CN115530179B - Carbon nano preparation capable of inhibiting rice pathogenic bacteria in broad spectrum, and preparation method and application thereof - Google Patents

Carbon nano preparation capable of inhibiting rice pathogenic bacteria in broad spectrum, and preparation method and application thereof Download PDF

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CN115530179B
CN115530179B CN202211188247.1A CN202211188247A CN115530179B CN 115530179 B CN115530179 B CN 115530179B CN 202211188247 A CN202211188247 A CN 202211188247A CN 115530179 B CN115530179 B CN 115530179B
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rice
preparation
carbon nano
bacteria
rice blast
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CN115530179A (en
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王莫
康振辉
陈秋实
黄健
时华
黄慧
陈凤平
王宗华
鲁国东
曾文治
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Suzhou University
Fujian Agriculture and Forestry University
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
    • C25B—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR
    • C25B1/00—Electrolytic production of inorganic compounds or non-metals
    • C25B1/01—Products
    • C25B1/135—Carbon
    • 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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Abstract

The invention discloses a carbon nano preparation capable of effectively inhibiting rice pathogenic bacteria in a broad spectrum, and a preparation method and application thereof. Placing a high-purity graphite rod which is cleaned by ultrapure water into a 50% phytic acid solution as a cathode and an anode, continuously electrolyzing to obtain a phytic acid dispersion liquid of graphene, filtering the phytic acid dispersion liquid of graphene by qualitative filter paper, centrifuging at a high speed, and dialyzing by ultrapure water to obtain the carbon nano preparation. The particle size of the nano particles in the carbon nano preparation is 2-5 nm, an unordered crystal structure exists, and oxygen-containing and phosphorus-containing functional groups are arranged on the surface of the nano particles, so that the nano particles can effectively inhibit the growth of Pyricularia oryzae, ustilaginoidea virens and bacterial blight bacteria. The nano preparation can be targeted to a microfilament skeleton consisting of filiform actin in rice blast bacteria cells, so that the polarity of the rice blast bacteria cells is lost, and the rice blast bacteria cells are abnormally expanded to rupture. Therefore, the carbon nano preparation is utilized to pretreat rice, so that the infection of rice blast bacteria can be effectively reduced, and the occurrence of rice blast can be prevented.

Description

Carbon nano preparation capable of inhibiting rice pathogenic bacteria in broad spectrum, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of nano materials and the research and development field of novel pesticides, and relates to a protective carbon nano preparation capable of effectively inhibiting growth of rice pathogenic bacteria in a broad spectrum, preventing and controlling occurrence of rice diseases, and a preparation method and application thereof.
Background
Although the traditional chemical pesticide can also effectively prevent and treat diseases and protect crop production, the traditional chemical pesticide has the characteristics of high toxicity, high residue, unfriendly ecological environment and the like, and can not meet the development needs of modern green agriculture at present. Along with the gradual enhancement of the awareness of people on environmental protection and food safety, the development of efficient, low-toxicity and environment-friendly green biopesticide has become the main trend of development in the field of prevention and control of agricultural diseases of various countries in the world.
The continuous development of nano technology makes it possible to nano various kinds of material and to make the material possess physical and chemical characteristic different from that of material. To date, although there have been many developments of nano-sized heavy metal ion bactericides, the accumulation of heavy metal ions in the ecological environment caused by the nano-sized bactericides has been reported. Therefore, the environment-friendly nano bactericide is developed, and the selection and innovation of raw material substances are key. The biological source nontoxic micromolecular organic matter is used as a raw material to prepare the bacteriostatic agent in a nanometer dosage form, which is a new idea for developing safe and environment-friendly novel biological pesticides.
Rice [ (Oryza sativa L.)Oryza sativa) Is the most important grain crop in China and even worldwide, and the stable yield of rice is also an important guarantee for the grain safety in China. From rice blast bacteriaMagnaporthe oryzae) The induction of rice blast has been the most serious fungal disease that jeopardizes rice production. The yield loss of the rice, which is directly caused by rice blast in China, is up to 30 hundred million kilograms each year, and the yield of the rice can be reduced by 40% -50% even is out of order when the disease is serious. The current pesticides commonly used for preventing and treating rice blast in rice production comprise: tricyclazole, isoprothiolane, prochloraz, pyraclostrobin and difenoconazoleCyproconazole, kasugamycin, etc., depending on their components, the transitional application of these chemical pesticides all causes agroecological safety problems. Several nano materials have been reported in recent years to have the effect of preventing and treating rice blast, such as nano silver, nano silicon, nano chitosan and the like. Only nano silver has proved to have the effect of directly inhibiting the growth of rice blast bacteria, however, the application safety of the heavy metal nano material is still to be further measured. Is prepared from Xanthomonas oryzaeXanthomonas oryzae pv. oryzae) Bacterial leaf blight is widely regarded as the bacterial disease most severely affecting rice yield. Bacterial blight bacteria are mainly infected from the wound, stomata and other parts of rice, and the production control mainly depends on the application of chemical pesticides such as metconazole, thiabendazole suspending agent and the like. Is prepared from ustilaginoidea virensUstilaginoidea virens) The false smut is a spike disease which seriously affects the quality of rice, and has a tendency of large-area popularity and increasing year by year in China in recent years. Due to the lack of disease-resistant varieties and the concealment of occurrence of the diseases, the existing rice production lacks an effective green prevention and control technology aiming at the occurrence of false smut. The invention successfully creates a target rice blast fungus microfilament skeleton actin, thereby effectively inhibiting the growth of rice blast fungus and preventing rice infection, and the carbon nano preparation can prevent and control rice blast. In addition, the carbon nano preparation has good inhibition effect on the growth of rice false smut bacteria and bacterial leaf blight bacteria.
Disclosure of Invention
The invention aims to provide a carbon nano preparation capable of effectively inhibiting the growth of rice blast germ, ustilaginoidea virens and bacterial blight germ and a preparation method thereof, and the carbon nano preparation can prevent and control the occurrence of rice blast.
In order to achieve the technical purpose and the technical effect, the invention is realized by the following technical scheme:
a preparation method of a carbon nano preparation capable of inhibiting rice pathogenic bacteria in a broad spectrum comprises the following steps:
1) Ultrasonically cleaning two high-purity graphite rods with ultrapure water for 5min, wherein the ultrasonic power is 300W, the ultrasonic frequency is 40KHz, and then, absorbing the surface water with absorbent paper;
2) Taking 1000ml of a phytic acid aqueous solution with the mass fraction of 50% as an electrolyte, placing a cleaned high-purity graphite rod as a cathode and an anode in the electrolyte, keeping the distance between the two electrodes at 3-10 cm, and continuously electrolyzing for 25-30 days under the condition of adding a constant-voltage direct-current power supply at 15-30V to obtain a phytic acid dispersion liquid of graphene;
3) Filtering the phytic acid dispersion liquid of the graphene with medium-speed qualitative filter paper for three times, centrifuging for 45min under the condition of 22000rcf by adopting a centrifuge, discarding the precipitate, and collecting the supernatant;
4) Pouring the supernatant into a dialysis bag with the molecular weight cut-off of 500-1000D, and dialyzing in ultrapure water for 24h to obtain the carbon nano preparation capable of effectively and widely inhibiting rice pathogenic bacteria.
The carbon nano preparation prepared by the preparation method is provided.
The application of the carbon nano preparation in inhibiting rice pathogenic bacteria is provided.
Further, the inhibition of rice pathogenic bacteria is inhibition of hypha growth and spore germination of rice pathogenic bacteria.
Further, the rice pathogenic bacteria are selected from Pyricularia oryzae, aspergillus oryzae and Rhizoctonia solani.
The invention has the following beneficial effects:
the invention can simply and rapidly prepare the carbon nano preparation containing the nano particles with high purity and uniform particle size, and the carbon nano preparation can effectively inhibit the growth of rice blast germ, ustilaginoidea virens and bacterial blight germ and can prevent the occurrence of rice blast.
Drawings
FIG. 1 is a transmission electron microscope image, a particle size distribution diagram, an infrared spectrum and a Raman spectrum of a carbon nano preparation. a, a transmission electron microscope image; b, particle size distribution map; c, infrared spectrum; d, raman spectrum diagram.
Fig. 2 is an ultraviolet absorption diagram of a carbon nano-formulation.
Fig. 3 is an X-ray photoelectron spectrum of a carbon nano-formulation.
FIG. 4 shows inhibition of Pyricularia oryzae by carbon nanoformulations and phytic acid.
FIG. 5 shows the mycelium morphology (scale: 20 μm) of rice blast bacteria treated with a carbon nanopreparation and phytic acid for 15 hours.
FIG. 6 is the effect of carbon nanoformulations on conidia of Pyricularia oryzae. a, the spore state of rice blast bacteria (scale 50 μm) after 24 hours of treatment of the carbon nano preparation; b, germination rate of conidium of Pyricularia oryzae after 24 hours of treatment of the carbon nano preparation.
FIG. 7 is a co-localization observation of autofluorescence of a carbon nano-preparation and rice blast fungus F-actin (scale 20 μm).
FIG. 8 shows the final onset of rice leaves treated with carbon nanoagent spray 12 hours before and after rice blast inoculation.
FIG. 9 shows the inhibition of Ustilago virens growth by carbon nanoformulations in PDA medium.
FIG. 10 is a graph showing the statistics of inhibition of bacterial growth by carbon nanoformulations.
The specific embodiment is as follows:
the present invention will be described in further detail with reference to the following embodiments.
The experimental materials involved in the invention are as follows:
high-purity graphite rod: the graphite content is more than 99%.
Ultrapure water: the resistivity is 18.4M Ω cm -1 。
Phytic acid solution: the mass fraction is 50%.
Example 1 preparation of carbon nanoformulations
Step 1: taking two high-purity graphite rods, ultrasonically cleaning the two high-purity graphite rods with ultrapure water at 25 ℃ for 5min, wherein the ultrasonic power is 300W and the ultrasonic frequency is 40KHz, and then using water absorbing paper to absorb water on the surface;
step 2: taking 1000ml of a phytic acid aqueous solution with the mass fraction of 50% as an electrolyte, placing a cleaned high-purity graphite rod as a cathode and an anode in the electrolyte, keeping the distance between the two electrodes at 3-10 cm, and continuously electrolyzing for 25-30 days under the condition of adding a constant-voltage direct-current power supply at 30V to obtain a phytic acid dispersion liquid of graphene;
step 3: filtering the phytic acid dispersion liquid of the graphene with medium-speed qualitative filter paper for three times, centrifuging for 45min under the condition of 22000rcf by adopting a centrifuge, discarding the precipitate, and collecting the supernatant;
step 4: pouring the supernatant into a dialysis bag with the molecular weight cut-off of 500-1000D, and dialyzing in ultrapure water for 24h to obtain the carbon nano preparation capable of effectively and widely inhibiting rice pathogenic bacteria.
Example 2 characterization parameters of carbon nanoformulations
The carbon nanofabricated formulation prepared in example 1 has the following characteristics (fig. 1 to 3):
as shown by the characterization of a transmission electron microscope, the particle size of the nano particles in the carbon nano preparation prepared by the invention is 2-5 nm. As can be seen from Raman spectrum, there are two peaks in the pulled-up spectrum of the carbon nano-preparation, which correspond to D-bands of the carbon nano-particles (1334 and 1334 cm -1 ) G belt (1601 cm) -1 ). If the peak intensity of the D band is higher than that of the G band in the raman spectrum, this indicates that a carbon nano-preparation has a large number of defects, and the ratio (ID/IG) of the peak intensities of the D band and the G band can measure the degree of disorder thereof. The ID/IG value of the prepared carbon nanofabric was calculated to be 1.11, suggesting the presence of an disordered crystal structure in the carbon nanofabric of the present invention. As can be seen from infrared and X-ray photoelectron spectroscopy tests, the surface of the carbon nano preparation provided by the invention has oxygen-containing functional groups, namely-OH, -COOH and C-O-C, P-O, C-C.
EXAMPLE 3 inhibitory Effect of carbon nanoformulations on Pyricularia oryzae
In order to test the inhibition effect of the carbon nano preparation on the growth of rice blast fungus hypha, the invention adds 75 mug/mL and 150 mug/mL of carbon nano preparation or phytic acid SYM culture medium (each liter of SYM culture medium contains 10 g soluble starch, 2 g yeast extract, 3 g sucrose, 20 g agar powder and pH=6.5), transfers rice blast fungus Guy11 bacterial cakes with the same size, and cultures for 5 days at 28 ℃ to calculate the growth inhibition rate, and takes SYM culture medium without adding the carbon nano preparation or phytic acid as a control. As a result, it was found (fig. 4) that carbon nano-preparation treatment was more effective in inhibiting the hypha growth of rice blast fungus than the original material phytic acid, and the difference was found to be extremely remarkable by statistical analysis.
The cultured rice blast fungus Guy11 bacterial cakes with the same size are respectively placed into CM liquid culture medium containing 75 mug/mL of carbon nano preparation or phytic acid, the CM liquid culture medium without the carbon nano preparation or phytic acid is used as a control, after the culture is carried out for 15 hours at the speed of 28 ℃ of 150 rpm, the phenomenon that the rice blast fungus hypha cells treated by the carbon nano preparation are expanded and vacuolated can be found through microscopic observation (shown in figure 5 and indicated by white arrows), and the rice blast fungus hypha treated by the phytic acid is not obviously different from the control.
Inoculating Pyricularia oryzae Guy11 into rice bran culture medium (containing 40 g rice bran and 20 g agar powder per liter of rice bran culture medium, pH=6.5), washing conidium of Guy11 with 0.02% Tween20 from rice bran culture medium cultured for 10 days, and filtering with double-layer filter paper to adjust spore concentration to 5×10 4 And adding 75 mug/mL or 150 mug/mL of the carbon nano preparation, dripping the carbon nano preparation on a hydrophobic glass slide, and counting the spore germination rate after the carbon nano preparation is placed for 24 hours under the conditions of moisture preservation and darkness at the temperature of 22 ℃. One treatment was performed at least three times, 100 or more conidia at a time. * Representative differences are very significant. As can be seen from fig. 6, the carbon nano-preparation also has a significant inhibitory effect on germination of conidia of rice blast germ.
Example 4 carbon nanoformulations inhibit target subcellular structures of Pyricularia oryzae
After culturing Pyricularia oryzae strain Guy carrying F-actin-labeled red fluorescent protein in 150 rpm shaker for 11 hr using CM liquid medium containing 75 μg/mL carbon nanofabric, it was observed using confocal microscope. Since the carbon nanofabric spontaneously fluoresces, co-localization of the carbon nanofabric with F-actin was observed as shown in fig. 7. The results show that the carbon nano preparation can target a microfilament skeleton consisting of filiform actin in rice blast fungus hypha cells to destroy the polarity of the hypha cells, thereby achieving the effect of inhibiting the growth of the rice blast fungus. This result is consistent with the carbon nanopreparation of fig. 5, which causes the expansion of hyphal cells and vacuoles.
EXAMPLE 5 control Effect of carbon nanoformulations on Pyricularia oryzae
In order to further explore the control effect and the use method of the carbon nano preparation on rice blast, the invention uses 150 mug/mL of the carbon nano preparation to uniformly spray and treat rice seedlings according to 0.5 mL/plant for 12 hours before and after rice seedlings are inoculated with rice blast fungus Guy11, and the disease condition is observed after 6 days. As shown in FIG. 8, the rice blast can be effectively prevented and controlled by using the carbon nano preparation treatment before the rice blast inoculation; and the carbon nano preparation treatment after inoculation has no obvious effect on preventing and treating rice blast. The results show that the 150 mug/mL carbon nano preparation has better prevention and control effects on rice leaf blast.
Example 6 growth inhibitory Effect of carbon nanopreparation on Ustilago oryzae
The inhibition effect of 75 mug/mL and 150 mug/mL of carbon nano-preparation on the growth of the Ustilago virens strain UV-FJ-1 is tested in a PDA solid culture medium, and the PDA solid culture medium without the carbon nano-preparation is used as a control. As shown in fig. 9, after 30 days of culture at 28 ℃, the calculated growth inhibition rate shows that the carbon nano preparation has a remarkable inhibition effect on the growth of ustilaginoidea virens compared with the control, and the higher the concentration, the stronger the inhibition effect.
EXAMPLE 7 growth inhibitory Effect of carbon nanoformulations on bacterial leaf blight bacteria
The invention further tests the inhibition effect of the carbon nano preparation on rice bacterial blight bacteria in NAA solid culture medium. The specific method comprises the following steps: the bacterial strain PX099 is cultured in liquid culture medium (20 g/L sucrose+5 g/L peptone+0.5 g/L FeSO 4 . 7H 2 O + 0.5 g/L Ca(NO 3 ).4H 2 O + 2 g/L Na 2 HPO 4 Ph=6.8) at 28 ℃ to OD 600 =0.4, then 5 μl was added dropwise to NAA solid medium containing 75 μg/mL and 150 μg/mL carbon nanofabrication, respectively. After two days of incubation at 28℃the bacteria were washed with 1mL of sterile water, then 10 gradients were diluted 10 times each time, and added dropwise to NAA solid medium, after 2 days the number of countable colonies was counted, and the growth index was calculated. As shown in FIG. 10, the carbon nano-preparation of 75. Mu.g/mL and 150. Mu.g/mL was remarkable for growth of bacterial leaf blight bacteriaThe inhibition effect is remarkable, and the higher the concentration is, the stronger the inhibition effect is. The parent representations of the different lowercase cases differ significantly from each other.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (4)

1. A preparation method of a carbon nano preparation capable of inhibiting rice pathogenic bacteria in a broad spectrum is characterized by comprising the following steps: the method comprises the following steps:
1) Ultrasonically cleaning two high-purity graphite rods with ultrapure water for 5min, wherein the ultrasonic power is 300W, the ultrasonic frequency is 40KHz, and then, absorbing the surface water with absorbent paper;
2) Taking 1000ml of a phytic acid aqueous solution with the mass fraction of 50% as an electrolyte, placing a cleaned high-purity graphite rod as a cathode and an anode in the electrolyte, keeping the distance between the two electrodes at 3-10 cm, and continuously electrolyzing for 25-30 days under the condition of adding a constant-voltage direct-current power supply at 15-30V to obtain a phytic acid dispersion liquid of graphene;
3) Filtering the phytic acid dispersion liquid of the graphene with medium-speed qualitative filter paper for three times, centrifuging for 45min under the condition of 22000rcf by adopting a centrifuge, discarding the precipitate, and collecting the supernatant;
4) And pouring the supernatant into a dialysis bag with the molecular weight cut-off of 500-1000D, and dialyzing in ultrapure water for 24 hours to obtain the carbon nano preparation capable of effectively and widely inhibiting rice pathogenic bacteria.
2. A carbon nano-preparation produced by the production method of claim 1.
3. The use of the carbon nano-preparation according to claim 2 for inhibiting rice blast germ, false smut germ and bacterial blight germ.
4. A use according to claim 3, characterized in that: the rice blast bacteria inhibition means the inhibition of the hypha growth and spore germination of rice blast bacteria, the rice false smut bacteria inhibition means the inhibition of the hypha growth of rice false smut bacteria, and the rice bacterial leaf blight bacteria inhibition means the inhibition of the colony growth of rice bacterial leaf blight bacteria.
CN202211188247.1A 2022-09-28 2022-09-28 Carbon nano preparation capable of inhibiting rice pathogenic bacteria in broad spectrum, and preparation method and application thereof Active CN115530179B (en)

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CN107793264A (en) * 2017-10-25 2018-03-13 中国矿业大学(北京) A kind of Nano Carbon water-retention fertilizer and its preparation method and application
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