CN116267989B - Nanometer iron antibacterial agent for fusarium trilineum - Google Patents
Nanometer iron antibacterial agent for fusarium trilineum Download PDFInfo
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- CN116267989B CN116267989B CN202211102339.3A CN202211102339A CN116267989B CN 116267989 B CN116267989 B CN 116267989B CN 202211102339 A CN202211102339 A CN 202211102339A CN 116267989 B CN116267989 B CN 116267989B
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 127
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 75
- 241000223218 Fusarium Species 0.000 title claims abstract description 45
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 title claims abstract description 43
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 42
- 239000002105 nanoparticle Substances 0.000 claims abstract description 19
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000001301 oxygen Substances 0.000 claims abstract description 4
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 4
- 239000011258 core-shell material Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 235000011164 potassium chloride Nutrition 0.000 claims description 2
- 239000012266 salt solution Substances 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims 1
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 15
- 239000012895 dilution Substances 0.000 abstract description 12
- 238000010790 dilution Methods 0.000 abstract description 12
- 230000035784 germination Effects 0.000 abstract description 5
- 238000005054 agglomeration Methods 0.000 abstract description 3
- 230000002776 aggregation Effects 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000002045 lasting effect Effects 0.000 abstract description 2
- 238000005507 spraying Methods 0.000 abstract 1
- 241000233866 Fungi Species 0.000 description 31
- 244000061456 Solanum tuberosum Species 0.000 description 16
- 235000002595 Solanum tuberosum Nutrition 0.000 description 16
- 239000012452 mother liquor Substances 0.000 description 16
- 239000001963 growth medium Substances 0.000 description 14
- 239000007853 buffer solution Substances 0.000 description 13
- 229920001817 Agar Polymers 0.000 description 12
- 239000008272 agar Substances 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 12
- 239000002609 medium Substances 0.000 description 10
- 238000012258 culturing Methods 0.000 description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 8
- 230000003213 activating effect Effects 0.000 description 8
- 239000008103 glucose Substances 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 238000009631 Broth culture Methods 0.000 description 4
- 230000005526 G1 to G0 transition Effects 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 239000003337 fertilizer Substances 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 239000011858 nanopowder Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000000575 pesticide Substances 0.000 description 4
- 239000001965 potato dextrose agar Substances 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- 238000003892 spreading Methods 0.000 description 4
- 230000007480 spreading Effects 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 235000002566 Capsicum Nutrition 0.000 description 2
- 239000006002 Pepper Substances 0.000 description 2
- 241000722363 Piper Species 0.000 description 2
- 235000016761 Piper aduncum Nutrition 0.000 description 2
- 235000017804 Piper guineense Nutrition 0.000 description 2
- 235000008184 Piper nigrum Nutrition 0.000 description 2
- 238000002441 X-ray diffraction Methods 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 239000004599 antimicrobial Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000012271 agricultural production Methods 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012459 cleaning agent Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 244000000004 fungal plant pathogen Species 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000003980 solgel method Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/16—Heavy metals; Compounds thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nanotechnology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Health & Medical Sciences (AREA)
- Dentistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Composite Materials (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Materials Engineering (AREA)
- Inorganic Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Manufacturing & Machinery (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
The invention relates to the technical field of antibacterial agents, in particular to a nano iron antibacterial agent for fusarium trilineum, which comprises the following steps: ultrasonic vibration treatment is carried out on the core-shell type iron nano-particles and phosphate buffer solution to obtain a nano-iron antibacterial agent; exploring the optimal dilution factor of fusarium trilineum; selecting the optimal dilution ratio of the fusarium tricorum, and respectively exploring the antibacterial rate of the fusarium tricorum on the fusarium tricorum by using nano iron antibacterial agents with different concentrations; treating seeds with nano iron antibacterial agents with different concentrations, observing germination rate, planting and spraying at a later stage; the beneficial effects are as follows: because the iron nano particles are spherical, have less agglomeration and good dispersibility, are composed of the iron with the body-centered cubic structure and the magnetic iron oxide with the face-centered cubic structure, only contain iron elements and oxygen elements, and have the contents of 91.45wt.% and 8.55wt.% respectively, the cost of the antibacterial agent is low, and the antibacterial agent has high-efficiency, lasting and broad-spectrum antibacterial property so as to improve the crop yield. For plant pathogenic fungi Fusarium tricuspidatum, the antibacterial rate can reach 72.97% when the concentration of the nano iron antibacterial agent is very low.
Description
Technical Field
The invention relates to the technical field of antibacterial agents, in particular to a nano iron antibacterial agent for fusarium trilineum.
Background
Nanotechnology is the leading edge and cross emerging discipline field which is developed gradually by the last 80 s and the beginning 90 s of the 20 th century, and the rapid development of the nanotechnology can promote a revolutionary change in the industrial and agricultural fields in the 21 st century. Examples of the method for preparing the metal nano powder include an electro-explosive wire method (EEW), a plasma synthesis method, a sol-gel method, an induction heating evaporation method (IHE), a laser induction composite heating evaporation method (LCHE), and the like. However, none of these methods can produce nano-powder on a large scale, and in order to solve the market demand, the applicant has independently developed a macro-scale preparation technology of metal nano-powder (MPNP). The technology is to convert high-purity metal-containing raw materials into a flowing state through a specific energy conversion method, react with reaction gases, control phase balance by utilizing a temperature field, and nucleate and agglomerate to obtain nano powder. The process has the advantages of high raw material utilization rate, high yield and environmental protection.
For agricultural production, the use of traditional chemical fertilizers and pesticides is greatly damaged, the chemical fertilizers mainly comprise various salt substances and nutrient substances, and the concentration of soil solution can be increased after long-term use, so that the osmotic pressure in the soil is uneven; the harm of pesticides directly acts on the environment to cause serious pollution to the environment, and the pesticides can indirectly harm the human body through ecological circulation. The simple substance nano micro-fertilizer can be used for crops to increase yield and efficiency, improve quality, increase stress resistance, effectively reduce the usage amount of agricultural chemical fertilizer and pesticide, improve soil quality and the like.
Disclosure of Invention
The invention aims to provide a nano iron antibacterial agent for fusarium trilineum, which improves the antibacterial rate of fusarium trilineum through the excellent antibacterial performance of iron nano particles so as to improve the crop yield.
In order to achieve the above object, the present invention provides the following technical solutions:
A nano-iron antibacterial agent for fusarium trilineum, which is characterized by comprising the following steps:
step one: ultrasonic vibration treatment is carried out on the core-shell type iron nano-particles and phosphate buffer solution to obtain a nano-iron antibacterial agent;
step two: exploring the optimal dilution factor of fusarium trilineum;
Step three: selecting the optimal dilution ratio of the fusarium tricorum, and respectively exploring the antibacterial rate of the fusarium tricorum on the fusarium tricorum by using nano iron antibacterial agents with different concentrations;
step four: the seeds are treated by nano iron antibacterial agents with different concentrations, the germination rate is observed, and the seeds are planted and sprayed in a later period.
Preferably, in the first step, the iron nanoparticle is composed of a body-centered cubic structure iron and a face-centered cubic structure magnetic iron oxide (Fe 3O4), as shown in fig. 1, three distinct diffraction peaks are visible, diffraction angles 2θ are 44.673 °, 65.021 ° and 82.333 °, respectively, corresponding to (110), (200) and (211) crystal planes of the body-centered cubic structure iron; in addition, there is a weak diffraction peak, the diffraction angle 2 theta is 36.820 degrees, corresponding to the (311) crystal face of the magnetic iron oxide (Fe 3O4) with face-centered cubic structure.
Preferably, in the first step, as shown in fig. 2, the iron nanoparticles are spherical, have less agglomeration and good dispersibility, and have an average particle diameter of 55.09nm.
Preferably, in the first step, as shown in fig. 3, the iron nanoparticle contains only iron element and oxygen element in the amounts of 91.45wt.% and 8.55wt.%, respectively.
Preferably, in the first step, the phosphate buffer solution is a buffer solution commonly used in biological research, and is a water-based salt solution containing sodium chloride, phosphate with pH of 7.2-7.4, and the main components comprise 137mM sodium chloride, 10mM disodium hydrogen phosphate, 1.76mM potassium dihydrogen phosphate and 2.7mM potassium chloride.
Preferably, in the first step, the ultrasonic frequency of the ultrasonic cleaning agent for preparing the nano-iron antibacterial agent is 40+/-1 KHz, and the antibacterial agent is prepared by the following steps:
Preparation of an antimicrobial agent with a nano-iron concentration of 1X 10 -6 g/ml: injecting 5ml PBS buffer solution into a centrifuge tube, adding 0.01g nano-iron into the centrifuge tube, carrying out ultrasonic oscillation treatment for 10min at 40KHz, sucking 0.5ml nano-iron solution into a new centrifuge tube by using a pipette, injecting 4.5ml PBS buffer solution into the centrifuge tube, carrying out ultrasonic treatment to obtain 5ml PBS solution which is diluted 10 times, namely contains 0.001g nano-iron, sucking 0.5ml and adding the 0.5ml PBS buffer solution into 99.5ml PBS buffer solution, and obtaining the nano-iron antibacterial agent with the concentration of 1X 10 -6 g/ml.
Preferably, in step two, the fusarium trilineum is a plant pathogenic fungus.
Preferably, in the second step, the optimal dilution factor is explored by selecting fusarium tricuspension with the dilution factor of 10 2、103、104, and the main steps are as follows:
(1) Preparation of bacterial suspension: the fusarium tricuspidatum strain inclined plane is inoculated with a certain amount of bacteria by an inoculating loop and added into 100ml potato dextrose broth culture medium, and the main components are 0.7g/l potato soaked powder, 1.4g/l peptone, 2.1g/l glucose and 0.7g/l sodium chloride. Placing into a constant temperature shaking table, and activating at 28deg.C for 24 hr at 180r/min to obtain fungus mother liquor.
(2) Activating fusarium trilineum: 100 μl of the fungus mother liquor was added to 100ml of nutrient broth medium, and the mixture was placed in a thermostatic shaker and activated at 28℃for 24 hours at 180r/min to obtain a fungus mother liquor at stationary phase.
(3) Diluting bacterial liquid and culturing: the pipette sucks 0.5ml of fungus mother liquor into a 10ml centrifuge tube, then sucks 4.5ml of PBS buffer solution into the centrifuge tube, shakes and shakes evenly to obtain 10 1 times diluted fungus solution, and the steps are repeated to obtain 10 2、103、104 times diluted fusarium tricuspension respectively.
3 Conical flasks of 250ml were prepared, 95ml of PBS buffer was added to each flask, 5ml of diluted bacterial solution was added to each flask, and after sealing, the flasks were placed in a shaker and incubated at 28℃for 24 hours at 180 r/min.
(4) Coating: the potato agar culture medium is prepared in advance, and the main components of the potato agar culture medium are 1.104g/l of potato soaked powder, 3.68g/l of agar, 3.68g/l of glucose and the pH value is 5.6. 100 μl of each of the cultured mixed solutions was sucked up and dropped onto the surface of the solid medium, smeared with a spreading bar, and each treatment was repeated three times, and placed in a 28℃incubator for culturing for 48 hours.
(5) Counting: the solid medium after 48 hours of culture was counted, each colony was counted as 1 fungus, and the number of 3 parallel dishes of fungi was counted and averaged, as shown in FIG. 4, to find that the optimal dilution was 10 3 times.
Preferably, in step three, the concentration of the nano-iron antibacterial agent is 0, 1X 10 -6g/ml、5×10-6g/ml、1×10-5 g/ml, respectively.
Preferably, in step three, the antibacterial test procedure is as follows:
(1) Preparation of bacterial suspension: the fusarium tricuspidatum strain inclined plane is inoculated with a certain amount of bacteria by an inoculating loop and added into 100ml potato dextrose broth culture medium, and the main components are 0.7g/l potato soaked powder, 1.4g/l peptone, 2.1g/l glucose and 0.7g/l sodium chloride. Placing into a constant temperature shaking table, and activating at 28deg.C for 24 hr at 180r/min to obtain fungus mother liquor.
(2) Activating fusarium trilineum: 100 μl of the fungus mother liquor was added to 100ml of nutrient broth medium, and the mixture was placed in a thermostatic shaker and activated at 28℃for 24 hours at 180r/min to obtain a fungus mother liquor at stationary phase.
(3) Diluting bacterial liquid and co-culturing: the pipette sucks 0.5ml of fungus mother liquor into a 10ml centrifuge tube, then sucks 4.5ml of PBS buffer solution into the centrifuge tube, shakes and shakes evenly to obtain fungus liquid diluted 10 1 times, and three-wire fusarium diluted 10 3 times can be obtained by repeating the steps.
Preparing 250ml conical flasks with concentration gradient, adding 90ml PBS buffer solution and 5ml nano-iron antibacterial agent with different concentrations, shaking uniformly, adding 5ml bacterial solution diluted by 10 3 times into each conical flask, sealing, placing into a shaking table, and co-culturing at 28 ℃ for 24h at 180 r/min.
(4) Coating: the potato agar culture medium is prepared in advance, and the main components of the potato agar culture medium are 1.104g/l of potato soaked powder, 3.68g/l of agar, 3.68g/l of glucose and the pH value is 5.6. 100 μl of each of the cultured mixed solutions was sucked up and dropped onto the surface of the solid medium, smeared with a spreading bar, and each treatment was repeated three times, and placed in a 28℃incubator for culturing for 48 hours.
(5) Counting: the solid medium after 48 hours of culture was counted, each colony was counted as 1 fungus, the number of fungi in 3 parallel dishes was counted and averaged, and then the antibacterial ratio was calculated. As shown in fig. 6, it is known that the antibacterial rate reaches 72.97% when the concentration of the nano-iron antibacterial agent is very low.
Preferably, the antibacterial rate calculation formula is:
Wherein:
R-antibacterial ratio%
Colony count of control samples after A-Co-culture
Colony count of test samples after B-Co-cultivation
Preferably, in step four, the nano-iron antimicrobial concentration of the treated seed is 0, 10, 20, 50, 100, 200mg/l, respectively.
By the process and the selected reagent, the nano iron antibacterial agent can greatly improve the antibacterial rate of fusarium trilineum.
Compared with the prior art, the invention has the following beneficial effects: because the iron nano particles are spherical, have less agglomeration and good dispersibility, the average particle diameter is 55.09nm, and the iron nano particles consist of body-centered cubic structure iron and face-centered cubic structure magnetic iron oxide (Fe 3O4), only contain iron element and oxygen element, and have the contents of 91.45wt.% and 8.55wt.% respectively, the cost of the antibacterial agent is low, and the antibacterial agent has high-efficiency, lasting and broad-spectrum antibacterial property so as to improve the crop yield. For plant pathogenic fungi Fusarium tricuspidatum, the antibacterial rate can reach 72.97% when the concentration of the nano iron antibacterial agent is very low.
Drawings
FIG. 1 is an X-ray diffraction pattern (XRD) of an iron nanoparticle according to the present invention;
FIG. 2 (a) is a Scanning Electron Microscope (SEM) of iron nanoparticles according to the present invention; FIG. 2 (b) is a graph showing a particle size distribution of iron nanoparticles obtained by Scanning Electron Microscopy (SEM) in accordance with the present invention;
FIG. 3 is an energy spectrum (EDS) of iron nanoparticles of the present invention;
FIG. 4 is a graph showing colonies of Fusarium tricuspidatum cultured for 48h at different dilution factors in the present invention;
FIG. 5 is a bar graph of colony count of different dilutions of Fusarium tricuspidatum cultured for 48h according to the present invention;
FIG. 6 is a graph showing colonies of different amounts of nano-iron antibacterial agents co-cultured with Fusarium tricuspidatum for 48 hours in the present invention;
FIG. 7 (a) is a histogram of colony count of different amounts of nano-iron antibacterial agent co-cultured with Fusarium tricuspidatum for 48h in the present invention; FIG. 7 (b) is a graph showing the antibacterial ratio of the antibacterial agent with different nano-iron content and Fusarium tricuspidatum co-cultured for 48 hours in the present invention;
fig. 8 is a germination chart of pepper seeds treated with different nano-iron content restoration agents according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
When the nano-iron antibacterial agent is prepared, 5ml of PBS buffer solution is injected into a centrifuge tube, 0.01g of nano-iron and 0.05g of nano-iron are respectively added into the centrifuge tube, and ultrasonic oscillation treatment is carried out for 10min at 40 KHz. Adding 0.5ml of nano-iron solution into a new centrifuge tube by using a pipetting gun, injecting 4.5ml of PBS buffer solution into the centrifuge tube, and carrying out ultrasonic treatment to obtain 10 times of diluted nano-iron solution, wherein 5ml of PBS solution containing 0.001g, 0.005g and 0.01g of nano-iron can be obtained by the dilution method, 0.5ml of each solution is respectively sucked and added into 99.5ml of PBS buffer solution, and finally the nano-iron antibacterial agent with the concentration of 1X 10 -6g/ml、5×10- 6g/ml、1×10-5 g/ml is obtained.
Example 2
The optimal dilution factor is explored by selecting fusarium tricuspension with the dilution factor of 10 2、103、104, and the main steps are as follows:
(1) Preparation of bacterial suspension: the fusarium tricuspidatum strain inclined plane is inoculated with a certain amount of bacteria by an inoculating loop and added into 100ml potato dextrose broth culture medium, and the main components are 0.7g/l potato soaked powder, 1.4g/l peptone, 2.1g/l glucose and 0.7g/l sodium chloride. Placing into a constant temperature shaking table, and activating at 28deg.C for 24 hr at 180r/min to obtain fungus mother liquor.
(2) Activating fusarium trilineum: 100 μl of the fungus mother liquor was added to 100ml of nutrient broth medium, and the mixture was placed in a thermostatic shaker and activated at 28℃for 24 hours at 180r/min to obtain a fungus mother liquor at stationary phase.
(3) Diluting bacterial liquid and culturing: the pipette sucks 0.5ml of fungus mother liquor into a 10ml centrifuge tube, then sucks 4.5ml of PBS buffer solution into the centrifuge tube, shakes and shakes evenly to obtain three-wire fusarium diluted 10 1 times, and the steps are repeated to obtain three-wire fusarium diluted 10 2、103、104 respectively.
3 Conical flasks of 250ml were prepared, 95ml of PBS buffer was added to each flask, 5ml of diluted bacterial solution was added to each flask, and after sealing, the flasks were placed in a shaker and incubated at 28℃for 24 hours at 180 r/min.
(4) Coating: the potato agar culture medium is prepared in advance, and the main components of the potato agar culture medium are 1.104g/l of potato soaked powder, 3.68g/l of agar, 3.68g/l of glucose and the pH value is 5.6. 100 μl of each of the cultured mixed solutions was sucked up and dropped onto the surface of the solid medium, smeared with a spreading bar uniformly, and each treatment was repeated four times, and placed in a 28℃incubator for culturing for 48 hours.
(5) Counting: the solid culture medium after 48h of culture is taken for counting, each colony is marked as 1 fungus, as shown in figure 4, the number of the fungi in 4 parallel culture dishes diluted by 10 2 times is not counted, the number of the fungi in 4 parallel culture dishes diluted by 10 3 times is respectively 10, 13, 11 and 28, the average colony number is 16, the number of the fungi in 4 parallel culture dishes diluted by 10 4 times is respectively 1,3 and 3, the average colony number is 2, and the optimal dilution of fusarium trilineum is 10 3 times.
Example 3
(1) Preparation of bacterial suspension: the fusarium tricuspidatum strain inclined plane is inoculated with a certain amount of bacteria by an inoculating loop and added into 100ml potato dextrose broth culture medium, and the main components are 0.7g/l potato soaked powder, 1.4g/l peptone, 2.1g/l glucose and 0.7g/l sodium chloride. Placing into a constant temperature shaking table, and activating at 28deg.C for 24 hr at 180r/min to obtain fungus mother liquor.
(2) Activating fusarium trilineum: 100 μl of the fungus mother liquor was added to 100ml of nutrient broth medium, and the mixture was placed in a thermostatic shaker and activated at 28℃for 24 hours at 180r/min to obtain a fungus mother liquor at stationary phase.
(3) Diluting bacterial liquid and co-culturing: the pipette sucks 0.5ml of fungus mother liquor into a 10ml centrifuge tube, then sucks 4.5ml of PBS buffer solution into the centrifuge tube, shakes and shakes evenly to obtain diluted 10 1 three-wire fusarium, and the step is repeated to obtain diluted 10 3 times three-wire fusarium. 3 conical flasks of 250ml were prepared, 90ml of PBS buffer and 5ml of an antimicrobial agent containing 1X 10 -4g、5×10-4、1×10-3 g of nano-iron were added thereto, and the mixture was shaken well, and 1 conical flask of 250ml was prepared, and 95ml of PBS buffer was added thereto as a control group. 5ml of diluted bacterial liquid is added into each conical flask, a shaking table is put into the conical flask after sealing, and co-culture is carried out for 24 hours at 28 ℃ and 180 r/min.
(4) Coating: the potato agar culture medium is prepared in advance, and the main components of the potato agar culture medium are 1.104g/l of potato soaked powder, 3.68g/l of agar, 3.68g/l of glucose and the pH value is 5.6. 100 μl of each of the cultured mixed solutions was sucked up and dropped onto the surface of the solid medium, smeared with a spreading bar uniformly, and each treatment was repeated four times, and placed in a 28℃incubator for culturing for 48 hours.
(5) Counting: counting the solid culture medium after 48 hours of culture, wherein each colony is marked as 1 fungus, the fungus numbers of 3 parallel culture dishes of a blank control group are respectively 27,2 and 50, and the average colony number is 37; 3 parallel dishes of 1X 10 -6 g/ml antimicrobial had fungus counts of 24, 26, 29, respectively, and an average colony count of 27; the fungus number of 3 parallel culture dishes of 5X 10 -6 g/ml antibacterial agent is 16, 14 and 14 respectively, and the average colony number is 15; the fungus number of 3 parallel culture dishes of the antibacterial agent with the concentration of 1 multiplied by 10 -5 g/ml is 10, 11 and 8 respectively, and the average colony number is 10; as shown in FIG. 6, it was found that the antibacterial agent having a nano-iron concentration of 1X 10 -6g/ml、5×10-6g/ml、1×10-5 g/ml had antibacterial properties of 27.03%, 59.46% and 72.97% when co-cultured with Fusarium tricuspidatum for 24 hours.
Example 4
The antibacterial agents with the nano iron concentration of 0,10 mg/l, 20mg/l, 50mg/l, 100mg/l and 200mg/l are used for cultivating pepper seeds, and the germination rates of 80.00%, 93.33%, 90.00%, 83.33% and 60.00% are respectively known, and as shown in fig. 8, the application study of soil cultivation and germination bags is carried out in the later stage.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (3)
1. The nano iron antibacterial agent for the fusarium trilineum is characterized by being obtained by ultrasonic oscillation treatment of core-shell iron nano particles and phosphate buffer solution, wherein the iron nano particles consist of body-centered cubic iron and face-centered cubic magnetic iron oxide (Fe 3O4); the iron nano-particles only contain iron element and oxygen element, the content is 91.45wt.% and 8.55wt.%, respectively, the average particle diameter of the iron nano-particles is 55.09nm, and the concentration of the iron nano-particles in the nano-iron antibacterial agent is 1X 10 -6g/ml、5×10-6g/ml、1×10-5 g/ml respectively.
2. The nano-iron antibacterial agent for fusarium trilineum according to claim 1, wherein the preparation method of the nano-iron antibacterial agent comprises the following steps: injecting 5ml phosphate buffer solution into a centrifuge tube, adding 0.01g of iron nano particles into the centrifuge tube, carrying out ultrasonic oscillation treatment for 10min at 40KHz, sucking 0.5ml of nano iron solution into a new centrifuge tube by using a pipette, injecting 4.5ml of phosphate buffer solution into the centrifuge tube, carrying out ultrasonic treatment to obtain 5ml of nano iron solution diluted by 10 times, namely, phosphate solution containing 0.001g of nano iron, sucking 0.5ml of nano iron solution, and adding the nano iron solution into 99.5ml of phosphate buffer solution to obtain nano iron antibacterial agent with the concentration of 1X 10 -6 g/ml; the phosphate buffer solution is a water-based salt solution containing sodium chloride, phosphate with the pH of 7.2-7.4, and the main components of the phosphate buffer solution comprise 137mM sodium chloride, 10mM disodium hydrogen phosphate, 1.76mM monopotassium phosphate and 2.7mM potassium chloride.
3. Use of the nano-iron antibacterial agent of claim 1 or 2 for inhibiting fusarium trilineum.
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