A method for reducing the ability of citrus psyllids to acquire Huanglongbing bacteria using water-soluble ZnO quantum dots

Abstract

The present invention discloses a method for reducing the ability of citrus psyllids to acquire Huanglongbing bacteria using water-soluble ZnO quantum dots, belonging to the technical field of controlling the spread of citrus Huanglongbing disease. The present invention discloses a method for reducing the ability of citrus psyllids to acquire Huanglongbing bacteria using water-soluble ZnO quantum dots. The method utilizes the antibacterial properties of the water-soluble ZnO quantum dots and feeds them into the bodies of citrus psyllids. The results demonstrate that the water-soluble ZnO quantum dots not only penetrate the bodies of citrus psyllids, reducing their ability to acquire Huanglongbing bacteria, but also have no toxic effects on plants and can even promote plant growth to a certain extent. Based on these results, the method can effectively control the spread of Huanglongbing disease from citrus psyllids to healthy citrus plants, reducing the risk of healthy plants becoming infected, and addressing the adverse environmental and ecological impacts of existing technologies for controlling citrus psyllids.

Description

Technical Field

[0001] The present invention relates to the technical field of controlling the spread of citrus Huanglongbing disease, and more particularly to a method for reducing the ability of citrus psyllids to acquire Huanglongbing disease bacteria by utilizing water-soluble ZnO quantum dots. Background Art

[0002] Citrus Huanglongbing (HLB) is a devastating disease that occurs on citrus. It is also known as yellow tip disease and yellow wilt disease and is extremely widely distributed worldwide. Symptoms include mottled yellowing of leaves and "red nose" fruit, which affect the yield and quality of citrus. In severe cases, the disease can cause plant death. The citrus psyllid (Diaphorina citri Kuwayama) is an insect of the Psyllidae family (Hemipetra) in the order Hemipetra. It is the main insect vector for spreading citrus Huanglongbing in the field, mainly harming Rutaceae plants such as citrus, orange, tangerine, pomelo and lemon. The citrus psyllid acquires the citrus Huanglongbing pathogen by feeding, then migrates to healthy citrus plants to feed, transmitting the pathogen into the plants and causing the plants to become sick. Therefore, reducing the acquisition of citrus Huanglongbing by citrus psyllids is of great significance for reducing the spread of citrus Huanglongbing. However, there is currently no effective method in the art that can be applied to citrus psyllids and reduce their acquisition of Huanglongbing.

[0003] Therefore, providing a method for using water-soluble ZnO quantum dots to reduce the ability of citrus psyllids to acquire Huanglongbing bacteria is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides a method for reducing the ability of citrus psyllids to acquire Huanglongbing bacteria by using water-soluble ZnO quantum dots. The method can significantly reduce the ability of citrus psyllids to acquire Huanglongbing bacteria, hinder the spread of citrus psyllids to a certain extent, reduce the risk of healthy plants becoming ill, and also reduce the spread of citrus Huanglongbing disease.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for reducing the ability of citrus psyllids to acquire Huanglongbing bacteria by utilizing water-soluble ZnO quantum dots, wherein the water-soluble ZnO quantum dots are utilized to interfere with the ability of citrus psyllids to acquire Huanglongbing bacteria.

[0007] Furthermore, the concentration of the water-soluble ZnO quantum dots is 5 mg / mL and 2 mg / mL.

[0008] It can be seen from the above technical solution that compared with the existing technology, the present invention provides a method for using water-soluble ZnO quantum dots to reduce the ability of citrus psyllids to obtain Huanglongbing bacteria. According to the transmission medium of citrus Huanglongbing, by interfering with the acquisition efficiency of the transmission medium - citrus psyllids, the spread of citrus Huanglongbing is reduced, and the risk of healthy citrus plants being infected is reduced, providing technical support for the effective control of citrus Huanglongbing. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0010] Figure 1 The accompanying figure shows the yellow fluorescence observed when the water-soluble ZnO quantum dot solution of the present invention is excited by ultraviolet light;

[0011] The left picture shows a water-soluble ZnO quantum dot solution; the right picture shows a water-soluble ZnO quantum dot solution that produces yellow fluorescence after being excited by ultraviolet light.

[0012] Figure 2 The accompanying figure shows the water-soluble ZnO quantum dots observed by transmission electron microscopy in the present invention, confirming that the size of the water-soluble ZnO quantum dots is about 5 nm;

[0013] Figure 3 The accompanying figure shows the present invention treating citrus Huanglongbing trees in an orchard by cutting branches, bringing them back to the laboratory for hydroponics, and placing them in insect breeding cages;

[0014] Figure 4 The accompanying figure shows that a citrus tree grafted with citrus greening disease was moved into a laboratory, its branches were placed in an insect breeding cage, and citrus psyllids were placed therein for breeding;

[0015] Figure 5 The accompanying figure shows the detection of citrus Huanglongbing disease on branches and citrus psyllids reared on citrus trees infected with Huanglongbing disease at 7, 12, and 18 days of age;

[0016] Among them, in vitro: branches; in vivo: citrus trees;

[0017] Figure 6 The accompanying figure shows that healthy citrus plants treated with different concentrations of zinc oxide according to the present invention are used to feed citrus psyllids, confirming that water-soluble ZnO quantum dots can enter the body of citrus psyllids;

[0018] Figure 7The accompanying figure shows the detection of citrus Huanglongbing disease bacteria after the citrus psyllid of the present invention is fed on citrus plants that are infected with Huanglongbing disease bacteria;

[0019] Figure 8 The accompanying figure shows the present invention using different concentrations of water-soluble ZnO quantum dots to irrigate the roots of healthy citrus plants, confirming that the water-soluble ZnO quantum dots have no toxic effects;

[0020] Among them, A: 5 mg / mL water-soluble ZnO quantum dots treatment; B: 2 mg / mL water-soluble ZnO quantum dots treatment; C: water treatment. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Example 1

[0023] A method for reducing the ability of citrus psyllids to acquire Huanglongbing bacteria by using water-soluble ZnO quantum dots comprises the following steps:

[0024] 1) testing citrus trees in a citrus orchard to detect trees infected with citrus Huanglongbing; and testing grafted citrus plants in a citrus orchard to detect grafted citrus plants infected with citrus Huanglongbing;

[0025] In the citrus orchard, leaves from plants suspected of being infected with citrus Huanglongbing and leaves from grafted citrus plants were collected. The midribs of the leaves were ground into powder using liquid nitrogen, and the total DNA was extracted and the DNA concentration was measured.

[0026] Real-time fluorescence quantitative PCR was used to detect citrus Huanglongbing disease. DNA was quantified to 80 ng. The primers and probes used were:

[0027] HLBas: 5'-TCGAGCGCGTATGCAATACG-3'; SEQ ID NO.1;

[0028] HLBr: 5'-GCGTTATCCCGTAGAAAAAGGTAG-3'; SEQ ID NO.2;

[0029] HLBp: 5'-AGACGGGTGAGTTAACGCG-3'; SEQ ID NO.3;

[0030] The probe was labeled with FAM at the 5' end and BHQ-1 at the 3' end.

[0031] The reaction system is shown in Table 1:

[0032] Table 1 Huanglong disease detection reaction system

[0033]

[0034]

[0035] The reaction procedure is shown in Table 2:

[0036] Table 2 Huanglong disease reaction program

[0037]

[0038] 2) Synthesis of water-soluble ZnO quantum dots

[0039] Weigh 5.54g of Zn(Ac)2·2H2O and dissolve it in 150mL of anhydrous ethanol. Place it in a round-bottom flask and reflux it at 80°C for 2h using a magnetic stirrer. Weigh 2.06g of KOH and 20ml of anhydrous ethanol into a 50ml centrifuge tube and ultrasonicate it in an ultrasonic bath for 30min until the KOH is completely dissolved. Remove the round-bottom flask and cool it down. Adjust the temperature of the magnetic stirrer to 0°C and place an ice pack inside to maintain the low temperature. Return the round-bottom flask to the magnetic stirrer and slowly add the KOH solution using a separatory funnel at a rate of 1 drop / second. After the addition is complete, react at 0°C and 650rpm for 30min. Remove the biological ice bag and adjust the temperature of the magnetic stirrer to 25°C. After the temperature rises, take 500 μL of APTES and mix it evenly with 1.5 mL of double-distilled water in a 50 mL centrifuge tube. Use a separatory funnel to add it dropwise to the round-bottom flask at a rate of 1 drop / second. After the addition is completed, react at 25°C and 650 rpm for 5 hours. Collect the product in a 50 mL centrifuge tube at 8000 rpm for 10 minutes, then wash it three times with anhydrous ethanol and dry it at 65°C.

[0040] 3) By exciting the water-soluble ZnO quantum dot solution with ultraviolet light, yellow fluorescence was observed ( Figure 1 );

[0041] The water-soluble ZnO quantum dots were prepared into a 1 mg / mL solution, 1 mL of which was excited by ultraviolet light (365 nm), and the particle morphology of the water-soluble ZnO quantum dots was analyzed by TEM.

[0042] The water-soluble ZnO quantum dots were observed by transmission electron microscopy and the size of the water-soluble ZnO quantum dots was confirmed to be about 5 nm ( Figure 2 ).

[0043] 4) Cut branches from the citrus Huanglongbing trees in step 1) and bring them back to the laboratory for hydroponics. Place them in insect breeding cages and place 30 citrus psyllids ( Figure 3 );

[0044] The citrus trees grafted with citrus greening disease in step 1) were moved into the laboratory, and their branches were placed in insect breeding cages, where 30 citrus psyllids ( Figure 4 );

[0045] 5) After the citrus psyllids that have completed step 4) are reared, citrus Huanglongbing disease fungus detection is performed ( Figure 5 );

[0046] At 7, 12, and 18 days of rearing, individual citrus psyllids were collected into 1.5 mL centrifuge tubes. Ten citrus psyllids were collected for each treatment, with three replicates. The samples were ground into a homogenous slurry using an electric grinder, and total DNA was extracted. DNA concentration was determined, and real-time fluorescence quantitative PCR was performed, with DNA quantified to the nearest 80 ng. The results showed that the rate of citrus psyllid acquisition increased with the duration of exposure, but the rate of citrus psyllid acquisition in the live plant treatment was significantly higher than that in the detached branch treatment.

[0047] 6) Treating healthy citrus plants with different concentrations of water-soluble ZnO quantum dots;

[0048] The roots of healthy citrus plants were immersed in 5, 2, and 0 mg / mL water-soluble ZnO quantum dots (add them to a 50 mL centrifuge tube, add 25 mL of water-soluble ZnO quantum dot solution, and seal with a parafilm), and citrus psyllids were reared. After 48 hours, 0.1 g of citrus psyllids were taken from each tube and placed in a 5 mL centrifuge tube for ICP-MS to confirm the presence of water-soluble ZnO quantum dots in the citrus psyllids ( Figure 6 At the same time (i.e., after 48 hours of rearing the citrus psyllids at room temperature), the citrus psyllids were transferred to living plants (infected with Huanglongbing bacteria) and reared for 18 days.

[0049] Citrus psyllids were collected after 18 days of feeding with the virus. Single citrus psyllids were collected in 1.5 mL centrifuge tubes. Ten citrus psyllids were collected for each treatment and three replicates were set. The samples were ground into a homogenous slurry using an electric grinder. Total DNA was extracted and the DNA concentration was determined. Real-time fluorescence quantitative PCR was performed. The DNA was quantified to 80 ng. After the citrus psyllids were raised on living plants, the citrus Huanglongbing disease was detected ( Figure 7 ); The results showed that water-soluble ZnO quantum dots treatment significantly reduced the ability of citrus psyllids to acquire toxins (P<0.01), and the ability of high concentration (5 mg / mL) of water-soluble ZnO quantum dots to inhibit toxin acquisition was also significantly higher than that of low concentration treatment (2 mg / mL), proving that water-soluble ZnO quantum dots have the ability to effectively inhibit citrus psyllids from acquiring Huanglongbing fungus.

[0050] 7) Root irrigation of healthy citrus plants was performed using 5, 2, and 0 mg / mL water-soluble ZnO quantum dots at 0, 1, 3, 5, and 7 days (50 mL each time). The same water and fertilizer management was performed for the rest of the time until 45 days. The growth status of the plants was observed to confirm that the water-soluble ZnO quantum dots had no toxic effect and had a certain promoting effect on plant growth ( Figure 8 ).

[0051] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for reducing the ability of citrus psyllids to acquire Huanglongbing bacteria using water-soluble ZnO quantum dots, characterized in that: Water-soluble ZnO quantum dots are used to interfere with the ability of citrus psyllids to acquire Huanglongbing bacteria; the concentrations of the water-soluble ZnO quantum dots are 5 mg / mL and 2 mg / mL.

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