A method for extracting a living body of citrus huanglongbing bacteria

Abstract

The application discloses a living extraction method of citrus Huanglongbing bacteria, relates to the technical field of plant pathogenic bacteria extraction, and solves the problem that the prior art cannot guarantee that the citrus Huanglongbing bacteria have complete biological activity and high purity at the same time. The method comprises four core steps of pretreatment of Rutaceae plant samples, extraction of bacterial liquid, initial enrichment of the bacterial liquid and re-enrichment of the bacterial liquid in sequence, the whole extraction process is carried out in a low-temperature environment, plant tissues infected with pathogenic bacteria are broken by freezing and grinding, and step-by-step extraction and enrichment of the citrus Huanglongbing bacteria are realized by combining step-by-step differential centrifugation and sucrose density gradient centrifugation. The method can effectively retain the complete biological activity of the citrus Huanglongbing bacteria, significantly improve the purity of the bacterial liquid, and the prepared bacterial liquid can be used for infection of healthy Rutaceae plants and microinjection transfection of citrus psyllids, the method is simple in operation and good in repeatability, and provides stable and reliable experimental materials for the research on the pathogenic mechanism, transmission rule and prevention and control technology of the citrus Huanglongbing disease.

Description

Technical Field

[0001] This invention relates to the field of plant pathogen extraction technology, specifically a method for the live extraction of Huanglongbing (HLB) pathogen from citrus. Background Technology

[0002] Citrus Huanglongbing (HLB) is a major disease of citrus plants caused by bacteria of the genus *Bacillus*. It occurs widely in many major citrus-producing areas worldwide, causing severe economic losses to the citrus industry. This pathogen is an obligate parasite, and artificial pure culture has not yet been achieved. Therefore, obtaining live pathogen material is a crucial prerequisite for conducting research on the pathogenesis, transmission patterns, and control technologies of HLB.

[0003] Currently, several methods for extracting Huanglongbing (HLB) bacteria from citrus trees have been disclosed. For example, some existing techniques involve grinding infected citrus leaves and then obtaining the supernatant as the bacterial solution through a single centrifugation. Other techniques involve preliminary enrichment of the extracted bacterial solution through a single high-speed centrifugation.

[0004] The problem with the existing technology is that it cannot simultaneously guarantee that the extracted Huanglongbing pathogen of citrus has complete biological activity and high purity. The existing extraction method does not strictly control the temperature throughout the extraction process, resulting in significant inactivation of the pathogen during grinding and centrifugation. Furthermore, it does not employ a stepwise gradient purification process, resulting in the extracted bacterial solution containing a large amount of plant tissue fragments and soluble impurities, which cannot meet the requirements of subsequent infection experiments and high-precision molecular biology experiments. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for the live extraction of Huanglongbing pathogens from citrus. By combining cryogenic grinding under low-temperature conditions with stepwise differential centrifugation and sucrose density gradient centrifugation, the method can simultaneously ensure the complete biological activity and high extraction purity of Huanglongbing pathogens from citrus, providing stable and reliable live experimental materials for research on Huanglongbing from citrus.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for live extraction of Huanglongbing (HLB) pathogen from citrus, the method comprising the following steps: Pretreatment steps for Rutaceae plant samples: Rutaceae plant tissues infected with Citrus Huanglongbing fungus were placed together with grinding beads and PBS buffer in a cryogenic grinding tube and crushed into a homogenate at a temperature below -20℃ using a cryogenic grinder. The homogenate was then preliminarily centrifuged and filtered to obtain a homogenized suspension. Bacterial suspension extraction steps: Centrifuge the homogenized suspension at 4℃ at a speed of 1000r / min to 3000r / min for 8min to 12min, discard the precipitate and foam, repeat the centrifugation operation until the supernatant is free of residual impurities, and collect the supernatant as crude extract. Initial enrichment of bacterial solution: Centrifuge the crude extract at 9000r / min to 11000r / min at 4℃ for 4 to 8 minutes, discard the supernatant, collect the bottom precipitate as concentrated bacterial solution, combine multiple concentrated bacterial solutions and resuspend to obtain primary enrichment solution. Bacterial solution re-enrichment step: The primary enrichment solution is subjected to density gradient centrifugation, and the solution of the 30% sucrose solution layer is collected as the secondary enrichment solution.

[0007] By employing a stepwise extraction and enrichment process combining low-temperature grinding with differential centrifugation, high-speed centrifugation, and 30% sucrose density gradient centrifugation, it is possible to extract live bacterial suspensions of citrus Huanglongbing bacteria with complete biological activity from the tissues of Rutaceae plants infected with citrus Huanglongbing.

[0008] Furthermore, in the pretreatment step of the Rutaceae plant samples, the tender leaves near the top and the veins of Newhall citrus plants infected with Huanglongbing (HLB) are selected as initial materials. The initial materials are chopped and added to a cryogenic grinding tube. A preset number of grinding beads and insect isotonic solution PBS buffer are added. After the cryogenic grinding tube is tightly capped, it is placed in a cryogenic grinder for grinding. During the grinding process, the temperature is continuously controlled to be below -20°C until the initial materials are completely broken down to form a uniform slurry.

[0009] By selecting tender leaves and veins near the top of Newhall citrus plants as extraction raw materials and strictly controlling the grinding temperature below -20℃, the biological activity of Huanglongbing pathogen in citrus can be preserved to the greatest extent, while improving the extraction efficiency of pathogens per unit mass of raw materials and reducing the interference of ineffective tissues on the subsequent extraction process.

[0010] Furthermore, in the pretreatment step of the Rutaceae plant sample, the homogenous material obtained by grinding is subjected to preliminary centrifugation and filtration, and large particles of plant tissue impurities in the homogenous material are removed by filtration with a sterile filter screen. The filtered liquid is collected as a homogenized suspension.

[0011] Large particles of plant tissue impurities were initially removed by sterile filtration, and differential centrifugation at speeds of 2000 to 3000 rpm was performed to effectively separate plant cell fragments from the Huanglongbing fungus, significantly reducing the impurity content in the crude extract and improving the purity of the bacterial solution and the accuracy of subsequent experiments.

[0012] Furthermore, in the bacterial culture extraction step, the homogenized suspension is dispensed into sterile centrifuge tubes and centrifuged in a 4°C constant temperature centrifuge at a speed of 2000 r / min to 3000 r / min for 8 min to 12 min. After removing the centrifuge tubes, the bottom precipitate and the upper foam are discarded, and the clear supernatant in the middle is retained. Transfer the retained intermediate clarified supernatant to a new sterile centrifuge tube and repeat the centrifugation operation twice with the same centrifugation parameters. After each centrifugation, discard the bottom precipitate and the upper foam. The supernatant collected at the end is the crude extract of citrus Huanglongbing bacteria.

[0013] By repeating the differential centrifugation operation twice, residual plant tissue fragments and cell debris in the crude extract can be completely removed, resulting in a clearer crude extract of citrus Huanglongbing pathogen, thus avoiding the adverse effects of impurities on the subsequent enrichment process and infection experiments.

[0014] Furthermore, in the initial enrichment step of the bacterial solution, the crude extract of citrus Huanglongbing bacteria is transferred to a sterile high-speed centrifuge tube and centrifuged at 9000 r / min to 11000 r / min for 4 to 8 minutes in a constant temperature high-speed centrifuge at 4°C. After centrifugation, the supernatant is slowly poured out, leaving only a small amount of precipitate at the bottom of the centrifuge tube. This small amount of precipitate at the bottom of the centrifuge tube is the concentrated bacterial solution. Multiple concentrated bacterial solutions are combined into the same sterile centrifuge tube, and a preset volume of PBS buffer is added for resuspending to obtain the first enrichment solution.

[0015] High-speed centrifugation at 9000 r / min to 11000 r / min was used to initially enrich the citrus Huanglongbing pathogen. The concentrated bacterial solutions from multiple tubes were then combined and resuspended, which can quickly obtain a high-concentration primary enrichment solution, meet the basic requirements of different experiments for bacterial solution concentration, and shorten the preparation cycle of experimental materials.

[0016] Furthermore, in the bacterial solution re-enrichment step, a 30% sucrose solution is prepared as a density gradient medium. The primary enrichment solution is slowly added above the sucrose density gradient solution, and density gradient centrifugation is performed at 4°C. After centrifugation, the citrus Huanglongbing bacteria are enriched in the 30% sucrose solution layer. The solution in the 30% sucrose solution layer is carefully aspirated using a sterile pipette and transferred to a new sterile centrifuge tube to obtain the secondary enrichment solution.

[0017] By using a 30% sucrose solution as the density gradient centrifugation medium and accurately collecting the bacterial solution layer in the 30% sucrose solution, it is possible to further purify the citrus Huanglongbing fungus, remove soluble plant proteins and small molecule impurities, and obtain a secondary enrichment solution with higher purity, which is suitable for high-precision cell and molecular biology experiments.

[0018] Furthermore, the method is carried out entirely in a low-temperature environment, and the biological activity retention time of all extracted citrus Huanglongbing bacteria solutions is 24 hours. The citrus Huanglongbing bacteria solutions must be used within 24 hours after extraction.

[0019] When enriching the bacterial solution of Huanglongbing (HLB) of citrus, the extraction and initial enrichment steps can be repeated multiple times according to actual experimental needs to further increase the concentration of the bacterial solution. By conducting the extraction operation at low temperature throughout and ensuring that the biological activity retention time of the bacterial solution is 24 hours, the HLB can be guaranteed to maintain its complete infectivity during extraction and use. By repeating the extraction and enrichment steps, the bacterial solution concentration can be flexibly adjusted according to experimental needs, improving the flexibility and applicability of the experiment.

[0020] Furthermore, when performing DNA extraction and detection on the extracted citrus Huanglongbing fungal culture, a microbial-specific DNA extraction kit was used to extract DNA from the citrus Huanglongbing fungal culture. The presence of the target gene fragment of citrus Huanglongbing in the DNA was verified by PCR electrophoresis, and the titer value of citrus Huanglongbing in the citrus Huanglongbing fungal culture was determined by real-time quantitative PCR.

[0021] By using a microbial DNA extraction kit to extract DNA from the bacterial culture and combining it with PCR electrophoresis and quantitative real-time PCR, the presence of citrus Huanglongbing pathogen in the bacterial culture can be accurately verified, and the pathogen titer in the bacterial culture can be precisely determined, providing reliable data support for subsequent infection experiments and quantitative studies.

[0022] Furthermore, the crude extract of citrus Huanglongbing was used for infection of healthy Rutaceae plants. The crude extract of citrus Huanglongbing was injected into the phloem of healthy Rutaceae plants, 3 to 5 times per plant, with each injection containing 100 μL of the crude extract. Infected Rutaceae plants were obtained 30 to 60 days after injection.

[0023] By directly injecting the crude extract of citrus Huanglongbing bacteria into the phloem of healthy Rutaceae plants, experimental materials of Rutaceae plants infected with citrus Huanglongbing can be obtained quickly, which greatly shortens the experimental cycle required by traditional grafting or insect-borne transmission methods, while reducing the safety risks of disease spread.

[0024] Furthermore, the extracted Huanglongbing fungal solution was used for microinjection transfection of citrus psyllids. The Huanglongbing fungal solution with a titer of 2×10^4 copies / μL to 2×10^6 copies / μL was selected and injected into the thorax and abdomen of 4th to 5th instar citrus psyllid nymphs and the abdomen of wingless citrus psyllid adults through a microinjection needle. The volume of Huanglongbing fungal solution injected into each citrus psyllid was 10nL to 30nL each time.

[0025] By microinjecting citrus Huanglongbing fungal suspension at specific titers into specific sites on citrus psyllid nymphs and wingless adults, artificial and controllable transfection of citrus Huanglongbing fungus into citrus psyllids can be achieved.

[0026] Compared with existing technologies, this method for live extraction of Huanglongbing fungus from citrus has the following advantages: I. This invention employs a cryogenic grinding process combined with stepwise differential centrifugation and sucrose density gradient centrifugation under low-temperature conditions throughout the extraction process. This allows for strict control of temperature conditions throughout the extraction, maximizing the preservation of the complete biological activity of the citrus Huanglongbing pathogen. Simultaneously, through multi-step gradient physical separation, large plant tissue fragments, cell debris, and soluble plant proteins are removed sequentially, effectively improving the purity of the bacterial solution. This solves the problem in existing technologies that cannot simultaneously ensure both the biological activity of the pathogen and the purity of the extraction, providing experimental materials with complete infectivity for research related to citrus Huanglongbing.

[0027] II. This invention employs purely physical separation methods to extract and enrich Huanglongbing (HLB) of citrus, eliminating the need for additional chemical reagents. The operation process is simple and standardized, and the experimental results are stable, reliable, and highly reproducible. This method allows for flexible adjustment of the number of repetitions of the extraction and enrichment steps to prepare bacterial solutions of varying concentrations and purities, suitable for plant infection experiments, citrus psyllid transfection experiments, and various high-precision molecular biology experiments. This significantly shortens the preparation cycle of experimental materials and reduces the safety risks associated with disease spread.

[0028] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0030] Figure 1 This is a flowchart illustrating the overall process of the live extraction method for Huanglongbing (HLB) of citrus in this invention. Figure 2 This is a schematic diagram of the differential centrifugation enrichment process of the present invention; Figure 3 This is a schematic diagram of the density gradient centrifugation stratification results of the present invention; Figure 4This is a schematic diagram of the homogenized state of a small sample after grinding according to the present invention; Figure 5 This is a schematic diagram of the state of the bacterial solution after one centrifugation process according to the present invention; Figure 6 This is a schematic diagram showing the state of the bacterial solution after two centrifugation processes according to the present invention. Figure 7 This is a schematic diagram showing the state of the bacterial solution after three centrifugation processes according to the present invention. Figure 8 This is a schematic diagram showing the state of the bacterial solution after four centrifugation processes according to the present invention. Figure 9 This is a schematic diagram of the state of the bacterial solution after five centrifugation processes according to the present invention. Detailed Implementation

[0031] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below. Example

[0032] This embodiment details the complete preparation process of crude extract of Huanglongbing (HLB) of citrus and the verification method for infecting healthy citrus plants with the crude extract. This embodiment prepares a biologically active crude extract of HLB by strictly controlling the temperature and centrifugation parameters throughout the extraction process, and verifies the infectivity of the crude extract through plant infection experiments.

[0033] like Figure 1 As shown, the overall process of the live extraction method of Huanglongbing fungus for citrus provided by the present invention includes four core steps: pretreatment of Rutaceae plant samples, extraction of bacterial solution, initial enrichment of bacterial solution, and re-enrichment of bacterial solution. Figure 1 The sequence of each step and the flow of materials are clearly shown. From the initial input of diseased leaf samples, through a series of physical separation operations, the final output is live bacterial solutions of citrus Huanglongbing bacteria of different purity levels.

[0034] like Figure 2 As shown, the differential centrifugation enrichment process of this invention separates substances of different densities sequentially by gradually increasing the centrifugation speed. First, large plant tissue fragments are removed by low-speed centrifugation, and then the citrus Huanglongbing fungal cells are precipitated by high-speed centrifugation, achieving preliminary separation of the fungal cells from soluble impurities.

[0035] like Figure 3 As shown, the density gradient centrifugation stratification results of the present invention show that after centrifugation, the solution forms a clear stratification interface. The citrus Huanglongbing pathogen is mainly enriched in the 30% sucrose solution layer, which is located in the middle of the centrifuge tube and is light milky white in color, clearly distinguishable from the transparent solutions above and below.

[0036] Specifically, Newhall citrus plants naturally infected with citrus Huanglongbing (HLB) in the field were selected. Tender leaves that had just unfolded 1-2 weeks prior to the top of the plant, along with the corresponding veins, were collected as initial material. Leaves with obvious mechanical damage or other pest or disease damage were avoided during collection. 20-30 leaves were collected from each plant, with a total weight of approximately 5 grams. The collected leaves were rinsed three times with sterile deionized water to remove dust and impurities from the leaf surface, and then the moisture was blotted dry with sterile filter paper.

[0037] Use sterile scissors to cut the dried leaves into small pieces approximately 0.5 cm on each side, and transfer them all to 50 mL cryogenic tubes. Add 20 zirconia beads (5 mm in diameter) and 20 mL of pre-cooled (4°C) insect isotonic PBS buffer to the cryogenic tubes. Tightly cap the cryogenic tubes and place them in a cryogenic grinder pre-cooled to below -20°C. Set the grinder parameters to 30 Hz and 3 minutes, continuously purging liquid nitrogen into the grinder during the process to maintain the grinder temperature below -20°C. After grinding, remove the cryogenic tubes and observe the state of the material inside until all leaf tissue is completely broken down into a homogeneous paste.

[0038] The homogenized slurry obtained from grinding was initially filtered through a sterile nylon filter with a pore size of 100 micrometers in a 4°C refrigerator. The filter was rinsed three times with a small amount of pre-cooled PBS buffer, and all the filtered liquid was collected to obtain a homogenized suspension. The homogenized suspension was evenly distributed into four 50 mL sterile centrifuge tubes, with each tube containing approximately 5 mL of liquid. The centrifuge tubes were placed in a pre-cooled 4°C incubator, and centrifuged at 2500 rpm for 10 minutes.

[0039] After centrifugation, carefully remove the centrifuge tube, avoiding any shaking of the liquid inside. At this point, the contents of the centrifuge tube will separate into three layers: a pale yellow foam on top, a clear supernatant in the middle, and a dark green plant tissue precipitate at the bottom. Slowly aspirate the foam using a sterile pipette, then transfer the clear supernatant to a new 50 mL sterile centrifuge tube, discarding the bottom precipitate. Centrifuge the transferred supernatant twice more using the same centrifugation parameters, discarding both the foam and the precipitate after each centrifugation. The final collected supernatant is the crude extract of *Citrus citrus Huanglongbing* (HLB). Transfer the crude extract to a sterile reagent bottle and store it at 4°C for later use.

[0040] Verification of the effect of centrifugation number on the clarity of crude extract: To further optimize the crude extract preparation process and verify the effect of centrifugation times on impurity removal, this embodiment also conducted a gradient centrifugation comparison experiment on a small sample. 0.4g of leaves from the same batch of infected Newhall citrus was taken, chopped, mixed with 2mm zirconia grinding beads, and 800μL of PBS buffer pre-cooled to 4℃ was added. The mixture was placed in a cryogenic grinding tube and ground evenly at -20℃ or below. Figure 4 (As shown in the figure). Subsequently, gradient centrifugation was performed at 1500 rpm at 4°C to remove the precipitate and upper layer foam sequentially. The states of the bacterial culture after different centrifugation cycles are shown in Table 1 and... Figures 5-9 As shown. In this comparative experiment, a rotation speed of 1500 rpm was used to more clearly observe the impurity removal gradient effect of different centrifugation times. The optimal parameter of "cumulative 3 centrifugations" was finally determined and is also applicable to the rotation speed range of 2000 r / min to 3000 r / min of this invention.

[0041] Table 1. Comparison of clarity of crude extract after different centrifugation cycles: ; Experimental results showed that when the number of centrifugations reached 3, the crude extract was free of visible impurities, and the clarity met the requirements for subsequent experiments. Increasing the number of centrifugations to 4-5 did not significantly improve the clarity of the bacterial solution, but resulted in a loss of approximately 5%-8% of pathogens. Therefore, this invention determined that repeating centrifugation twice (a total of 3 centrifugations) was the optimal process parameter, ensuring effective impurity removal while maximizing the retention of pathogens.

[0042] Take 1 ml of the crude extract of *Citrus citrus Huanglongbing* prepared above, and extract DNA from the crude extract using a plant DNA extraction kit. Detect the presence of the 16S rRNA gene fragment of *Citrus citrus Huanglongbing* in the DNA using PCR electrophoresis. The PCR reaction program was: 94℃ pre-denaturation for 5 minutes, 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 1 minute, for a total of 35 cycles, with a final extension at 72℃ for 10 minutes. Detection of the PCR product by 1% agarose gel electrophoresis showed a clear target band at approximately 1100 base pairs, confirming the presence of *Citrus citrus Huanglongbing* in the crude extract.

[0043] Ten healthy Newhall citrus seedlings of uniform growth and 6 months of age were selected as the infection targets. A crude extract of Huanglongbing (HLB) was drawn from the main stem of each plant and injected into the phloem using a sterile syringe. Each plant was injected four times, with each injection volume being 100 μL. After injection, the plants were placed in a greenhouse at 25°C and 60% relative humidity, maintaining normal light, water, and fertilizer management.

[0044] The effect of injection interval on plant infection efficacy and growth: To optimize plant infection procedures and reduce the damage to plant growth caused by injection wounds, this embodiment included a comparative experiment with five groups of different injection intervals. Each group consisted of ten healthy Newhall citrus plants with consistent growth stages. Each plant underwent four injections, with each injection containing 100 μL. The injection site was the phloem of the main stem. The pathogen detection time and plant growth under different injection intervals are shown in Table 2.

[0045] Table 2. Effect of different injection intervals on plant infection efficacy: Injection interval days 1 day 2 days 3 days 4 days 5 days The earliest detection time of pathogens in group 1 35d 35d 45d 55d 55d The earliest detection time of pathogens in group 2 35d 40d 40d 45d 50d The earliest detection time of pathogens in group 3 35d 35d 40d 55d 55d Impact on plant growth Symptoms began to appear after 30 days, but frequent wounding caused significant damage to the plants, with some plants exhibiting leaf wilting. Symptoms began to appear after 35 days, and the damage to the plant from the wounds significantly decreased. Symptoms begin to appear after 45 days, and the wounds cause relatively little damage to the plant. Symptoms begin to appear after 45 days, and the wounds cause relatively little damage to the plant. Symptoms begin to appear after 45 days. The wounds cause minimal damage to the plant, and the plant continues to grow well. Experimental results show that an injection interval of 2 days can ensure 100% plant infection rate within 35-40 days and significantly reduce the adverse effects of wounds on plant growth. This is the optimal injection interval that balances infection efficiency and plant health.

[0046] New leaves were collected from the plants on days 30, 45, and 60 post-infection, and leaf DNA was extracted for PCR testing. The results showed that on day 30 post-infection, citrus Huanglongbing (HLB) was detected in the leaves of 3 plants; on day 45 post-infection, it was detected in the leaves of 8 plants; and on day 60 post-infection, it was detected in the leaves of all 10 plants. Simultaneously, on day 60 post-infection, some plants began to show typical HLB mottling and yellowing symptoms in their new leaves.

[0047] The PCR product was detected by 1% agarose gel electrophoresis, and a clear target band appeared at approximately 1100 base pairs, confirming the presence of *Citrus Huanglongbing Fungicide* in the crude extract. Quantitative real-time PCR determined the titer of *Citrus Huanglongbing Fungicide* in the crude extract prepared in this example to be 3.6 × 10^4 copies per microliter.

[0048] A preliminary study on the effects of culture temperature on the survival rate and emergence rate of citrus psyllids after injection: To provide basic data for subsequent transfection experiments on citrus psyllids using the bacterial solution prepared in this invention, this example preliminarily investigated the effects of different culture temperatures on the survival rate and emergence rate of citrus psyllids after injection. Fifty fourth-instar, non-toxic citrus psyllid nymphs were selected, and each was injected with 10 nL of the crude extract of citrus Huanglongbing bacteria prepared above. They were then cultured in constant temperature and humidity incubators at 15℃, 20℃, 25℃, and 30℃ with a relative humidity of 50%. The survival and emergence of the psyllids were observed for 5 consecutive days, and the results are shown in Table 3.

[0049] Table 3. Survival rate and emergence rate of citrus psyllids at different culture temperatures: Incubation temperature Initial number of psyllids Day 1 survival count Day 2 survival count Day 3 survival count Survival count on day 4 Day 5 survival count 15℃ 50 45 40 39 37 36 20℃ 50 46 43 42 41 41 25℃ 50 45 43 43 42 40 30℃ 50 43 40 39 37 35 The experimental results show that under the culture condition of 25℃, the survival rate and emergence rate of citrus psyllids reach a good balance, which is suitable as the standard culture temperature for citrus psyllid transfection experiments.

[0050] This embodiment successfully prepared a crude extract of citrus Huanglongbing (HLB) with biological activity. The presence of HLB in the crude extract was verified by PCR detection, and its infectivity was verified by plant infection experiments. This embodiment demonstrates that the method of freezing and grinding at -20℃ combined with multiple differential centrifugations at 4℃ can effectively extract HLB with intact biological activity, solving the problem of significant pathogen inactivation during extraction in existing technologies.

[0051] Example 2 This embodiment details the initial and re-enrichment processes of *H. coli*, the pathogen of citrus Huanglongbing (HLB), and the method for determining the titer of bacterial solutions at different enrichment levels. This embodiment uses high-speed centrifugation for initial enrichment and density gradient centrifugation with a 30% sucrose solution for re-enrichment, gradually increasing the concentration and purity of *H. coli* in the bacterial solution. The pathogen titer at different enrichment stages is accurately determined using quantitative real-time PCR.

[0052] Specifically, 50 ml of the crude extract of Huanglongbing fungus prepared in Example 1 was divided into 10 sterile high-speed centrifuge tubes of 5 ml each, with each tube containing 5 ml of liquid. The centrifuge tubes were placed in a constant-temperature high-speed centrifuge pre-cooled to 4°C, and the centrifugation speed was set to 10,000 rpm for 6 minutes.

[0053] After centrifugation, slowly remove the centrifuge tubes, tilt them at a 45-degree angle, and slowly pour out most of the supernatant, retaining only about 0.1 ml of precipitate at the bottom of the tube. Combine the precipitates from 10 centrifuge tubes into a single 1.5 ml sterile centrifuge tube, add 0.5 ml of pre-chilled PBS buffer, and gently pipette to mix thoroughly, resuspending the precipitate completely to obtain the primary enrichment solution. Store the primary enrichment solution at 4°C for later use.

[0054] Take 0.5 mL of the prepared primary enrichment solution and extract DNA from it using a microbial DNA extraction kit. The titer of *Citrus citrus Huanglongbing* in the primary enrichment solution was determined by quantitative real-time PCR. The PCR reaction system was 20 μL, including 10 μL of 2×SYBR Green PCR Master Mix, 0.5 μL each of forward and reverse primers, 2 μL of DNA template, and 7 μL of sterile deionized water. The reaction program was 95℃ pre-denaturation for 30 seconds, 95℃ denaturation for 5 seconds, and 60℃ annealing for 30 seconds, for a total of 40 cycles. Using a plasmid containing a known copy number of the *Citrus citrus Huanglongbing* 16S rRNA gene fragment as a standard, a standard curve was plotted, and the titer of *Citrus citrus Huanglongbing* in the primary enrichment solution was calculated to be 5.2 × 10^6 copies per μL.

[0055] Prepare a 30% sucrose solution by weighing 30 g of analytical grade sucrose and adding it to 70 mL of pre-chilled PBS buffer. Stir until the sucrose is completely dissolved, then bring the volume to 100 mL. Autoclave at 121°C for 20 minutes and cool to 4°C for later use. Add 5 mL of the 30% sucrose solution to a 10 mL sterile centrifuge tube. Slowly add 0.5 mL of the primary enrichment solution on top of the sucrose solution, being careful not to disrupt the solution interface. Place the centrifuge tube in an ultracentrifuge pre-cooled to 4°C and set the centrifugation speed to 40,000 rpm for 2 hours.

[0056] After centrifugation, carefully remove the centrifuge tube, avoiding any shaking of the liquid inside. At this point, the solution in the centrifuge tube has formed clear layers, from top to bottom: a PBS solution layer, a residual layer of the primary enrichment solution, a 30% sucrose solution layer, and a precipitate layer. Using a sterile pipette, carefully aspirate the entire 30% sucrose solution layer and transfer it to a new 1.5 mL sterile centrifuge tube to obtain the secondary enrichment solution, with a total volume of approximately 0.5 mL.

[0057] 0.1 mL of the prepared secondary enrichment solution was taken, and DNA was extracted and detected by real-time PCR using the same method. The titer of *Citrus citrus Huanglongbing* in the secondary enrichment solution was calculated to be 2.8 × 10^7 copies per μL. Simultaneously, the protein impurity content in the secondary enrichment solution was detected by polyacrylamide gel electrophoresis. The results showed that the plant protein impurity content in the secondary enrichment solution was reduced by more than 85% compared to the primary enrichment solution.

[0058] A secondary enrichment solution with a titer of 1.2 × 10⁵ copies per μL was used for microinjection transfection experiments on citrus psyllids. Fifty non-toxic citrus psyllid nymphs (4th to 5th instar) were selected and fixed ventrally upwards onto a pre-prepared adhesive plate. The adhesive plate was prepared by attaching permanent double-sided adhesive transparent tape to a regular transparent petri dish, spraying the adhesive side with 75% alcohol, allowing it to dry, and then coating the adhesive area with PBS solution. Using a microsyringe, 10 nanoliters of bacterial suspension were injected into each psyllid at the thorax and abdomen. After injection, the psyllids were transferred to healthy Rutaceae plant shoots and cultured for 2 days in a constant temperature and humidity incubator at 25°C and 50% relative humidity. After culture, DNA was extracted from 20 random psyllids for PCR detection. The results showed that citrus Huanglongbing (HLB) was detected in 16 psyllids, with a transfection success rate of 80%.

[0059] This embodiment successfully achieved the initial and re-enrichment of *Citrus citrus Huanglongbing-fungus*, obtaining a high-concentration, high-purity bacterial suspension. The bacterial titer at different enrichment stages was accurately determined using quantitative real-time PCR, demonstrating that the enrichment method of this invention can effectively increase the concentration of *Citrus citrus Huanglongbing-fungus* in the bacterial suspension. Simultaneously, the bioactivity of the enriched bacterial suspension was verified through a microinjection transfection experiment using citrus psyllids, providing reliable experimental materials for the study of the interaction between citrus psyllids and *Citrus citrus Huanglongbing-fungus*.

[0060] Comparative Example This comparative example uses the existing technology disclosed in the background section to extract Huanglongbing (HLB) from citrus, namely, a method combining room temperature grinding with a single low-speed centrifugation. The initial materials, sample volume, and buffer solution volume are all consistent with those in Example 1; only the grinding temperature and the number of centrifugation cycles are changed. By comparing the experimental results, the technical advantages of this invention are highlighted.

[0061] Specifically, 5 grams of Newhall citrus leaves infected with Huanglongbing (HLB) were collected from the same batch as in Example 1. The leaves were rinsed three times with sterile deionized water, blotted dry with sterile filter paper, and cut into small pieces with sides approximately 0.5 cm. The pieces were then transferred to a 50 ml ordinary grinding tube. Twenty 5 mm diameter zirconia grinding beads were added, along with 20 ml of room temperature PBS buffer. The tube was capped tightly and placed in a regular tissue homogenizer. The tissue was homogenized at 30 Hz for 3 minutes at room temperature until the leaf tissue was completely broken down into a homogenate.

[0062] The homogenized slurry obtained from grinding was initially filtered through a sterile nylon filter with a pore size of 100 micrometers, and the filtered liquid was collected to obtain a homogenized suspension. The homogenized suspension was evenly distributed into four 50 ml sterile centrifuge tubes and centrifuged at 2500 rpm for 10 minutes at room temperature. After centrifugation, the upper foam and the bottom sediment were discarded, and the middle supernatant was collected as the extract of citrus Huanglongbing (HLB) and stored in a refrigerator at 4°C for later use.

[0063] Take 1 ml of the prepared extract and extract DNA using the same method as in Example 1, then perform PCR detection. The PCR product was detected by 1% agarose gel electrophoresis; a weak target band appeared at approximately 1100 base pairs, indicating that the extract contained a small amount of *Citrus citrus Huanglongbing* (CSC).

[0064] Ten healthy Newhall citrus seedlings, all from the same batch as in Example 1 and at 6 months of age, were selected as the infection targets. Infection was performed using the same method as in Example 1, with each seedling injected four times, each injection volume being 100 μL. The seedlings were then cultured in a greenhouse under the same conditions. New leaves were collected on days 30, 45, and 60 post-infection for PCR testing.

[0065] The test results showed that on the 30th day after infection, no citrus Huanglongbing (HLB) pathogen was detected in the leaves of any of the plants; on the 45th day after infection, HLB was detected in the leaves of only one plant; and on the 60th day after infection, HLB was detected in the leaves of only two plants. Furthermore, on the 60th day after infection, none of the plants showed obvious symptoms of citrus Huanglongbing.

[0066] This comparative example uses existing techniques to extract *Huanglongbing* (citrus citrus greening pathogen), resulting in an extract with low pathogen content and poor biological activity. Plant infection experiments show that the infection success rate of the extract prepared using existing methods is only 20%, far lower than the 100% success rate of Example 1 of this invention. This fully demonstrates that this invention, by strictly controlling the low-temperature conditions throughout the extraction process and employing multiple differential centrifugations, can significantly improve the extraction efficiency and biological activity of *Huanglongbing*, effectively solving the problems existing in the prior art.

[0067] Comparison of the effects of different extraction methods: In order to more intuitively demonstrate the differences between the present invention and the prior art, the key parameters and experimental results of Example 1, Example 2 and the comparative example are summarized and compared, as shown in Table 4.

[0068] Table 4 summarizes and compares the key parameters and experimental results of Examples 1, 2, and the comparative examples: Comparison Projects Example 1 Example 2 Comparative Example Grinding temperature -20℃ or below -20℃ or below room temperature Centrifugation times 3 times 3 times + 1 time high-speed centrifugation + 1 time density gradient centrifugation 1 time bacterial titer 3.6 × 10^4 copies / μL 2.8 × 10^7 copies / μL 1.2 × 10^3 copies / μL Plant infection success rate 100% 100% 20% Success rate of psyllid transfection Not detected 80% Not detected Protein impurity content high Low Extremely high As can be seen from the table above, the extraction method of the present invention is significantly superior to existing technologies in terms of pathogen extraction efficiency, bioactivity, and bacterial solution purity. Example 1, through low-temperature grinding and multiple differential centrifugations, increased the pathogen extraction efficiency by 30 times, raising the plant infection success rate from 20% to 100%. Example 2, based on this, further improved the pathogen titer through enrichment and purification, increasing it by nearly 800 times while significantly reducing the impurity content in the bacterial solution, meeting the requirements of high-precision molecular biology experiments and citrus psyllid transfection experiments.

[0069] In summary, this invention provides a method for the live extraction of *Huanglongbing Fungi* from citrus. By optimizing extraction process parameters and combining low-temperature grinding, stepwise differential centrifugation, and 30% sucrose density gradient centrifugation, it successfully achieves high-activity extraction and high-purity enrichment of *Huanglongbing Fungi*. This method is simple to operate, has good reproducibility, and can provide stable and reliable experimental materials for research related to citrus Huanglongbing, thus possessing significant scientific research application value.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for live extraction of Huanglongbing (HLB) of citrus, characterized in that, The method includes the following steps: Pretreatment steps for Rutaceae plant samples: Rutaceae plant tissues infected with Citrus Huanglongbing fungus were placed together with grinding beads and PBS buffer in a cryogenic grinding tube and crushed into a homogenate at a temperature below -20℃ using a cryogenic grinder. The homogenate was then preliminarily centrifuged and filtered to obtain a homogenized suspension. Bacterial suspension extraction steps: Centrifuge the homogenized suspension at 4℃ at a speed of 1000r / min to 3000r / min for 8min to 12min, discard the precipitate and foam, repeat the centrifugation operation until the supernatant is free of residual impurities, and collect the supernatant as crude extract. Initial enrichment of bacterial solution: Centrifuge the crude extract at 9000r / min to 11000r / min at 4℃ for 4 to 8 minutes, discard the supernatant, collect the bottom precipitate as concentrated bacterial solution, combine multiple concentrated bacterial solutions and resuspend to obtain primary enrichment solution. Bacterial solution re-enrichment step: The primary enrichment solution is subjected to density gradient centrifugation, and the solution of the 30% sucrose solution layer is collected as the secondary enrichment solution.

2. The method for live extraction of Huanglongbing fungus from citrus trees according to claim 1, characterized in that, In the pretreatment step of the Rutaceae plant samples, the tender leaves near the top and the veins of Newhall citrus plants infected with Huanglongbing (HLB) were selected as the initial materials. The initial materials were cut into small pieces and added to a cryogenic grinding tube. A preset number of grinding beads and insect isotonic solution PBS buffer were added. After the cryogenic grinding tube was tightly capped, it was placed in a cryogenic grinder for grinding. During the grinding process, the temperature was continuously controlled to be below -20°C until the initial materials were completely broken down to form a uniform slurry.

3. The method for live extraction of Huanglongbing fungus from citrus trees according to claim 1, characterized in that, In the pretreatment step of the Rutaceae plant sample, the homogenous material obtained by grinding is subjected to preliminary centrifugation and filtration. Large particles of plant tissue impurities in the homogenous material are removed by filtration with a sterile filter screen, and the filtered liquid is collected as a homogenized suspension.

4. The method for live extraction of Huanglongbing fungus from citrus trees according to claim 1, characterized in that, In the bacterial culture extraction step, the homogenized suspension is dispensed into sterile centrifuge tubes and centrifuged in a 4°C constant temperature centrifuge at a speed of 2000 r / min to 3000 r / min for 8 min to 12 min. After removing the centrifuge tubes, the bottom precipitate and the upper foam are discarded, and the clear supernatant in the middle is retained. Transfer the retained intermediate clarified supernatant to a new sterile centrifuge tube and repeat the centrifugation operation twice with the same centrifugation parameters. After each centrifugation, discard the bottom precipitate and the upper foam. The supernatant collected at the end is the crude extract of citrus Huanglongbing bacteria.

5. The method for live extraction of Huanglongbing fungus from citrus trees according to claim 1, characterized in that, In the initial enrichment step of the bacterial solution, the crude extract of citrus Huanglongbing bacteria is transferred to a sterile high-speed centrifuge tube and centrifuged at 9000r / min to 11000r / min for 4 to 8 minutes in a constant temperature high-speed centrifuge at 4℃. After centrifugation, the supernatant is slowly poured out, leaving only a small amount of precipitate at the bottom of the centrifuge tube. The small amount of precipitate at the bottom of the centrifuge tube is the concentrated bacterial solution. Multiple concentrated bacterial solutions are combined into the same sterile centrifuge tube, and a preset volume of PBS buffer is added for resuspending to obtain the first enrichment solution.

6. The method for live extraction of Huanglongbing fungus from citrus trees according to claim 5, characterized in that, In the bacterial solution re-enrichment step, a 30% sucrose solution is prepared as a density gradient medium. The primary enrichment solution is slowly added above the sucrose density gradient solution, and density gradient centrifugation is performed at 4°C. After centrifugation, the citrus Huanglongbing pathogen is enriched in the 30% sucrose solution layer. The solution in the 30% sucrose solution layer is carefully aspirated using a sterile pipette and transferred to a new sterile centrifuge tube to obtain the secondary enrichment solution.

7. The method for live extraction of Huanglongbing fungus from citrus trees according to claim 1, characterized in that, The entire process is carried out in a low-temperature environment. The biological activity retention time of all extracted citrus Huanglongbing bacteria solutions is 24 hours. The citrus Huanglongbing bacteria solutions must be used within 24 hours after extraction.

8. The method for live extraction of Huanglongbing fungus from citrus trees according to claim 1, characterized in that, When performing DNA extraction and detection on the extracted citrus Huanglongbing fungal culture, a microbial-specific DNA extraction kit was used to extract DNA from the citrus Huanglongbing fungal culture. The presence of the target gene fragment of citrus Huanglongbing in the DNA was verified by PCR electrophoresis, and the titer value of citrus Huanglongbing in the citrus Huanglongbing fungal culture was determined by real-time quantitative PCR.

9. The method for live extraction of Huanglongbing fungus from citrus trees according to claim 1, characterized in that, The crude extract of citrus Huanglongbing fungus was used for infection of healthy Rutaceae plants. The crude extract of citrus Huanglongbing fungus was injected into the phloem of healthy Rutaceae plants, 3 to 5 times per plant, with each injection containing 100 μL of the crude extract. Infected Rutaceae plants were obtained 30 to 60 days after injection.

10. The method for live extraction of Huanglongbing fungus from citrus trees according to claim 1, characterized in that, The extracted Huanglongbing fungal solution was used for microinjection transfection of citrus psyllids. The Huanglongbing fungal solution with a titer of 2×10^4 copies / μL to 2×10^6 copies / μL was selected. The Huanglongbing fungal solution was injected into the thorax and abdomen of 4th to 5th instar citrus psyllid nymphs and the abdomen of wingless citrus psyllid adults through a microinjection needle. The volume of Huanglongbing fungal solution injected into each citrus psyllid was 10nL to 30nL.

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