CN110741854A - Method for reducing content of CLas in pathogenic bacteria of citrus Huanglongbing disease - Google Patents
Method for reducing content of CLas in pathogenic bacteria of citrus Huanglongbing disease Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention belongs to the technical field of plant disease control, and particularly relates to a method for reducing the content of CLas (pathogen of citrus Huanglongbing) in pathogenic bacteria. The carbon quantum dot nano-oxytetracycline is prepared by applying the carbon quantum dot nano-particles, and the trunk is injected into the citrus plants suffering from the Huanglongbing. The preparation of the carbon quantum dot nano oxytetracycline comprises the preparation of carbon quantum dot nano particles and the preparation of the carbon quantum dot nano oxytetracycline. The carbon quantum dot nano oxytetracycline has the advantage of small size, is smaller than the aperture of the sieve tube, and has good sterilization effect because pathogenic bacteria parasitize in the sieve tube of plants; and the corresponding solution has stable and uniform properties and meets the injection requirements. The method for reducing the content of the CLas of the pathogenic bacteria of citrus greening disease has important significance for preventing and treating the citrus greening disease, and has the potential of large-scale application in agriculture.
Description
Technical Field
The invention belongs to the technical field of plant disease control, and particularly relates to a method for reducing the content of CLas (pathogen of citrus Huanglongbing) in pathogenic bacteria.
Background
Citrus Huanglongbing (HLB) is abused worldwide, and treatment is still lacking in typhoid areas or Citrus varieties requiring protection, and once infected, the tree bodies can only be cut off. Since the 21 st century, scientists have conducted researches on prevention and treatment of huanglongbing disease by small molecular targets of hormone, nutritional intervention, osmotic stress tolerance, CLas virulence, plant growth regulators and the like, and the effect is still not ideal. Although the united states is forced to release Oxytetracycline (Oxytetracycline) and streptomycin from being used in citrus orchard of fossa, the research and development of the novel antibiotic antibacterial drugs are delayed, and a need to find the novel antibacterial drugs to solve the serious problems is needed. Terramycin is concerned because pathogenic bacteria are not easy to generate resistance, and is widely applied to control pathogenic bacteria such as apple and pear fire blight pathogenic bacteria Erwinia amylovora, peach perforation pathogenic bacteria Xanthomonas arboricola and vegetable pathogenic bacteria Pseudomonas spp. In addition, oxytetracycline is also used to control phloem necrosis of palms and elms caused by phytoplasmas by injection into the trunk. Moreover, oxytetracycline is currently the only antibiotic that allows disease control using the trunk injection method.
The fluorescent carbon quantum dot is a novel carbon nano material, has the size of 1-10 nm, and has the characteristics of excellent fluorescence, low toxicity, good biocompatibility, low preparation cost and the like. The carbon quantum dots have more dangling bonds on the surfaces, show unsaturation, are easy to combine with surrounding atoms to form a more stable state, are widely applied to biosensing imaging and display technologies at present, and the fluorescence color of the carbon quantum dots can be simply adjusted by changing the excitation wavelength. The b-PEI coated carbon quantum dots prepared by Hu et al can be uniformly distributed in plasma, and the carbon quantum dots can be used for marking in organisms. The carbon quantum dot particles are used for the fluorescent probe and have obvious advantages, high water solubility, low toxicity and strong light stability, the surfaces of the carbon quantum dot particles are covered with hydrophilic groups such as carboxyl, hydroxyl and the like, so that the water solubility is extremely high, the study of ZHao et al shows that the carbon quantum dots do not damage human renal cells, the cytotoxicity of the carbon quantum dots functionalized by polyacrylic acid (PAA), polyethylene glycol (PEG) and the like is low, and the detection of PL attenuation curve shows that the fluorescence intensity of the carbon quantum dots is stable within 72 h. Therefore, in order to improve the application effect of the oxytetracycline in the citrus greening disease, the invention provides the carbon quantum dot nano oxytetracycline.
Disclosure of Invention
The invention aims to provide a method for reducing the content of CLas (pathogenic bacteria of Candidatus Liberibacter astricus) of citrus varieties suffering from citrus yellow shoot disease.
In order to achieve the purpose, the invention adopts the following scheme:
the method for reducing the content of the CLas of the pathogenic bacteria of the citrus Huanglongbing comprises the steps of drilling a hole with the depth of 0.6-1.5 cm at a position 10-15 cm away from the ground, and injecting carbon quantum dot nano-oxytetracycline by using an injector; the preparation of the carbon quantum dot nano oxytetracycline comprises the following steps:
1) preparing carbon quantum dot nanoparticles: preparing by a hydrothermal method, weighing citric acid and ethylenediamine, dissolving in deionized water, heating at 200 ℃ overnight, naturally cooling to room temperature after reaction, adding 10% polyacrylic acid into a carbon quantum dot solution, heating at 80 ℃ for 4h to obtain carbon quantum dot-polyacrylic acid, dialyzing, and vacuum drying to obtain carbon quantum dot nanoparticles;
2) preparing carbon quantum dot nano oxytetracycline: dispersing the prepared carbon quantum dot nano-particles in water, adding oxytetracycline for dispersion, stirring at 80 ℃ for 1h, and then stirring to normal temperature to obtain the carbon quantum dot nano-oxytetracycline.
Further, the carbon quantum dot nano-particles are connected with oxytetracycline in an adsorption manner.
Furthermore, the injection concentration of the carbon quantum dot nano oxytetracycline is 0.1-0.2 g/strain.
Preferably, the injection concentration of the carbon quantum dot nano oxytetracycline is 0.1 g/strain.
Further, the citrus variety is a summer orange plant.
Further, the mass volume ratio of the citric acid to the ethylenediamine is 1.05: 0.335.
Further, the volume ratio of the ethylenediamine to the polyacrylic acid was 0.335: 1.
Further, the size of the carbon quantum dot nanoparticle is 4 nm.
Furthermore, the surface of the carbon quantum dot nano particle is provided with hydrophilic groups-OH and-COOH.
Further, the content ratio of the carbon quantum dot nano-oxytetracycline in the plant stems, roots and leaves after being injected for 24 hours is 219:173: 83.
The invention has the beneficial effects that:
the carbon quantum dot nano oxytetracycline provided by the invention has the advantage of small size, is smaller than the aperture of a sieve tube, and has good sterilization effect because pathogenic bacteria parasitize in the sieve tube of plants; and the corresponding solution has stable and uniform properties and meets the injection requirements. The method for reducing the CLas content of the citrus varieties suffering from citrus greening disease has important significance for preventing and treating the citrus greening disease, and can be applied to agriculture in a large scale.
Drawings
Fig. 1 is a transmission electron micrograph of carbon quantum dot nanoparticles.
Fig. 2 is a transmission electron microscope image of iron oxide nanoparticles.
FIG. 3 is a fluorescence spectrum of carbon quantum dot nanoparticles at different excitation wavelengths.
Fig. 4 is a fourier transform infrared spectrum of carbon quantum dot nanoparticles.
FIG. 5 shows a carbon quantum dot nano oxytetracycline solution.
FIG. 6 shows a solution of iron oxide nano-oxytetracycline.
Fig. 7 shows the metastasis and distribution of the three oxytetracycline formulations in 5-year-old Valencia stem tissue, with a, b representing significance of difference at the 0.05% level (P < 0.05).
FIG. 8 shows the metastasis and distribution of three oxytetracycline formulations in 5-year-old Valencia root tissues, and a and b represent significance of difference at the 0.05% level (P < 0.05).
Fig. 9 shows the metastasis and distribution of the three oxytetracycline formulations in 5-year-old Valencia leaf tissue, with a, b representing significance of difference at the 0.05% level (P < 0.05).
FIG. 10 shows the level of xanthomonas campestris in leaves of 4-year old Valencia orange treated with different oxytetracycline formulations.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1 preparation of carbon Quantum dot Nanotetracycline
Preparing carbon quantum dot nanoparticles: the preparation method comprises the steps of weighing 1.0507g of citric acid and 335 mu L of ethylenediamine, dissolving in 10mL of deionized water, transferring the solution onto a flask (50mL), heating at 200 ℃ overnight, naturally cooling a reactor to room temperature after reaction to achieve a production yield of 50%, adding 1mL of 10% polyacrylic acid (PAA, Sigma) into a carbon quantum dot solution, heating at 80 ℃ for 4 hours to obtain carbon quantum dot-polyacrylic acid, dialyzing in a dialysis bag, and drying in vacuum to obtain carbon quantum dot nanoparticles;
preparing carbon quantum dot nano oxytetracycline: dispersing the prepared carbon quantum dot nano-particles in water, adding oxytetracycline for dispersion, stirring at 80 ℃ for 1h, and then stirring to normal temperature to obtain the carbon quantum dot nano-oxytetracycline, wherein the carbon quantum dot nano-oxytetracycline preparation is reddish brown, the solution is uniform and stable, and no precipitate is generated after long-term use (figure 5).
Example 2 preparation of iron oxide Nanotetracycline
Preparing iron oxide nanoparticles: the iron oxide nano-particles are prepared by a coprecipitation method, 8.1g FeCl3.6H is added2Dissolving O in deionized water, transferring the obtained solution to a three-neck cookerBottle, heat to 70 ℃ with stirring. 4.4g FeCl2·4H2Dissolving O in 10mL of deionized water, filtering, adding 75mL of the filtered solution into a three-neck flask, adding 18mL of 25% concentrated ammonia water under vigorous stirring, dropwise adding 4.66g of oleic acid after 1min, stirring for 1h at 70 ℃, obtaining a black jelly-shaped precipitate after the reaction is finished, separating the precipitate from a reaction system through an external magnetic field, washing with ethanol twice to remove excessive oleic acid, and washing with deionized water to be neutral.
Preparing iron oxide nano oxytetracycline: dispersing the prepared iron oxide nano-particles in water, respectively adding oxytetracycline for dispersion, stirring at 80 ℃ for 1h, and then stirring to normal temperature to obtain the iron oxide nano-oxytetracycline. The iron oxide nano oxytetracycline preparation is dark brown, the solution is relatively uniform, the color of the solution becomes light after the solution is stored for a long time, and brown precipitates are generated at the bottom of a container (figure 6).
Example 3 nanoparticle characterization
A scanning Electron microscope (Transmission Electron microscope, TEM) can observe the morphology and structure of the nano-particles with the particle size less than 0.2 μm. The TEM sample preparation process comprises the following steps: and cutting off the carbon conductive adhesive and attaching the carbon conductive adhesive to a sample table, dipping a small amount of powder sample and adhering the powder sample to the surface of the conductive adhesive, vacuumizing, and performing shape observation and component analysis after carbon spraying. And measuring the fluorescence spectrum of the sample by using a fluorescence photometer, and detecting the chemical groups by using Fourier transform infrared spectroscopy.
The characterization result shows that the transmission electron microscope characterizes that the fluorescent carbon quantum dot nanoparticles have good dispersibility (figure 1), the particle size distribution is uniform, the size of the carbon quantum dot nanoparticles is 4nm, and the size of the iron oxide nanoparticles is 6nm (figure 2); the fluorescence spectrum result of the carbon quantum dot nanoparticles shows that the carbon quantum dot nanoparticles are excited every 10nm from the excitation wavelength of 320nm to 360nm, when the excitation wavelength is 350nm, the fluorescence emission wavelength of the carbon quantum dot nanoparticles is strongest, and the emission wavelength is basically unchanged along with the increase of the excitation wavelength, which indicates that the carbon quantum dot nanoparticles do not have the wavelength dependence (fig. 3, which indicates that the fluorescence characteristic is not limited by the excitation wavelength); the Fourier transform infrared spectrum result of the carbon quantum dot nanoparticles shows that (figure 4), hydrophilic groups-OH and-COOH are arranged on the surfaces of the carbon quantum dot nanoparticles, so that the water solubility of the carbon quantum dot nanoparticles is ensured.
Example 4 detection of oxytetracycline profiles
The iron oxide nano-oxytetracycline, the carbon quantum dot nano-oxytetracycline and the common oxytetracycline preparation are injected, holes are drilled at the position 15cm away from the ground to a depth of 0.6cm, an injector is used for injecting the three medicines into the Valencia orange which is 5 years old, 0.1g of the three medicines are injected, and the amount of each injection treatment sample is 3. Collecting root, stem and leaf tissues 6h, 24h and 48h after the injection, wrapping with tinfoil paper, recording, storing in a refrigerator at-80 deg.C, and detecting oxytetracycline distribution by ELISA method.
The detection sample collection method comprises the following steps:
1) and (3) stem: collecting an injection hole injected by a trunk of the Valencia orange plant, and cutting stem tissues with xylem by using a scalpel at the position of 40cm upwards;
2) a blade: collecting mature leaves (1 year leaves) in 3 different directions randomly, and mixing;
3) root: removing surface soil, and collecting fibrous roots about 5cm below the surface of the earth.
And 2d after the carbon quantum dot nano oxytetracycline is injected, collecting root, stem and leaf tissues, and positioning by using a confocal fluorescence microscope after a frozen section is manufactured. The root, stem and leaf tissue collection methods were as above.
In addition, three preparations of iron oxide nano-oxytetracycline, carbon quantum dot nano-oxytetracycline and ordinary oxytetracycline are injected into 5-year-old Valencia orange, 0.1g of active ingredients are injected into each tree, and the injection treatment sample volume is 3 plants for the sick plant control by injecting clear water. Leaf samples were collected before injection and at 5d and 10d after injection, respectively, and the CLas content was determined, the leaf collection method being as above.
Test results show that the transfer speeds of the carbon quantum dot nano-oxytetracycline, the iron oxide nano-oxytetracycline and the ordinary oxytetracycline in stems, leaves and roots of the Valencia orange are different.
1) And (3) stem: 6h after the trunk is injected with the oxytetracycline, ELISA detection shows that the content of the nano oxytetracycline with carbon quantum dots in the stem is the highest, wherein the content of the nano oxytetracycline is 134.73 mug/kg per stem, and the balance of the nano oxytetracycline is ferric oxide and the common oxytetracycline in sequence; the content of the three preparations in the stem is increased within 48 hours after injection, and the content of the carbon quantum dot nano oxytetracycline is 197.66 mug/kg of stem and 219.09 mug/kg of stem respectively 12 hours and 24 hours after injection.
The SPSS20.0 significance analysis result shows that the content of the carbon quantum dot nano-oxytetracycline in the stem is remarkably higher than that of the iron oxide nano-oxytetracycline and the ordinary oxytetracycline 6h, 24h and 48h after the injection of the medicament; the content of the iron oxide nano-oxytetracycline in the stem is obviously higher than that of the common oxytetracycline preparation 6 hours and 24 hours after injection; 48h after injection, the content of the iron oxide nano-oxytetracycline in the stem is equivalent to that of the ordinary oxytetracycline (figure 7).
The carbon quantum dot nano-oxytetracycline fluorescence microscopic observation result shows that oxytetracycline is distributed more uniformly in plant stem tissues and mostly concentrated in fiber and phloem tissues. In comparison, the blue fluorescence of the carbon quantum dot nano oxytetracycline 24h after injection is not obviously increased compared with that of the carbon quantum dot nano oxytetracycline 6h after injection, but ELISA detection shows that the oxytetracycline content in stems is increased 24h after injection compared with 6 h.
2) Root: the oxytetracycline can be detected in root tissues 6 hours after the trunk is injected with the oxytetracycline, and the content range is 125-144 mug/kg root; 24h after injection, the terramycin content in the root is increased, wherein the concentration of the carbon quantum dot nano terramycin is increased to 173.23 mug/kg root; 48 hours after injection, the terramycin content in the roots is improved compared with 24 hours, and the concentration of the carbon quantum dot nano terramycin with the maximum content can reach 234.45 mu g/kg per root.
SPSS20.0 significance analysis shows that the content of carbon quantum dot nano oxytetracycline in roots is obviously higher than that of oxytetracycline oxide and ordinary oxytetracycline 6 hours after injection; 24h after injection, the three oxytetracycline preparations have no difference significance in roots; 48h after injection, the content of the carbon quantum dot nano oxytetracycline in the roots is obviously higher than that of the common oxytetracycline, but the carbon quantum dot nano oxytetracycline has no obvious difference with the iron oxide nano oxytetracycline (figure 8).
3) A blade: 6 hours after the trunk injection, the terramycin content of the leaf tissue is 45-65 mug/kg per leaf; 24 hours after injection, the average content of the carbon quantum dot nano oxytetracycline is 83.24 mug/kg per leaf; 48 hours after injection, the oxytetracycline content in the leaves is improved compared with 24 hours, and the average content of the carbon quantum dot nano oxytetracycline is 118.49 mug/kg per leaf. The temperature during the test is supposed to be low (11-16 ℃), and the transpiration effect is poor, so that the speed of transporting to the leaves is slow, the plants are high, and the amount of transporting to the leaves is small.
SPSS significance analysis shows that the content of carbon quantum dot nano oxytetracycline is significantly higher than that of iron oxide nano oxytetracycline and common oxytetracycline preparations 6h, 24h and 48h after injection, and the nano preparations move fast due to small particle size in a long-distance transportation process and have certain transportation and diffusion advantages (figure 9).
Example 5 bacteriostatic effect of carbon quantum dot nano oxytetracycline
The CLas content of morbid summer orange 7 days after the carbon quantum dot nano-oxytetracycline and the ordinary oxytetracycline are injected, wherein the CLas content of the carbon quantum dot nano-oxytetracycline treated is 1.55 multiplied by 107 +/-1.02 multiplied by 107cell/g.blade is reduced to 2.87 multiplied by 106±1.20×106cells/g blade, reduced by 81.48%; after the patient is injected with the ordinary terramycin, the CLas content is from 1.67 multiplied by 107±1.13×107cell/g.blade is reduced to 5.35X 106 +/-2.93X 106cells/g.blade, reduced by 67.96%; the CLas content in the sick plant injected with clear water is 1.52 × 107±8.80×106cells/g.blade drop to 1.34X 107±7.03×106cells/g.leaf, after which the content of CLas increased to 1.47X 10 by 15d6±1.25×106cells/g.vane. Compared with the two, the carbon quantum dot nano-oxytetracycline has more advantages than the common oxytetracycline in the aspect of preventing and treating the CLas (figure 10).
Comprehensive analysis shows that the carbon quantum dot nano-oxytetracycline has better therapeutic action on HLB than the common oxytetracycline preparation, and has development potential. Because the carbon quantum dot nano oxytetracycline has an obvious advantage compared with the common oxytetracycline, the carbon quantum dot nano particle mediated oxytetracycline is presumed to enter the sieve tube tissues of plants due to small particle size, but whether HLB is relapsed after drug withdrawal needs to be observed and verified for a long time. In addition, no adverse effect was observed on the test Valencia plants, as observed by the internal structure and external characteristics of the plants during the test.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (9)
1. A method for reducing the content of CLas in pathogenic bacteria of citrus Huanglongbing is characterized in that holes are drilled at a position 10-15 cm away from the ground for 0.6-1.5 cm in depth, and carbon quantum dot nano-oxytetracycline is injected into a trunk; the preparation of the carbon quantum dot nano oxytetracycline comprises the following steps:
1) preparing carbon quantum dot nanoparticles: preparing by a hydrothermal method, weighing citric acid and ethylenediamine, dissolving in deionized water, heating at 200 ℃ overnight, naturally cooling to room temperature after reaction, adding 10% polyacrylic acid into a carbon quantum dot solution, heating at 80 ℃ for 4h to obtain carbon quantum dot-polyacrylic acid, dialyzing, and vacuum drying to obtain carbon quantum dot nanoparticles;
2) preparing carbon quantum dot nano oxytetracycline: dispersing the prepared carbon quantum dot nano-particles in water, adding oxytetracycline for dispersion, stirring at 80 ℃ for 1h, and then stirring to normal temperature to obtain the carbon quantum dot nano-oxytetracycline.
2. The method of claim 1, wherein the carbon quantum dot nanoparticles are attached to oxytetracycline by adsorption.
3. The method of claim 1, wherein the injection concentration of the carbon quantum dot nano oxytetracycline is 0.1-0.2 g/strain.
4. The method of claim 1, wherein the citrus variety is a 4-year old potamon Valencia plant.
5. The method of claim 1, wherein the mass to volume ratio of citric acid to ethylenediamine is 1.05: 0.335.
6. The method of claim 1, wherein the volume ratio of ethylenediamine to polyacrylic acid is 0.335: 1.
7. The method of claim 1, wherein the carbon quantum dot nanoparticles are 4nm in size.
8. The method of claim 1, wherein the carbon quantum dot nanoparticles have hydrophilic groups-OH and-COOH on the surface.
9. The method as claimed in claim 1, wherein the content ratio of the carbon quantum dot nano-oxytetracycline in the plant stems, roots and leaves after 24h of injection is 219:173: 83.
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