occurrence of scab can be prevented, the DON content in wheat grains can be reduced, and the quality of wheat can be improved.
Table 2 field experiment results of biocontrol effect of NF002 bacterial liquid on wheat scab
| Grouping | Disease Index (DI) | DON (μg/kg) |
| Normally spraying medicine | 34.95±1.26% | 268.5±12.6 |
| Does not need to be sprayed | 64.60±3.68% | 1469.6±65.6 |
| No-drug-spraying + NF002 | 38.66±2.89% | 158.9±22.3 |
Sequence listing
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<400>1
agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60
ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa 120
cctgcctgta agactgggat aactccggga aaccggggct aataccggat ggttgtttga 180
accgcatgat tcagacataa aaggtggctt ccgctaccac ttacagatgg acccgcggcg 240
cattagctag ttggtgaggt aacggctcac caaggcgacg atgcgtagcc gacctgagag 300
ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg 360
gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt 420
cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg gcggcacctt 480
gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540
gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt tcttaagtct 600
gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa cttgagtgca 660
gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc 720
agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg 780
aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg 840
tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc 900
aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960
ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc ctagagatag 1020
gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1080
agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt 1140
tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc 1200
atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacagaaca aagggcagcg 1260
aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc agtctgcaac 1320
tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380
tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt 1440
gaggtaacct tttaggagcc agccgccgaa ggtgggacag atgattgggg tgaagtcgta 1500
acaaggtagc cgta 1514
<210>2
<211>20
<212>DNA
<213> Artificial sequence
<221>gene
<400>2
agagtttgat cctggctcag 20
<210>3
<211>22
<212>DNA
<213> Artificial sequence
<221>gene
<400>3
tacggctacc ttgttacgac tt 22
Claims (3)
1. Fusarium graminearum (A) with efficient antagonismFusariumgraminearum) Bacillus belgii (B.), (B.)Bacillus velezensis ) The method is characterized in that: the bacillus beilesensis is bacillus beilesensis NF002, which is preserved in China center for type culture Collection in 12 months and 03 days in 2019, and the preservation number is as follows: CCTCC NO: M20191000;
the strain NF002 can inhibit the hypha growth and spore germination of fusarium graminearum, inhibit the synthesis of the DON toxin of fusarium graminearum and degrade the DON toxin in wheat grains, and is used for preventing and treating wheat scab.
2. A method of preparing a bacillus belgii spray formulation for the efficient antagonism of fusarium graminearum according to claim 1, wherein: the method comprises the following steps:
(1) the NF002 single colony obtained in the method of claim 1 is picked from a flat plate, inoculated into a sterilized LB liquid culture medium, cultured for 24 hours at the temperature of 28 ℃ and under the condition of 180 r/min, and activated;
(2) transferring the activated bacterial liquid obtained in the step (1) into a sterilized fermentation medium according to the inoculation amount of 5%, and culturing for 2 days at the temperature of 28 ℃ and under the condition of 180 r/min to obtain fermentation liquor;
(3) adding Tween 20 with volume fraction of 0.1% into the fermentation liquid obtained in the step (2) for dilution, wherein the final concentration of the diluted fermentation liquid is 1.0 × 108CFU/mL to obtain the spray preparation.
3. The method for preparing a bacillus belgii spray formulation for efficiently antagonizing fusarium graminearum as claimed in claim 2, wherein the method comprises the steps of: the fermentation medium of the step (2) is as follows: 10.0g of sucrose, 3.0 g of yeast powder, 5.0 g of peptone, 5.0 g of NaCl and K2HPO4 0.5 g，MgSO4 0.1 g，MnSO4 0.01 g，FeSO40.001 g, adding deionized water to 1.0L, pH 7.0, sterilizing at 115 deg.C for 30 min.
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| CN114231432A (en) * | 2021-10-08 | 2022-03-25 | 河南工业大学 | Bacillus belgii capable of degrading vomitoxin and zearalenone simultaneously and application of bacillus belgii |
| CN113999801B (en) * | 2021-11-25 | 2023-05-02 | 山东省农业科学院作物研究所 | Bacillus belicus capable of modifying wheat bran and application thereof |
| CN114703096B (en) * | 2022-04-01 | 2023-10-13 | 天津科技大学 | Bacillus bailii strain, fermented feed degradation microbial toxin thereof and application |
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| CN107236693B (en) * | 2017-07-04 | 2020-12-04 | 中国农业科学院农产品加工研究所 | Bacillus belgii JS25R and application thereof |
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