A method for removing the endosymbiotic bacterium Wolbachia from the South American leafminer fly.
By irrigating the soil of kidney bean plants with rifampicin solution, a strain of South American serpentine leafminer uninfected with Wolbachia was screened and established, solving the problem of removing the endosymbiotic fungus Wolbachia from South American serpentine leafminer in existing technologies, and achieving rapid, stable and low-cost removal results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-05
- Publication Date
- 2026-03-13
AI Technical Summary
Existing technologies are insufficient for the rapid and stable removal of the endosymbiotic fungus Wolbachia from the South American leafminer fly, and direct use of antibiotics or high-temperature treatment is harmful and ineffective to the insect.
By irrigating the soil of kidney bean plants with rifampicin solution, South American serpentine leafminers were continuously reared, and male and female flies not infected with Wolbachia were screened. A South American serpentine leafminer strain not infected with Wolbachia was established by PCR detection.
This method enables rapid and stable removal of the endosymbiotic bacterium Wolbachia from the South American serpentine leafminer, establishing a Wolbachia-free strain of the South American serpentine leafminer. It avoids the toxicity of direct antibiotic feeding to the host, and is simple and low-cost.
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Figure CN118489628B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for removing the endosymbiotic bacterium Wolbachia from the South American serpentine leafminer, and more particularly to a plant-mediated method for removing the endosymbiotic bacterium Wolbachia from the South American serpentine leafminer based on soil irrigation with rifampicin, belonging to the field of biotechnology. Background Technology
[0002] Wolbachia is a symbiotic bacterium widely found in arthropod cells. It has been found in some important agricultural pests such as whiteflies, spider mites, and leaf miners. Wolbachia plays an important role in the reproduction and development of its host. Its reproductive regulation of the host is mainly divided into cytoplasmic incompatibility (CI), feminization, male killing, and parthenogenesis. CI refers to the inability of offspring produced by the mating of uninfected females and infected males to develop normally. This phenotype is of great importance for the development of green pest control technologies that combine symbiotic bacteria.
[0003] Currently, direct high-temperature treatment or feeding insects with antibiotics can remove Wolbachia from insects such as whiteflies, planthoppers, and spider mites. However, direct feeding with antibiotics or high-temperature treatment is quite harmful to the insects themselves and is ineffective against certain insects. Therefore, exploring rapid and stable methods to remove Wolbachia and constructing insect strains that are not infected with Wolbachia is of great significance for studying the function of symbiotic bacteria and pest control.
[0004] The South American serpentine leafminer, *Liriomyza huidorbrensis* (Blanchard), is a significant invasive pest of vegetables and flowers in my country. Its natural population is 100% infected with the symbiotic fungus *Wolbachia*. *Wolbachia* enhances the host's cold tolerance and fitness, which is key to the rapid invasion, adaptation, and outbreak of this pest in my country's cold-climate regions. Developing a rapid and stable method to remove the symbiotic fungus *Wolbachia* from the South American serpentine leafminer will allow for the acquisition of Wolbachia-free strains to investigate the biological functions of *Wolbachia* on the host. Furthermore, removing *Wolbachia* from the South American serpentine leafminer significantly reduces the host's reproductive capacity and cold tolerance, thus facilitating the control of the spread and damage caused by the South American serpentine leafminer to my country's cold-climate regions.
[0005] In conclusion, removing the symbiotic bacterium Wolbachia from the South American serpentine leafminer is extremely important for studying its biological functions and for developing new technologies for controlling the South American serpentine leafminer using the symbiotic bacterium Wolbachia. However, there is currently no simple and effective method to remove the symbiotic bacterium Wolbachia from the insect. Summary of the Invention
[0006] Purpose of the invention: This invention provides a method for removing the endosymbiotic bacterium Wolbachia from the South American serpentine leafminer. The method used in this invention can quickly and effectively remove the endosymbiotic bacterium Wolbachia without affecting the subsequent reproduction of the population, thereby establishing a Wolbachia-free South American serpentine leafminer strain.
[0007] Technical Solution: The present invention describes a method for removing the symbiotic bacterium Wolbachia from the South American serpentine leafminer. Rifampicin solution is used to irrigate the soil of kidney bean plants. These kidney bean plants are then used to continuously feed South American serpentine leafminers infected with Wolbachia. Uninfected, negative male and female flies are selected and fed with ordinary kidney bean plants. Multiple generations of testing show negative results, thus successfully removing Wolbachia from the South American serpentine leafminer.
[0008] Furthermore, the method for removing the endosymbiotic bacterium Wolbachia from the South American serpentine leafminer includes the following steps:
[0009] (1) Collection of adult South American serpentine leafminer: Collect bean leaves with long and narrow tunnels and wait for the South American serpentine leafminer to pupate. Collect the pupae and wait for them to hatch into adults. Adults with 100% infection with Wolbachia are used as parent adults.
[0010] (2) Rifampicin treatment of soil: Place the pot with green bean plants and the parent adult South American leafminer into the insect rearing cage, and irrigate the soil in the pot with green bean plants with rifampicin solution until the leaf surface forms a long and narrow tunnel. Remove the leaves and wait for them to pupate. Collect the pupae and wait for the pupae to hatch into adults, which is the first generation of offspring.
[0011] (3) Screening of South American serpentine leafminer: Adults of each generation were collected for identification of the symbiotic fungus Wolbachia. If the identification results showed that Wolbachia infection still existed, the next generation was treated with rifampicin solution. When all the identification results were negative, the treatment with rifampicin solution was stopped, and the soil in the pot with kidney bean plants was irrigated with clean water. The process was repeated for three generations. The symbiotic fungus Wolbachia was identified in time for each generation. If there was no Wolbachia symbiosis within three generations, the strain could be stably inherited. After three generations, a South American serpentine leafminer strain that was not infected with Wolbachia was established.
[0012] Furthermore, in step (2), the concentration of the rifampicin solution is 0.05%-0.15%, preferably 0.1%.
[0013] Furthermore, in step (2), the pot containing the bean plants contains 3-6 bean plants, and the amount of rifampicin solution applied is 100-200 mL / pot each time. 6-8 pots of bean plants are used to treat 100 adult South American leafminers, and the number of times the rifampicin solution is applied is 3-5 times / day.
[0014] Furthermore, in step (3), the identification of the symbiotic bacterium Wolbachia in the South American serpentine leafminer is performed by PCR detection, including the following steps:
[0015] (1) Using the DNA of the South American leafminer fly as a template, a pair of primers targeting the wsp gene sequence of Wolbachia were used to detect the infection status of the symbiotic bacterium Wolbachia.
[0016] Furthermore, the primers targeting the wsp gene sequence of Wolbachia are:
[0017] wsp-F:5'-GTCCAATARSTGATGARGAAAC-3'
[0018] wsp-R:5'-CYGCACCAAYAGYRCTRTAAA-3'.
[0019] Furthermore, the amplification products were subjected to agarose gel electrophoresis, and the Wolbachia test was negative, indicating that the samples were from South American serpentine leafminers uninfected with Wolbachia. The concentration of the agarose gel was 1%.
[0020] Furthermore, in step (3), the operation of watering with clean water is the same as watering with rifampicin solution.
[0021] Furthermore, the method also includes the detection of the vertical transmission rate of Wolbachia, comprising the following steps: mating adult South American serpentine leafminer flies whose parental generation (G0) is 100% infected with Wolbachia, performing PCR detection on the Wolbachia infection status of the offspring, and continuously detecting for 3 generations, thereby determining the vertical transmission rate of Wolbachia and the stability of the symbiotic relationship.
[0022] Furthermore, the method also includes continuous or alternate-generation testing of the infection status of Wolbachia in the remaining offspring, thereby determining the stability of the new strain of South American leafminer fly that is not infected with Wolbachia.
[0023] Furthermore, after two consecutive generations of treatment with rifampicin solution, the symbiotic bacterium Wolbachia can be removed.
[0024] Beneficial effects: Compared with the prior art, the present invention has some significant advantages:
[0025] (1) In this invention, the soil of kidney bean plants is irrigated with rifampicin solution. The kidney bean plants are then continuously fed with two generations of South American serpentine leafminer infected with Wolbachia. Uninfected male and female flies are selected from these plants. The uninfected South American serpentine leafminers are then mated. PCR is used to detect the infection status of Wolbachia in South American serpentine leafminers in the G3, G6 and G9 generations to confirm the stability of Wolbachia and South American serpentine leafminers, thereby establishing a South American serpentine leafminer strain without Wolbachia.
[0026] (2) This invention is the first successful method to remove the symbiotic fungus Wolbachia from the body of the South American serpentine leafminer. It combines the use of plant-mediated removal of Wolbachia from the South American serpentine leafminer, improves the existing method of directly feeding antibiotics, and effectively avoids the toxic effects of direct feeding of antibiotics on the host. The method of removing Wolbachia in this invention can quickly establish a new strain of South American serpentine leafminer that is not infected with Wolbachia, and realize the possibility of stable reproduction and propagation of South American serpentine leafminers that are not infected with Wolbachia.
[0027] (3) This method is simple to operate and highly controllable. Once the rifampicin solution is prepared, it can be directly applied to the soil, saving significant costs and time. The South American serpentine leafminer is infected with Wolbachia in nature. PCR testing of rifampicin-treated South American serpentine leafminers can confirm their infection status without false negatives. This method is of great significance for studying the biological functions of the South American serpentine leafminer and for developing new technologies for its control using the symbiotic bacterium Wolbachia.
[0028] (6) The method described in this invention can quickly establish new strains of South American serpentine leafminer that are not infected with Wolbachia. Their reproductive capacity and hatching rate are reduced to a certain extent. This provides a new idea for the biological control of South American serpentine leafminer and also provides a model for the rapid removal of other symbiotic bacteria. Attached Figure Description
[0029] Figure 1 This is a flowchart of the method for removing the endosymbiotic bacterium Wolbachia from the South American leafminer fly according to the present invention.
[0030] Figure 2 Figure showing the titer changes of rifampicin in two generations of Wolbachia leafminer.
[0031] Figure 3 The infection efficiency of Wolbachia in two generations of South American serpentine leafminer populations after continuous treatment with rifampicin.
[0032] Figure 4To remove the Wolbachia leafminer strain, the infection status of female and male flies was tested at G3, G6, and G9 to determine the removal effect.
[0033] Figure 5 Bands showing the infection status of Wolbachia in second-generation adult South American serpentine leafminer when rifampicin solution concentrations were 0.01%, 0.05%, 0.15%, and 0.20%. Detailed Implementation
[0034] The following embodiments are further illustrations of the present invention, but not limitations thereof.
[0035] Example 1
[0036] (1) Collection of adult South American serpentine leafminer flies:
[0037] Collect the kidney bean leaves that already have slender tunnels into a plastic bag, place a few paper towels in the bag to absorb moisture, and wait 2-3 days for them to pupate.
[0038] Collect the pupae into 10mL centrifuge tubes without caps using a fine brush. Cover the tube openings with a piece of 200-mesh gauze cut to the appropriate size and secure with a rubber band. Each tube contains approximately 100 pupae. Then place the centrifuge tubes under constant conditions of (24±1)℃, (60±5)% humidity, and a light-to-dark ratio of 16:8 and wait for a week until the adult larvae hatch.
[0039] Adults 100% infected with Wolbachia (W+) were used as parent adults (G0).
[0040] (2) Soil treatment with rifampicin:
[0041] A rifampicin solution was prepared by mixing rifampicin powder (Sangon Biotech (Shanghai) Co., Ltd., rifamycin (product code A600812-0025)) with pure water and storing it in a glass bottle away from light. The concentration of the rifampicin solution was 0.1%.
[0042] One hundred parental adult South American serpentine leafminers were placed in a 30cm x 30cm x 50cm rearing cage (the parental (G0) adults were 100% infected with Wolbachia serpentine leafminers and mated). A basket (31.6cm x 21.3cm x 7.5cm) containing six pots (top base: 10cm x 10cm, bottom base: 7.2cm x 7.2cm, height: 8.5cm) of bean seedlings were placed in the rearing cage. Each pot contained five bean seedlings. The soil of the bean seedlings was watered with rifampicin solution at a rate of 100mL per pot, three times a day. Watering was stopped after long, narrow tunnels formed on the leaves. The leaves were then removed and placed in a plastic bag for the South American serpentine leafminers to pupate.
[0043] Soil irrigated with rifampicin solution should not be reused and should be discarded.
[0044] (3) The soil of the bean seedlings was continuously irrigated with rifampicin solution for two generations (adult parent G0 and F1) until the third generation of adults (i.e. F2) began to feed and lay eggs. The soil of the bean seedlings was then irrigated with clean water in the same way as the rifampicin solution irrigation method, and the third generation G3 was obtained.
[0045] The specific implementation steps are as follows: Figure 1 As shown.
[0046] (4) Screening of South American serpentine leafminer:
[0047] Adults from each generation are collected for identification of the symbiotic bacterium Wolbachia. If the identification results show that Wolbachia infection still exists, the next generation is treated with rifampicin solution. When all identification results are negative, the treatment with rifampicin solution is stopped, and the soil is irrigated with clean water. The soil is then irrigated with clean water for three generations, and each generation is tested in a timely manner. If the absence of Wolbachia within three generations can be stably inherited, a South American leafminer strain that is not infected with Wolbachia (W-) is established after three generations.
[0048] The symbiotic bacterium Wolbachia was identified using PCR detection. DNA was extracted from the South American leafminer fly using the FastPureCell / Tissue DNAIsolation Mini Kit (Vazyme). The specific steps were as follows:
[0049] ① Transfer a single end to a 1.5 mL centrifuge tube. Add 230 μL of Buffer GA and 20 μL of Proteinase K, and vortex to mix.
[0050] ②Incubate in a 55℃ water bath until the tissue is completely enzymatically hydrolyzed to obtain a digestive solution.
[0051] ③ If the digestion solution is cloudy or contains obvious particles, centrifuge at 12000 rpm (13400×g) for 3 min and transfer the supernatant to a new 1.5 mL centrifuge tube.
[0052] ④ Add 250 μL of Buffer GB to the digestion solution, vortex at the highest speed for 20 seconds, and incubate in a 70°C water bath for 10 minutes.
[0053] ⑤ Add 180 μL of anhydrous ethanol to the digestion solution, vortex and mix for 15-20 seconds to obtain the mixture.
[0054] ⑥ Place the gDNA Columns adsorption column into a 2mL Collection Tube. Transfer the mixture (including the precipitate) obtained in the previous step into the adsorption column. Centrifuge at 12000 rpm (13400×g) for 1 min. If column blockage occurs, centrifuge at the highest speed for another 3-5 min. If the mixture exceeds 750 μL, it needs to be filtered through the column in multiple batches.
[0055] ⑦ Discard the filtrate, place the adsorption column in the collection tube, and add 500 μL of Washing Buffer A to the adsorption column. Centrifuge at 12000 rpm (13,400 × g) for 1 min.
[0056] ⑧ Discard the filtrate, place the adsorption column in the collection tube, and add 650 μL of Washing Buffer B to the adsorption column. Centrifuge at 12000 rpm (13400 × g) for 1 min.
[0057] 9. Repeat step 8.
[0058] ⑩ Discard the filtrate and place the adsorption column in the collection tube. Centrifuge the empty tube at 12000 rpm (13400 × g) for 2 min.
[0059] Place the adsorption column in a new 1.5 mL centrifuge tube, add 30-100 μL of preheated Elution Buffer (70 °C) to the center of the membrane in the adsorption column, incubate at room temperature for 3 min, and centrifuge at 12000 rpm (13400 × g) for 1 min.
[0060] Discard the adsorption column and store the DNA at 2–8°C. For long-term storage, store at -30–-15°C.
[0061] PCR detection of infection status in the South American leafminer fly was performed using a pair of primers targeting the wsp gene sequence of *Wolbachia*: wsp-F and wsp-R. The sequences of wsp-F and wsp-R are as follows:
[0062] wsp-F:5'-GTCCAATARSTGATGARGAAAC-3'
[0063] wsp-R:5'-CYGCACCAAYAGYRCTRTAAA-3'
[0064] Reaction system:
[0065]
[0066] Total volume 25μL
[0067] Amplification conditions:
[0068] Pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 45 s, extension at 72℃ for 1 min, 35 cycles; extension at 72℃ for another 5 min; store at 4℃.
[0069] The PCR products were detected by 1% agarose gel electrophoresis, with a 2kb marker as a control. 5 μL of PCR product and marker were added to a 1% agarose gel and immediately placed in 1x TAE buffer for electrophoresis. The electrophoresis conditions were 110V, 90mA, and 25min.
[0070] The gel imaging system is used to photograph the gel, and the results are obtained by saving the images, such as the band diagram. Figure 2 As shown. Figure 2 This is a band diagram showing the change in titer of the endosymbiotic bacterium Wolbachia in the South American serpentine leafminer after two generations of continuous treatment during the experiment. The presence of a band indicates host infection with Wolbachia, while the absence of a band indicates host non-infection with Wolbachia. Figure 2 It can be seen that Wolbachia bands were detected in all three pairs of samples from the G0 generation of South American serpentine leafminer, only half of the three pairs of samples from the G1 generation of South American serpentine leafminer showed Wolbachia bands, and no Wolbachia bands were detected in any of the three pairs of samples from the G2 generation of South American serpentine leafminer. Therefore, South American serpentine leafminers that were not infected with Wolbachia were obtained.
[0071] Figure 3 This is a graph showing the infection efficiency of two generations of the endosymbiotic bacterium Wolbachia treated with *Leymus chinensis* during the experiment. Figure 3 It is evident that 100% of the parents (G0) were infected with Wolbachia, 50% of the first-generation offspring (G1) were infected with Wolbachia, and none of the second-generation offspring (G2) were infected with Wolbachia.
[0072] Depend on Figure 2 and Figure 3 It can be seen that after two generations of treatment, South American leafminer flies that are not infected with Wolbachia can be screened out.
[0073] (5) Detection of the vertical propagation rate of Wolbachia and the stability of the symbiotic relationship:
[0074] The infection status of female and male Wolbachia larvae in the first three generations (G0, G1, G2) was detected to determine the vertical transmission rate of Wolbachia in the host. In addition to detecting the infection status of the first three generations, the infection status of the remaining generations was also detected. Since no Wolbachia band was detected in the second generation, the soil of the bean seedlings was irrigated with water in the same way until the offspring G4-G9 were obtained. In this experiment, three generations, G3, G6 and G9, were selected, and PCR detection was performed using wsp-F and wsp-R to determine the stability of Wolbachia larvae with the South American leafminer.
[0075] PCR detection was performed using wsp-F and wsp-R to detect three generations (G3, G6, and G9). The PCR reaction procedure, conditions, and 1% agarose gel electrophoresis detection were the same as in step (4). Each generation contained 20 individuals, 10 males and 10 females, to determine the stability of the new strain. A gel imaging system was used to photograph the strains, and the results were obtained by saving the images. The bands were as follows: Figure 4 As shown. Figure 4 The graph shows the infection status of female and male serpentine leafminer strains in G3, G6, and G9 after removing Wolbachia. None of the strains in G3, G6, and G9 were infected with Wolbachia.
[0076] Depend on Figure 4 It can be seen that no Wolbachia band was detected in any of the 60 samples from the three generations of South American serpentine leafminer (G3, G6, and G9), indicating that a new strain of South American serpentine leafminer that is stable and uninfected by Wolbachia was obtained.
[0077] Example 2
[0078] The experimental procedure was the same as in Example 1, except that the concentrations of rifampicin solution were 0.01%, 0.05%, 0.15%, and 0.20%, respectively. PCR detection revealed that the second-generation adults treated with 0.01% rifampicin still exhibited Wolbachia bands, indicating that Wolbachia was not completely eliminated and a second generation treatment was necessary. However, the second-generation adults treated with 0.05%, 0.15%, and 0.20% rifampicin did not show any Wolbachia bands, indicating that Wolbachia was completely eliminated. The banding diagram is shown below. Figure 5 As shown. However, the 0.20% treatment affected the kidney bean seedlings; at this concentration, the seedlings easily wilted, leading to reduced feeding and egg-laying by the South American serpentine leafminer, which in turn made it difficult for them to complete the entire development process. Therefore, the suitable concentration of the Fuping solution in this experiment was 0.05%-0.15%, with 0.1% being optimal.
[0079] Comparative Example 1
[0080] In the prior art:
[0081] 1. Ahmed M, Ren SX, Xue
[0082] 2. Noda H, Koizumi Y, Zhang Q, Deng K, 2001. Infection density of Wolbachia and incompatibility level in two planthopper species, Laodelphax striatellus and Sogatella furcifera. Insect Biochem Mol Biol, 31(6-7):727-737;
[0083] 3. Mochiah MB, Ngi-Song AJ, Overholt WA, Stouthamer R, 2002. Wolbachiainfection in Cotesia sesamiae (Hymenoptera: Braconidae) causes cytoplasmicincompatibility:implications for biological control. Biol Control, 25(1):74-80.
[0084] The aforementioned literature records studies on the removal of the symbiotic bacterium Wolbachia from insects using antibiotics. These methods involved soaking plant seeds in rifampicin antibiotic solution or adding antibiotics to artificial feed for insects. The concentration of the rifampicin solution used ranged from a maximum of 0.25% to a minimum of 0.05%. Although Wolbachia was not detected after 2-3 consecutive generations of treatment, these methods all required artificial feed or seed soaking, making them cumbersome, costly, and harmful to insects when antibiotics were directly administered. In contrast, this invention uses a simple plant-mediated method that can also remove the symbiotic bacterium Wolbachia from insects and ensure stable genetic inheritance.
Claims
1. A method for removing endosymbiotic bacteria from the South American leafminer fly. Wolbachia The method is characterized by, The soil of the kidney bean plant is irrigated with a rifampicin solution, and the kidney bean plant is continuously fed with the Liriomyza huidobrensis infected Wolbachia , and uninfected negative male and female insects are screened out, and the common kidney bean plant is fed, and multiple generations are detected to be negative, so that the Liriomyza huidobrensis in the body of the Liriomyza huidobrensis is successfully removed Wolbachia ; comprising the steps of: (1) Collection of adult L. capitata: Collecting the bean leaves with long tunnels where L. capitata pupate, collecting the pupae, and waiting for the pupae to hatch into adults, with 100% of the adults infected as parental adults Wolbachia ; (2) treating the soil with rifampicin: put the pots with kidney bean plants and the parent generation of Liriomyza huidobrensis in a rearing cage, irrigate the soil in the pots with kidney bean plants with a rifampicin solution until the leaves form long tunnels, and then remove the leaves for pupation; collect the pupae, and when the pupae hatch into adults, the F1 generation is obtained; (3) Selection of Liriomyza sericata: collect adults of each generation for symbiotic bacteria Wolbachia identification, if the identification result shows that there is still Wolbachia infection, continue to use rifampicin solution to treat the next generation, when the identification result is all negative, stop using rifampicin solution, replace it with water to irrigate the soil in the pot where the kidney bean plants grow, restore for three generations, and timely perform symbiotic bacteria Wolbachia identification for each generation, if there is no Wolbachia symbiosis within three generations, it can be stably inherited, and after three generations, a strain of Liriomyza sericata that is not infected is established. Wolbachia 2. The method of removing the endosymbiont bacteria of Liriomyza huidobrensis of claim 1, characterized in that, Wolbachia In step (2), the concentration of the rifampicin solution is 0.05%-0.15%, the pots with kidney bean plants contain 3-6 kidney bean plants, the irrigation amount of the rifampicin solution is 100-200 mL / pot each time, 6-8 pots are used to treat 100 Liriomyza huidobrensis adults, and the irrigation frequency of the rifampicin solution is 3-5 times / day. 3. The method of removing the endosymbiont bacteria of Liriomyza huidobrensis of claim 1, characterized in that, Wolbachia In step (3), the Liriomyza huidobrensis is subjected to symbiotic bacteria Wolbachia The identification is by PCR detection, comprising the following steps: (1) The DNA of Liriomyza huidobrensis was used as template, and a pair of primers targeting the wsp gene sequence were used to detect the infection status of the endosymbiont. Wolbachia Wolbachia The DNA of Liriomyza huidobrensis was used as template, and a pair of primers targeting the wsp gene sequence were used to detect the infection status of the endosymbiont. 4. The method of removing the endosymbiont bacteria of Liriomyza huidobrensis of claim 3, characterized in that, Wolbachia The pair of target Wolbachia The primers for the wsp gene sequence are: wsp-F: 5'-GTCCAATARSTGATGARGAAAC-3' wsp-R: 5'-CYGCACCAAYAGYRCTRTAAA-3'.
5. The method of removing the endosymbiont bacteria of Liriomyza huidobrensis of claim 3, characterized in that, Wolbachia The amplification products were subjected to agarose gel electrophoresis, Wolbachia Detection was negative, indicating no infection Wolbachia of Liriomyza huidobrensis. 6. The method of removing the endosymbiont bacteria of Liriomyza huidobrensis of claim 1, characterized in that, Wolbachia In step (3), the operation of irrigating with water is the same as that of irrigating with the rifampicin solution. 7. The method of removing the endosymbiont bacteria of Liriomyza huidobrensis of claim 1, characterized in that, Wolbachia The method further comprises Wolbachia The detection of vertical transmission rate comprises the following steps: mating the parent 100% infected Wolbachia adults of Liriomyza huidobrensis, PCR detecting the infection of the offspring Wolbachia generated, and continuously detecting 3 generations, so as to determine Wolbachia the vertical transmission rate and the stability of the symbiotic relationship. 8. The method for removing endosymbiotic bacteria from the South American leafminer fly according to claim 1 Wolbachia The method is characterized by, The method also comprises the successive or alternate detection of the infection of the remaining offspring Wolbachia , thus determining the stability of the new strain of Liriomyza huidobrensis not infected Wolbachia .
9. The method of removing the endosymbiont bacteria of Liriomyza huidobrensis of claim 1, characterized in that, Wolbachia The removal of the symbiotic bacteria can be achieved after two successive treatments with a rifampicin solution Wolbachia Wolbachia .