CN112501053A - Bacillus amyloliquefaciens HBNS-1, application thereof and obtained agricultural fertilizer - Google Patents
Bacillus amyloliquefaciens HBNS-1, application thereof and obtained agricultural fertilizer Download PDFInfo
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Abstract
The invention provides bacillus amyloliquefaciens HBNS-1, application thereof and an obtained agricultural fertilizer, belonging to the technical field of microorganisms. The Bacillus amyloliquefaciens HBNS-1 has a preservation number of CGMCC No.20380 and is preserved in China general microbiological culture Collection center (CGMCC) at 7, 16 months in 2020. The bacillus amyloliquefaciens provided by the invention can inhibit peanut pathogenic bacteria, has good conversion efficiency on sodium selenite, and the nano selenium fertilizer prepared by the bacillus amyloliquefaciens can effectively improve the selenium content of peanuts and improve the nutritional quality of the peanuts.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus amyloliquefaciens HBNS-1, application thereof and an obtained agricultural fertilizer.
Background
Selenite has extremely strong biological toxicity, and some microorganisms have the capacity of reducing the toxic selenite into selenium, so that the selenite is considered to be an ideal means for producing the selenium. The researchers have succeeded in obtaining the microorganisms capable of reducing sodium selenite to generate nano-selenium from soil or plant rhizosphere through enrichment culture and separation, and have shown the potential of developing into excellent strong selenium agent.
Disclosure of Invention
The invention provides bacillus amyloliquefaciens HBNS-1, application thereof and an obtained agricultural fertilizer, wherein the bacillus amyloliquefaciens can inhibit peanut pathogenic bacteria and has good conversion efficiency on sodium selenite, and the nano selenium fertilizer prepared from the bacillus amyloliquefaciens can effectively improve the selenium content of peanuts and improve the nutritional quality of the peanuts.
In order to achieve the aim, the invention provides the Bacillus Amyloliquefaciens HBNS-1 with the preservation number of CGMCC No.20380, which is preserved in China general microbiological culture Collection center at 7-16.2020, and the Latin article thereof is named as Bacillus Amyloliquefaciens.
Preferably, the Bacillus amyloliquefaciens HBNS-1 has a nucleotide sequence shown as SEQ ID NO. 1.
The invention also provides a PCR amplification method of the Bacillus amyloliquefaciens HBNS-1 according to the technical scheme, which comprises the following steps:
1) extracting genomic DNA of a bacterial strain isolated from the soil suspension;
2) using the genome DNA of the bacterial strain as a template, and performing PCR amplification by using Taq Mix enzyme by using a primer;
wherein: the PCR reaction system is as follows: template DNA 0.5. mu.l, ddH2O 12μl，Taq Mix 12.5 μl。
Preferably, the amplification conditions are: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 1 min; annealing at 53 deg.C for 1 min; extension at 72 ℃ for 90 s; extension was carried out for 5min at 72 ℃ for a total of 30 cycles.
Preferably, the primer pair is:
a forward primer: 5'-AGAGTTTGATCATGGCTCAG-3', respectively;
reverse primer: 3 '-CGCTTACCTTGTTACGACTT-5'.
The invention also provides application of the bacillus amyloliquefaciens HBNS-1 in inhibiting peanut root rot.
The invention also provides an application of the bacillus amyloliquefaciens HBNS-1 in reducing sodium selenite into monomer selenium according to the technical scheme.
The invention also provides an agricultural fertilizer, the bacillus amyloliquefaciens HBNS-1 in the technical scheme is used as a main effective component, and the bacillus amyloliquefaciens HBNS-1 can reduce sodium selenite into monomer selenium.
The invention also provides a nano selenium fertilizer, which is prepared by expanding propagation of the bacillus amyloliquefaciens HBNS-1 in the technical scheme, inoculating the bacillus amyloliquefaciens HBNS-1 into a container containing 500ml of LB culture medium and 1mM of sodium selenite, and performing shake culture at 28-30 ℃ and 180-200rpm for 48-72 hours.
The invention also provides application of the nano selenium fertilizer in improving selenium content and nutritional quality of peanuts.
Compared with the prior art, the invention has the advantages and positive effects that:
the invention separates a strain of bacteria from the selenium-enriched soil in Enshi in Hubei, is identified as bacillus amyloliquefaciens through molecular biology and physiological biochemistry, can inhibit peanut pathogenic bacteria, and has good conversion efficiency on sodium selenite. The researched biological fertilizer based on the nano-selenium synthesized by the Bacillus Amyloliquefaciens HBNS-1(Bacillus Amyloliquefaciens) can effectively improve the selenium content of the peanuts and the nutritional quality of the peanuts, and has good application prospect.
Drawings
FIG. 1 is a schematic diagram showing the 16s rDNA amplification result of Bacillus amyloliquefaciens HBNS-1 strain according to example 2 of the present invention;
FIG. 2 is a schematic diagram of a cladogram constructed by Bacillus amyloliquefaciens HBNS-1 and other bacteria provided in example 2 of the present invention;
FIG. 3 shows the inhibitory effect of HBNS-1 on flower root rot, provided in example 3 of the present invention, wherein A is the normally growing root rot, and B is the growth of root rot after HBNS-1 inhibits;
FIG. 4 shows the growth of the strain HBNS-1 provided in example 4 of the present invention on a medium, wherein A is no sodium selenite added; b is sodium selenite; red colonies are indicated by selenite reduction and elemental selenium formation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 isolation and identification of the Strain
Collecting sources: from soil in the selenium-rich plots in Enshi of Hubei province.
The culture mode is as follows: weighing 5g of rhizosphere soil, putting the rhizosphere soil into a triangular flask containing 45ml of sterile water, uniformly mixing, and sequentially diluting the rhizosphere soil in a gradient manner by 10-1-10-6And (3) sucking 50 mu L of soil suspension with different dilution times respectively by using a micropipette to the LB plate, uniformly coating the soil suspension by using a sterilization coater, sealing the plate, and then inversely placing the plate in a constant-temperature incubator at 28 ℃ for culturing for 48 hours. And (3) selecting a batch of single colonies on the plate, drawing lines on an LB solid culture medium plate, and inversely placing the plate in a constant temperature incubator at 28 ℃ for culturing for 1-2 d.
And (3) morphological identification: the colony is circular, the colony is milky translucent, and the surface of the colony is protruded and semi-wet. The diameter is 1.2-2.1mm, and the shape of the thallus observed by a microscope is a gram-positive bacterium and a rod.
Naming and preserving strains: the bacillus amyloliquefaciens HBNS-1 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number as follows: CGMCC No.20380, the preservation time is 2020, 7 and 16 days, and the preservation address is as follows: the microbial research institute of the national academy of sciences, No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
And (3) strain identification: bacillus Amyloliquefaciens HBNS-1 is named Bacillus Amyloliquefaciens in Latin, gram-positive, aerobic bacteria, weak in growth under anaerobic condition, positive in contact enzyme activity and positive in V-P experiment, can degrade starch and xylan, and can utilize saccharides such as glucose, arabinose, mannose, xylose and the like.
TABLE 1 physio-biochemical characteristics of the active Strain HBNS-1
Note: "+" indicates that the reaction result was positive; "-" indicates that the reaction result was negative.
Example 2 molecular characterization of 16S rRNA of HBNS-1
Selecting bacterial strains with antagonistic activity on fungi, selecting 10 identical colonies, and extracting genome DNA by mainly referring to the instruction of a TIANGEN TIANAmp BACTERIA DNA Kit, wherein the steps are as follows:
(1) centrifuging 1mL of the bacterial culture solution at 10000rpm for 1min, and completely sucking the supernatant as much as possible;
(2) adding 200 mu L of buffer solution GA into the thallus precipitate, and shaking until the thallus is completely suspended;
(3) adding 4 μ LRNAase (100mg/mL) solution, shaking for 15s, standing at room temperature for 5 min;
(4) adding 20 mu L of proteinase K solution into the tube, and uniformly mixing;
(5) adding 220 μ L buffer solution GB, shaking for 15s, standing at 70 deg.C for 10min, centrifuging to remove water droplets on the inner wall of the tube cover;
(6) adding 220 μ L of anhydrous ethanol, shaking thoroughly, mixing for 15s to obtain flocculent precipitate, and centrifuging briefly to remove water drop on the inner wall of the tube cover;
(7) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column GB3 (the adsorption column is put into a collecting pipe), centrifuging at 12000rpm for 30s, pouring off waste liquid, and putting the adsorption column CB3 into the collecting pipe;
(8) adding 500 μ L buffer GD (checking whether absolute ethanol is added before use) into adsorption column CB3, centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
(9) adding 700 μ L of rinsing solution PW (added with anhydrous ethanol before use) into adsorption column CB3, centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing adsorption column CB3 into a collecting tube;
(10) putting 500 microliter rinsing liquid PW into an adsorption column CB3, centrifuging at 12000rpm for 30s, pouring off waste liquid, and putting the adsorption column CB3 into a collecting pipe;
(11) putting the adsorption column CB3 back into the collecting pipe, centrifuging at 12000rpm for 2min, pouring off waste liquid, and placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;
(12) transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 2-5min, centrifuging at 12000rpm for 2min, collecting the solution into the centrifuge tube, and storing at-20 ℃.
PCR and sequencing
The amplification method comprises the following steps: the extracted DNA was subjected to PCR amplification with reference to Kit instructions of 16s rDNA Bacterial Identification PCR Kit of TaKaRa, wherein the forward primers were: 5'-AGAGTTTGATCATGGCTCAG-3' (27F), the reverse primer is: 3 '-CGCTTACCTTGTTACGACTT-5' (1492R).
PCR amplification conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 1 min; annealing at 53 deg.C for 1 min; extension at 72 ℃ for 90 s; extension was carried out for 5min at 72 ℃ for a total of 30 cycles.
The PCR products were separated by electrophoresis on a 1% agarose gel, stained with EB, and observed under 3 UVTMTransill. mu. Minator (UVP, USA). The size of the 16s rDNA fragment of the HBNS-1 strain is about 1500bp through electrophoresis detection, the size of the fragment is consistent with the expected design of the kit, and the amplification result is shown in figure 1.
According to the size of a pre-designed primer and an expected amplified fragment, combining a Marker, cutting a target fragment under an ultraviolet lamp, and recovering DNA by using a recovery Kit Silica Bead DNA GelExtraction Kit. 16s rDNA sequencing was performed by Qingdao Okagaku Biotechnology Co. The sequence determination result shows that the 16s rDNA fragment of the HBNS-1 strain has 1511nt base pair composition, and the sequence is shown in SEQ ID NO. 1.
The determined sequence was compared with the sequences in GenBank (NCBI, Website http:// BLAST. NCBI. nlm. nih. gov/BLAST. cgi) by BLAST program, and then the 16s rDNA sequence of the species and genus similar to the test strain sequence was obtained from GenBank for determination. The alignment result is shown in figure 2, the 16s rDNA sequence of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) of the Paenibacillus in the GenBank gene library of the 16s rDNA sequence of the active strain is highly homologous, and the homology rate reaches 100%.
The results of the development tree construction and homology analysis show (figure 2), the strain HBNS-1 and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) (Accession No: MN795904) of the Bacillus form a branch independently, the evolutionary distance is nearest, the genetic relationship between the strain and the Bacillus amyloliquefaciens is reflected to be nearest, and the homology between the strain and the Bacillus amyloliquefaciens is 99% by analyzing with DANMAN software. And determining the strain HBNS-1 as bacillus amyloliquefaciens by combining the results of traditional physiological and biochemical characteristic identification and 16S rDNA sequence analysis.
Example 3 inhibition of peanut pathogens by HBNS-1
Inoculating a fungus cake with the diameter of about 0.5cm of peanut root rot fungus to the center of a PDA culture dish on a clean bench, inoculating HBNS-1 single colony at a distance of 2cm from the fungus, and continuously culturing at 28 ℃ to observe the bacteriostasis condition.
As can be seen in FIG. 3, HBNS-1 has a significant inhibitory effect on flower root rot. Obvious bacteriostatic zones appear around HBNS-1 bacterial colonies, and the inhibition rate is 50%. Wherein, the formula for calculating the bacteriostasis rate is as follows:
the inhibition rate is [ (control fungus growth radius-treated fungus growth radius)/control fungus growth radius ] × 100%
Example 4 reducing Effect of HBNS-1 on sodium selenite
The HBNS-1 strain grows as shown in FIG. 4, and no red single colony appears when no sodium selenite is added; a single red colony appeared upon addition of 5mM sodium selenite, indicating that the strain was able to reduce the colorless selenite to elemental selenium in red color with the addition of sodium selenite, thereby rendering the colony red.
Example 5 Effect of selenium Fertilizer on peanut quality
Streaking HBNS-1 strain stored at 4 deg.C on LB medium, culturing at 28 deg.C for 1-2 days until single colony appears, inoculating the single colony in LB medium containing 500ml and 1mM sodium selenite (Na)2SeO3) The flask is shake-cultured for 48h at 28 ℃ and 200rpm to obtain the primary selenium fertilizer.
In a further optimization treatment, bacterial liquid can be collected after culture, centrifuged at 12000g for 10min, and precipitated into a mixture of thalli and selenium. Adding 20mg/ml lysozyme into the thallus, cracking at 37 ℃ for 20min, taking out every 5min, and turning upside down for mixing. After the cleavage, the cells were sonicated on ice for 30min (sonication for 30s, stop for 40 s). And (3) centrifuging for 10min at 6000g, taking the supernatant to a new centrifuge tube, centrifuging for 10min at 13000g, discarding the supernatant, and dissolving and washing the precipitate for 3 times by using ultrapure water for later use.
1. Influence of nano selenium fertilizer on growth of peanut plants
Culturing peanut seeds by a water culture method for 20 mu M, setting the selenium concentration of a culture solution to be 10 mu M, 20 mu M and 50 mu M, taking the whole peanut plant after the peanut seedlings emerge for 2 weeks, cleaning and drying the whole plant, and measuring the selenium content by using an inductively coupled plasma emission mass spectrometer (7900).
TABLE 1 Effect of hydroponics on selenium content in peanut tissue
2. Application of nano selenium fertilizer in peanut field
The test is carried out on Laxi test farm of peanut research institute in Shandong province, and adopts random block design, wherein each block of peanuts is 70m24 repeats. Clear water was used as a blank CK, and the selenium concentration of the culture broth was set at 10. mu.M, 20. mu.M, and 50. mu.M. Irrigating roots 10d, 20d and 30d after emergence, and each plant is about 50m L each time. And (5) performing other field management to the normal production, and measuring the selenium content in the peanuts according to the measuring method after harvesting.
TABLE 2 selenium content in peanut fruits in field trial
The results of indoor tests and field tests show that the nano-selenium synthesized by the strain HBNS-1 can be effectively absorbed and utilized by peanuts through the transformation of peanut plants, and selenium-rich agricultural products meeting the national standard can be produced.
Sequence listing
<110> institute for peanut research in Shandong province
<120> Bacillus amyloliquefaciens HBNS-1, application thereof and obtained agricultural fertilizer
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1511
<212> DNA
<213> Bacillus amyloliquefaciens HBNS-1
<400> 1
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC 60
GGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAA 120
CCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGG 180
ACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCG 240
CATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAG 300
GGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGG 360
GAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTT 420
CGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTT 480
GACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG 540
GTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCT 600
GATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCA 660
GAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACC 720
AGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCG 780
AACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGG 840
TTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGC 900
AAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAA 960
TTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAG 1020
GACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTG 1080
AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGT 1140
TGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATC 1200
ATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCG 1260
AAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAAC 1320
TCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGT 1380
TCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGT 1440
GAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTA 1500
ACAAGGTAGCC 1511
Claims (10)
1. The bacillus amyloliquefaciens HBNS-1 is characterized in that the preservation number is CGMCC No.20380, and is preserved in China general microbiological culture Collection center (CGMCC) at 7, 16 months in 2020.
2. The bacillus amyloliquefaciens HBNS-1 according to claim 1, wherein the bacillus amyloliquefaciens HBNS-1 has a nucleotide sequence as shown in SEQ ID No. 1.
3. The method for PCR amplification of Bacillus amyloliquefaciens HBNS-1 according to claim 1 or 2, characterized in that it comprises the following steps:
1) extracting genomic DNA of a bacterial strain isolated from the soil suspension;
2) using the genome DNA of the bacterial strain as a template, and performing PCR amplification by using Taq Mix enzyme by using a primer;
wherein: the PCR reaction system is as follows: template DNA 0.5. mu.l, ddH2O 12μl，Taq Mix 12.5μl。
4. The PCR amplification method of claim 3, wherein the amplification conditions are: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 1 min; annealing at 53 deg.C for 1 min; extension at 72 ℃ for 90 s; extension was carried out for 5min at 72 ℃ for a total of 30 cycles.
5. The PCR amplification method of claim 3, wherein the primer pair is:
a forward primer: 5'-AGAGTTTGATCATGGCTCAG-3', respectively;
reverse primer: 3 '-CGCTTACCTTGTTACGACTT-5'.
6. The use of bacillus amyloliquefaciens HBNS-1 according to claim 1 or 2 for inhibiting peanut root rot.
7. Use of the bacillus amyloliquefaciens HBNS-1 according to claim 1 or 2 for reducing sodium selenite to monomeric selenium.
8. An agricultural fertilizer characterized in that the bacillus amyloliquefaciens HBNS-1 according to claim 1 or 2 is used as a main active ingredient, and the bacillus amyloliquefaciens HBNS-1 can reduce sodium selenite to selenium monomer.
9. The nano selenium fertilizer is characterized in that the nano selenium fertilizer is prepared by expanding propagation of the bacillus amyloliquefaciens HBNS-1 as claimed in claim 1 or 2, inoculating the bacillus amyloliquefaciens HBNS-1 into a container containing 500ml of LB culture medium and 1mM of sodium selenite, and performing shake culture at 28-30 ℃ and 180-200rpm for 48-72 hours.
10. The application of the nano selenium fertilizer as claimed in claim 9 in improving selenium content of peanuts and improving nutritional quality of peanuts.
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| CN113373180A (en) * | 2020-12-18 | 2021-09-10 | 中国农业科学院烟草研究所 | Nano-selenium synthetic active bacterial liquid of bacillus amyloliquefaciens, preparation method and application thereof |
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| CN101948771A (en) * | 2010-08-27 | 2011-01-19 | 黑龙江省科学院微生物研究所 | Bacillus amyloliquefaciens growing in disease-preventing and growth-promoting plant and application thereof |
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| CN106591193A (en) * | 2016-12-23 | 2017-04-26 | 河北省农林科学院遗传生理研究所 | Bacillus amyloliquefaciens with broad spectrum growth-promoting and stress-resisting effects |
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| CN111197017A (en) * | 2020-01-13 | 2020-05-26 | 中化农业（临沂）研发中心有限公司 | Bacillus amyloliquefaciens CGMCC No.17844 and application thereof |
| CN111197017B (en) * | 2020-01-13 | 2021-09-17 | 中化农业（临沂）研发中心有限公司 | Bacillus amyloliquefaciens CGMCC No.17844 and application thereof |
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