Method for establishing the circulation of phytoplasmas between vectors and plants by immunolabeling

Abstract

The present invention discloses a method for establishing the circulation of phytoplasma between vectors and plants through immune labeling. The phytoplasma is wheat blue dwarf disease phytoplasma, and the vector is heterosporus; the method comprises: at different times after heterosporus feeding, using antibodies to surface immune membrane proteins of wheat blue dwarf phytoplasma to immunolabel heterosporus leafhopper tissues infected with wheat blue dwarf phytoplasma, observing the distribution of wheat blue dwarf phytoplasma in different tissues of heterosporus leafhopper at different time periods, and clarifying the circulation process. The present invention has explored the circulation pathway of wheat blue dwarf phytoplasma in the vector heterosporus leafhopper, laying a foundation for subsequent research on the interaction between pathogens and vectors.

Description

Technical Field

[0001] The invention belongs to the technical field of plant pathology, and in particular relates to a method for establishing cyclic transmission of phytoplasmas between vectors and plants through immune marking. Background Art

[0002] Phytoplasma is a single-cell prokaryotic organism that relies on insect vectors for transmission and is a parasite on the phloem of plants. It has no cell wall and belongs to the class Mollicutes, phylum Tenericutes. When a new phytoplasma disease is discovered, the identification of the vector is mainly carried out on the insect species found near the diseased plants. The common method is to capture them using sticky traps, net sweeps, etc., and focus on selecting insects with piercing-sucking mouthparts for testing to determine whether they contain phytoplasmas. In the early days, phytoplasmas in insect vectors were observed by electron microscopy. Later, with the development of PCR technology, infected insects were identified by extracting insect DNA.

[0003] Wheat blue dwarf disease (WBD) is one of the important diseases in wheat production in my country. Studies have shown that the disease mainly occurs in intercropping, wheat straw-covered dryland areas and wheat irrigation areas. The widespread occurrence of wheat blue dwarf disease seriously threatens my country's wheat production. It is another important wheat disease after yellow dwarf disease, causing symptoms such as stunting, clumps, and sterility in wheat. In recent years, the area of ​​returning straw to the field has expanded, providing a favorable farmland microenvironment for the reproduction of transmission vectors; the rise in global temperature has increased the activity cycle of transmission vectors. Various factors have caused the number of vectors to increase sharply, causing the disease to spread widely with the migration of vectors, leading to periodic outbreaks. The number of virus vectors determines the degree of disease, and vector transmission has become an important central link in the spread of diseases.

[0004] The alien leafhopper (Psammotettix alienus L.) is an important vector of pests that harms wheat, barley, oats and other grass plants in the hilly arid regions of northern China. Its piercing-sucking stylets can feed on the vascular tissues of the stems and leaves of wheat plants, causing yellow spots on the affected parts, which in turn leads to large-scale chlorosis of the tissues, affecting plant photosynthesis and hindering plant growth and development. The vector leafhopper can spread a variety of diseases, including viral diseases and phytoplasmas. Currently, wheat dwarf disease (WDV) and wheat blue dwarf disease (WBD) have been studied in depth. Relevant studies have shown that once the vectors multiply in large numbers, it will directly lead to a large-scale outbreak and pandemic of the disease, seriously affecting wheat production and threatening wheat food security.

[0005] Wheat blue dwarf disease is transmitted by the specialized leafhopper, however, the interaction mechanism between wheat blue dwarf disease and its vector, the leafhopper, is not clear. Researchers generally believe that the infected parent leafhopper transmits WBDp to its offspring through the reproductive system. Therefore, the infection cycle of wheat blue dwarf phytoplasma (WBDp) and the transmission of wheat blue dwarf phytoplasma through the eggs of the leafhopper have become the key to exploring the interaction mechanism between phytoplasma and vector. Summary of the invention

[0006] The present invention aims to reveal the transmission pathway of wheat blue dwarf phytoplasma (WBDp) in heteroptera leafhoppers, laying a foundation for subsequent research on the interaction between pathogens and vectors.

[0007] Based on the above purpose, the present invention provides a method for establishing the cyclic transmission of phytoplasmas between vectors and plants through immune labeling. The phytoplasma involved in the method of the present invention is wheat blue dwarf disease phytoplasma, and the vector is the heteroptera leafhopper. The technical idea of ​​the present invention is: at different times after the heteroptera leafhopper is fed with poison, the heteroptera leafhopper tissue infected with wheat blue dwarf phytoplasma is immunolabeled with antibodies to the surface immune membrane proteins of wheat blue dwarf phytoplasma, the distribution of wheat blue dwarf phytoplasma in different tissues of heteroptera leafhopper in different time periods is observed, and the cyclic transmission process is clarified.

[0008] Furthermore, in a method for establishing the cyclic transmission of phytoplasmas between vectors and plants through immune labeling, starved leafhoppers were transferred to wheat carrying wheat blue dwarf phytoplasmas to feed with poison, and after feeding with poison, leafhopper tissues were dissected to obtain the leafhopper tissues, which were the digestive tract, reproductive system and salivary gland tissues of the leafhopper.

[0009] Furthermore, in the method of establishing the cyclic transmission of phytoplasmas between vectors and plants through immune labeling, the heteroptera leafhopper tissue is labeled with a cross-linked product of wheat blue dwarf phytoplasma surface immune membrane protein antibody and fluorescein FITC.

[0010] The localization of WBDp in the vector at different times showed that the wheat blue dwarf phytoplasma was distributed in the digestive tract and salivary gland tissues of the leafhopper at different times after feeding with the leafhopper through immunolabeling.

[0011] Based on the technical idea of ​​the present invention, the cyclic transmission process is determined as follows: the mouth needle of the heterosand leafhopper pierces and sucks the wheat vascular tissue infected with the wheat blue dwarf phytoplasma, the wheat blue dwarf phytoplasma enters the digestive system of the heterosand leafhopper along with the food through the mouth needle of the heterosand leafhopper, reaches the midgut after passing through the filter chamber, infects the midgut epithelial cells and enters the muscle layer, is released into the blood cavity and finally reaches the salivary gland, and then the wheat blue dwarf phytoplasma is released into the new wheat plant through the mouth needle of the heterosand leafhopper, completing the systemic transmission of the wheat blue dwarf phytoplasma.

[0012] The present invention uses immunofluorescence labeling technology to immunolabel the digestive system and ovary of leafhoppers infected with WBDp at different periods after feeding with the virus using surface immune membrane protein (IMP) antibodies, observes the distribution of WBDp in different tissues of leafhoppers at different time periods, clarifies the infection cycle process and egg transmission issues, and lays a foundation for subsequent research on the interaction between pathogens and vectors. Therefore, the present invention further requests protection for the use of the method in revealing the interaction between wheat blue dwarf phytoplasma and heterosand leafhopper, and the use of the method in the prevention and control of phytoplasma diseases.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] Wheat blue dwarf disease is an important phytoplasma disease in the northwest wheat region of my country, causing wheat dwarfing and bushy branches. It is spread by the specialized leafhopper, and vector transmission has become an important central link in the spread of the disease. Phytoplasmas are prokaryotes without cell walls and are wrapped by a layer of membrane. Therefore, the interaction between the immune membrane proteins on the surface of the bacteria and the vector insects is of great significance for virus transmission. The present invention creatively uses IMP protein to explore the interaction between wheat blue dwarf phytoplasma and the vector leafhopper.

[0015] The present invention discloses the circulation transmission pathway of wheat blue dwarf phytoplasma in the vector heterospora leafhopper. Immunofluorescence labeling technology was used to label wheat blue dwarf phytoplasma IMP (FITC labeled IMP is green) and heterospora leafhopper cytoskeleton Actin (rhodamine labeled phalloidin is red), and WBDp was located at different positions in the leafhopper at different times after feeding with the poison. Laser confocal microscopy was used to observe for the first time that WBDp was distributed in the midgut epithelial cells, muscle layer and salivary glands of the vector. It was clarified for the first time that the circulation transmission process of WBDp in the leafhopper is that the heterospora leafhopper's mouth needle pierces the vascular tissue of wheat, the source of the poison, and WBDp enters the digestive system of the vector insect through the mouth needle along with the food, reaches the midgut after passing through the filter chamber, infects the midgut epithelial cells and enters the muscle layer, is released into the blood cavity and finally reaches the salivary glands, and releases the phytoplasma into the new plant through the mouth needle feeding, completing the systemic transmission of the phytoplasma.

[0016] The present invention conducts a virus transmission experiment on poisonous leafhoppers, detects WBDp in parent leafhopper females, offspring leafhopper adults and fed wheat by PCR, and immunofluorescence labels the ovaries and eggs of parent leafhopper females, indicating that the poisonous parent leafhoppers cannot transmit WBDp to offspring leafhoppers through the reproductive system, that is, WBDp is not transmitted through leafhopper eggs. It can be seen that the present invention also eliminates the technical bias of many researchers. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the circulation pathway of wheat blue dwarf phytoplasma.

[0018] Figure 2 This is the PCR detection diagram of the leafhopper fed with WBDp; M is the DNA size marker, 1 is a healthy leafhopper, and 2-11 are leafhoppers fed with poison.

[0019] Figure 3 This is a picture of wheat disease caused by WBDp spread by the heteroptera leafhopper; A is susceptible wheat (wisps-brooming symptoms), B is healthy wheat, C is a group of leafhoppers feeding on poison, and D is a leafhopper feeding on poisoned wheat.

[0020] Figure 4 These are Rhodamine-phalloidin and FITC-IMP immunolabeling images; A is Rhodamine-phalloidin labeling of the digestive system cytoskeleton, B is Rhodamine-phalloidin labeling of the salivary gland cytoskeleton, and C is FITC-IMP labeling of WBDp.

[0021] Figure 5 This is the anatomical diagram of the leafhopper; A is the dissected leafhopper, B is the adult of the leafhopper, C is the Thermo Scientific Nunc cell culture dish, D is the ovary, E is the salivary gland, and F is the digestive system.

[0022] Figure 6 These are the immune localization diagrams of WBDp in the digestive system and salivary glands at different times after feeding with poison in leafhoppers; A shows the distribution of WBDp in the esophagus and filter chamber cavity of the leafhopper 0 h after feeding with poison, B shows the distribution of WBDp in the filter chamber cavity and midgut epithelial cells of the leafhopper 8 h after feeding with poison, C shows the distribution of WBDp in the midgut muscle layer 16 h after feeding with poison, D shows the distribution of WBDp in the posterior midgut muscle layer, and E shows the distribution of WBDp in the salivary glands 24 h after feeding with poison.

[0023] Figure 7 This is a diagram to verify the transmission of WBDp without ovary; A is the mating of leafhoppers, B is the development of compound eyes from eggs, C is the rupture of egg membranes, D is the ovary, and E is the egg.

[0024] Figure 8 This is the result of PCR detection of WBDp; M is a DNA size marker, A-1 to A-10 are female adults of the parent leafhopper, B is an adult of the offspring leafhopper, and C is the wheat fed by the offspring leafhopper. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is described below in conjunction with embodiments; however, the present invention is not limited to the following embodiments.

[0026] This example provides an application test of the method for establishing the circulation of phytoplasmas between vectors and plants through immunological labeling.

[0027] (1) Test materials

[0028] The source of wheat blue dwarf virus in this experiment came from diseased plants in Heyang, Shaanxi. It was collected and brought back to the laboratory. After being fed by non-toxic leafhoppers, it was transmitted to healthy wheat and vinca roseus. The virus source was stored on wheat and vinca roseus and isolated and planted in a greenhouse. Wheat variety: Xiaoyan No. 6. Rearing conditions: temperature 25℃; humidity 60%, photoperiod 16:8 (L:D).

[0029] The vector of wheat blue dwarf disease (Heterophyllum sphaeroides) was collected from the wheat fields of Yangling Agricultural High-tech Industrial Demonstration Zone, Xianyang City, Shaanxi Province, and bred in a greenhouse. The leaves with eggs were taken and hatched separately to obtain non-toxic larvae. After multiple generations of breeding, the non-toxic leafhoppers were raised on healthy wheat seedlings of Xiaoyan No. 6. The breeding conditions were: 25℃~30℃ during the day, sufficient light; 18℃~25℃ at night; humidity 30%, photoperiod 16:8 (L:D).

[0030] (2) Wheat breeding

[0031] Seed germination: Take wheat seeds and rinse them with clean water several times to remove surface impurities, soak them in 70% alcohol for 90 seconds and then rinse them thoroughly. Add sterile water to the germination box, soak the seeds for 48 hours to fully absorb water, then pour out the water, put filter paper at the bottom of the germination box, wet it with water and evenly sprinkle the soaked wheat seeds, cover the lid and place it in the tissue culture box for germination, and rinse with sterile water every 24 hours.

[0032] Soil sterilization: Sterilize the soil with high temperature and high pressure (15 pounds, 121°C, 2 hours) the day before planting. After sterilization, let it cool to room temperature, add organic soil (ratio 3:1) and appropriate amount of water, mix them together, and the soil will be moist after mixing.

[0033] Disinfection of square basin: The size of the square basin is 10cm×10cm. Wash it with clean water several times, then soak it in NaClO solution (1000mg / L) for 3h, rinse it 3 times after soaking, and dry it for later use.

[0034] Sowing: Wet the surface soil with a watering can, evenly sow 30 seeds per pot, cover the surface with a thin layer of soil and compact it, then wet the surface of the soil with a watering can. Place in an incubator, set the temperature to 25℃; humidity to 60%, and the photoperiod to 16:8 (L:D). Add water to the tray once every 2 days, with a water layer height of 1 cm. Keep the culture environment dry.

[0035] (3) Rearing of leafhoppers

[0036] The size of the insect cage is 60cm×60cm×80cm, and the material is 100 mesh gauze. 18 pots of wheat seedlings are placed in each insect cage, and the number of insects raised is: 2000 adults / cage. The breeding temperature is 28℃; the humidity is 30%, and the photoperiod is 16:8 (L:D).

[0037] (4) Leafhopper poisoning and dissection

[0038] Several adult leafhoppers raised in the laboratory were taken and starved for 4 hours before being transferred to wheat with WBDp for 24 hours. After feeding, they were transferred to a 15 mL centrifuge tube and placed in a -20 ° C refrigerator for 4 minutes for freezing anesthesia. Then they were taken out and placed in PBS buffer and dissected with pointed forceps under a dissecting microscope. One forceps clamped the tail of the leafhopper, and the other forceps carefully pulled the head of the leafhopper. The leafhopper head was removed and placed in a clean Thermo Scientific Nunc cell culture dish (PBS buffer was added in advance). The esophagus was pulled out with the head, along with the filter chamber, foregut, midgut, hindgut and Malpighian tubules, and transferred to another cell culture dish (PBS buffer was added in advance). The control was a healthy leafhopper, and the dissection method was the same.

[0039] (5) IgG (Immunoglobulin G) purification

[0040] Protein A Sepharose affinity column (Thermo) was used to purify the antiserum to obtain IgG. The purification process was as follows:

[0041] Remove the caps at both ends of the adsorption column, remove the storage solution, and add 5mL BindingBuffer; after equilibrating the adsorption column, slowly add 3mL of IMP antiserum; when the antiserum passes through the column, the IgG in it will bind to the adsorption column; after the adsorption process is completed, add BindingBuffer to wash the impurities that are not bound to the adsorption column; measure the OD280 absorbance value once with 1mL of the effluent, and stop adding BindingBuffer when the value is 0; elute the antiserum on the adsorption column with ElutionBuffer, measure the OD280 of 0.5mL of the collected solution once with a UV spectrophotometer, and add 10mL of ElutionBuffer to elute the column when the absorbance value is 0; add 5mL of 0.02% sodium azide to wash the adsorption column, retain 2mL in the adsorption column, cover the caps at both ends of the adsorption column, and store at 4°C.

[0042] (6) Preparation of cross-linked products of IMP antibody and fluorescein FITC

[0043] The fluorescein FITC and IMP membrane protein antibody crosslinked product (FITC-IMP) used in the experiment marked the WBDp in the leafhopper. Fluorescence microscopy observed that the leafhopper tissue emitted significant green fluorescence, indicating that the prepared FITC-IMP has strong specificity and can be used in subsequent experiments. The specific preparation steps are as follows:

[0044] Cut a 10cm dialysis bag, soak it in boiling water for 3 minutes, and then rinse it with distilled water twice; clamp one end of the dialysis bag with a clamp, add the IgG to be cross-linked and purified into the dialysis bag, and clamp the other end with a clamp; add 0.1molNaHCO3 to the beaker, put the dialysis bag containing IgG, stir at 4℃, change the dialysate every 4h, and change it 3 times in total; take a 1.5mL centrifuge tube, collect the dialyzed IgG in it, take a new centrifuge tube, add an equal amount of dimethylformamide and an appropriate amount of fluorescein, and shake to mix; add the mixed fluorescein to the 1.5mL centrifuge tube containing IgG, and vortex to shake and mix. Put the cross-linked IgG in a shaker at room temperature and incubate it in the dark for 2h; prepare a beaker with PBS solution, put the incubated IgG into the dialysis bag and then into the beaker, stir and dialyze at 4℃, change the dialysate every 4h, and change it 3 times in total; collect the cross-linked IgG, divide it into portions and store it in a -20℃ refrigerator.

[0045] (7) Immunofluorescence labeling

[0046] The digestive tract and salivary glands of the dissected leafhopper were used for immunofluorescence experiments. The specific antibody incubation steps were as follows:

[0047] Fixation: Add the dissected leafhopper tissues to 4% PFA fixative and place in a 25°C environment for 2 hours. After fixation, remove the excess PFA and wash with PBS solution 2-3 times, 5 minutes each time.

[0048] Permeabilization: After removing PFA, add 2% Triton solution for permeabilization for 0.5 h (room temperature). After removing the permeabilization solution, wash with PBS solution for 3 times, 5 min each time.

[0049] Incubate the antibody: add 10μL BSA liquid to a 2mL centrifuge tube, and clamp the leafhopper digestive tract and salivary gland. Add 0.1μL IgG to the centrifuge tube and incubate at 37℃ for 2h. After incubation, wash the sample repeatedly with PBS solution 5 times, 4min each time. Add the secondary antibody and incubate at 37℃ for 2h. After incubation, wash for 5min each time. The antibody incubation process needs to be protected from light.

[0050] Observation and photography: Take a clean glass slide, drop glycerol, select the digestive tract, reproductive system and salivary gland tissues, place them in place, put the cover slip, and finally seal the slide. Observe the green fluorescence emitted by IMP-FITC when stimulated by the 488nm exciter, and the red fluorescence emitted by Rhodamine-phalloidin when stimulated by the 542nm exciter.

[0051] (8) Leafhopper DNA extraction

[0052] DNA extraction uses the CTAB method, and the specific steps are as follows:

[0053] Weigh 0.5g of leafhoppers in a mortar, add liquid nitrogen and grind thoroughly, and transfer the ground powder to a 2mL centrifuge tube. Add 800μL of CTAB lysis buffer (1.4M NaCl, 0.5M EDTA, 1M Tris-HCl, 2% CTAB pH 8.0) to the centrifuge tube in advance. Then add 12μL of β-mercaptoethanol and shake immediately. Preheat the water bath to 65℃, place the sample in a water bath for 1h, and mix it upside down every 10min. Ice the sample for 10min, cool it, add the same volume of phenol chloroform isoamyl alcohol (pH>7.8), then shake for 20s, and centrifuge at 4℃, 12000rpm for 15min. Use a pipette to transfer 650μL of supernatant to a 1.5mL centrifuge tube, add the same volume of phenol chloroform isoamyl alcohol, vortex and mix, and centrifuge at 4℃, 12000rpm for 15min. Transfer the supernatant to a new centrifuge tube, add 2 times the volume of anhydrous ethanol and 1 / 10 volume of NaAc (pH5.2, 3M), shake gently upside down; place in a -20℃ refrigerator to precipitate for 2 hours. After the sample is precipitated, centrifuge at 4℃, 12000rpm for 5 minutes, wash the precipitate with pre-cooled 75% ethanol twice, wash it with anhydrous ethanol once, and blow it in a clean bench for 10 minutes after purification to fully evaporate the ethanol. Add sterile ultrapure water (DDW) to the obtained DNA precipitate sample to fully dissolve it, add RNase to remove impurities, and place it in a 37℃ water bath for 1 hour. Place the sample in a -20℃ refrigerator for storage.

[0054] (9) PCR amplification and product purification

[0055] The total DNA of the single-headed leafhopper was extracted after being infected with the virus, and the 16SrDNA fragment of the phytoplasma was amplified by nested PCR to detect the infection.

[0056] Amplification Primers:

[0057] First round: P1: 5′-AAGAGTTTGATCCTGGCTCAGGATT-3′;

[0058] P7: 5′-CGTCCTTCATCGGCTCTT-3′

[0059] Second round: R16F2n: 5′-GAAACGACTGCTAAGACTGG-3′;

[0060] R16R2: 5'-TGACGGGCGGTGTGTACAAACCCCG-3'

[0061] The PCR amplification reaction system is shown in Table 1.

[0062] Table 1. PCR amplification reaction system

[0063] Reagents volume 2×PCR Master Mix 12.5μL Prey-F 1μL Prey-R 1μL RNase-Free Water 10.5μL Total Volume 25μL

[0064] The PCR reaction conditions were as follows: denaturation at 98°C for 10 s, annealing at 55-60°C for 7 s, extension at 72°C for 15-30 s, 34 cycles, and the PCR products were stored at 4°C.

[0065] The PCR product was recovered and purified using the Biotech (DP1701) kit. The specific steps are as follows:

[0066] Use 1.2% agarose gel to separate the PCR products obtained in the experiment. Use a blade to cut out the gel containing the target fragment under ultraviolet light and put it into a 2mL centrifuge tube and weigh it; add DB buffer (2 times volume) to the centrifuge tube, heat it in a 56°C water bath until the gel is completely melted, and vortex it once every 2 minutes; after the gel is completely melted, place it in a recovery purification column, centrifuge it at 12,000rpm at room temperature for 1 minute, and discard the waste liquid; add 500μL WB buffer to the purification column, after 2 minutes, centrifuge it at 12,000rpm at room temperature for 1 minute, and discard the waste liquid; repeat the previous step once; empty the purification column at 12,000rpm, centrifuge it for 2 minutes to dry the residual liquid; put the purification column into a 2mL centrifuge tube, dry it at room temperature for 5-10 minutes to allow the ethanol to evaporate completely, preheat the elution buffer to 65°C, take 50μL of elution sample, and centrifuge it at 12,000rpm for 60s.

[0067] (10) Ligation and transformation of T vector

[0068] The product was recovered and the absorbance was measured, then connected to the T vector at 4°C overnight. The reaction system is shown in Table 2.

[0069] Table 2, ligation and transformation T vector reaction system

[0070] Reagents volume DNA solution 1μL Solution I 2.5μL T vector 0.25μL <![CDATA[ddH2O]]> 1.25μL Total Volume 5μL

[0071] (11) Preparation of DH5α competent cells

[0072] Prepare LB solid plate, streak culture of E. coli DH5α, place in 37℃ incubator for 16-20h; prepare a conical flask and add LB liquid culture medium, randomly pick a single clone and put it into the conical flask, shake and culture at 220rpm, 37℃ for 10h; then inoculate the initial culture into liquid LB culture medium, shake and culture at 18℃, 220rpm overnight; measure the OD600 value of the bacterial solution every 30min until the culture value is 0.55, ice bath for 10min, then divide into pre-cooled centrifuge tubes, centrifuge at 4℃, 2500rpm, collect the bacteria; discard the supernatant and use absorbent paper to dry the remaining liquid in the tube. Add pre-cooled CaCl2 solution to the centrifuge tube and vortex the bacteria, centrifuge at 4℃, 2500rpm, collect the bacteria for 10min; repeat the above step once; add pre-cooled CaCl2 solution and vortex the precipitate to fully suspend. Add 1.5 mL of sterile glycerol and place on ice for 10 min. Use a sterile pipette tip to dispense the bacterial suspension into cooled PCR tubes, seal them and immerse them in liquid nitrogen to freeze competent cells. Store at -80°C for later use.

[0073] (12) WBDp trans-oocyte delivery verification

[0074] Male and female parent leafhoppers carrying WBDp mate, and egg masses are laid on the front of wheat leaves. After hatching, the offspring leafhopper nymphs are immediately transferred to healthy wheat for feeding. After 30 days, PCR is used to detect the infection of phytoplasmas in offspring leafhoppers and fed wheat. The experimental operation refers to the above steps; after the parent leafhopper lays eggs, the ovaries and eggs of the parent leafhopper female are marked with immunofluorescence to observe phytoplasmas, and PCR is used to detect whether the parent female carries WBDp. Each treatment is repeated 10 cages.

[0075] (13) Detection of virus in wheat and virus vectors

[0076] The batches of leafhoppers were transferred to WBDp wheat and fed with poison for 7 days. DNA was extracted from a single poisoned leafhopper. After nested PCR amplification, 1% agarose gel electrophoresis was performed to detect that 80% of the leafhoppers had obtained WBDp. The results were as follows: Figure 2 The virus-feeding leafhoppers transferred to non-virus wheat and transmitted the virus for 15 days. Obvious symptoms were seen 30 days later, with the plants shrinking significantly and increasing tillering. Figure 3 -A. The leaves showed chlorotic spots and yellow-green stripes, while the healthy control wheat had no symptoms and grew well. Figure 3 -B.

[0077] (14) Localization of WBDp in the mediator at different times

[0078] Non-toxic leafhoppers were starved for 4 h, fed on a poisonous wheat source infected with WBDp for 24 h, and then samples were taken at intervals of 0 h, 8 h, 16 h, and 24 h. Thirty leafhoppers were used for each treatment, and the digestive tract was dissected out. Figure 5 -D), salivary glands ( Figure 5 -E), ovary ( Figure 5 -F) In PBS buffer, FITC-IMP and Rhodamine-phalloidin were used to label WBDp (green) and insect cell Actin (red), respectively, which can be seen under a laser confocal microscope:

[0079] 0 hours after the feeding of the vector Heteropsida on the wheat poison source, 25% of the Heteropsida leafhoppers showed green fluorescence in the esophagus and filter chamber cavity, indicating the presence of WBDp at this time ( Figure 6 -A);

[0080] 8 h after the end of the poison feeding, a small amount of green fluorescence was observed in the lumen of the leafhopper filter chamber, and a large amount of green fluorescence was observed in the midgut epithelial cells of 41% of the leafhoppers, indicating that WBDp had broken through the midgut invasion barrier and infected the midgut epithelial cells ( Figure 6 -B);

[0081] 16 hours after the end of the poisoning, a large amount of green fluorescence was observed in the midgut, post-midgut circular muscle and longitudinal muscle tissue of 55% of the leafhoppers. Figure 6 -C and Figure 6 -D), indicating that the phytoplasma has spread from the epithelium to the midgut muscle layer;

[0082] 24 hours after the end of the feeding, fluorescence was found in 57% of the salivary glands ( Figure 6 -E), indicating that WBDp has now broken through the intestinal release barrier and reached the salivary glands;

[0083] WBDp was consistently observed in the salivary glands, suggesting that WBDp proliferated in the salivary gland tissue.

[0084] (15) Localization of WBDp in the mediator at different times

[0085] The leafhopper uses a piercing-sucking mouthpart to feed on the vascular tissue of infected wheat. The phytoplasma enters the insect's esophagus through the mouth needle along with the sap, moves from the esophagus to the filter chamber cavity and enters the midgut cavity, infects the epithelial cells of the midgut, then breaks through the leafhopper's midgut barrier and invades the midgut muscle layer. After releasing the blood cavity, it breaks through the blood barrier and moves to the salivary glands, proliferates in the salivary glands, breaks through the salivary gland barrier, and releases the phytoplasma into new plants through the mouth needle, completing the systemic transmission of the phytoplasma. Based on the above results, the circulation pathway of WBDp ( Figure 1 ).

[0086] (16) Verification of WBDp transmission in parental leafhopper females without egg transmission

[0087] After mating, the female leafhopper will pierce the ovipositor into the mesophyll tissue and lay eggs on the front of the wheat leaf in rows, with 2 to 6 eggs in each row. Figure 7-A and Figure 7 -C), leafhopper eggs obtain nutrients and water from the mesophyll tissue. Mark the leaves with egg masses, and after 5-6 days of incubation, dark red compound eyes will gradually develop ( Figure 7 -B), transfer the leaves with egg masses to healthy wheat for further incubation, and after the nymphs hatch, promptly transfer them to healthy wheat to start the virus transmission experiment. After 30 days, the PCR results showed that the WBDp test in the offspring leafhopper adults and the fed wheat was negative, and the parent leafhopper female was positive ( Figure 8 ); Immunofluorescence labeling of the ovaries and eggs of the parent leafhopper female showed no WBDp particles ( Figure 7 -D and Figure 7 -E), indicating that the parent leafhopper females carry WBDp, but their ovaries and eggs do not, and the offspring leafhopper adults and the wheat they feed on do not have WBDp. The experimental results show that the infected parent leafhopper cannot pass WBDp to the offspring leafhoppers through the reproductive system, that is, WBDp is not transmitted through leafhopper eggs.

[0088] In summary, the method provided by the present invention for establishing the circulation transmission of phytoplasma between vectors and plants through immunolabeling, using immunofluorescence labeling technology, using IMP antibodies to immunolabel the digestive system and ovaries of leafhoppers infected with WBDp at different periods after feeding, observing the distribution of WBDp in different tissues of leafhoppers at different time periods, and revealing the circulation transmission pathway of wheat blue dwarf phytoplasma in the vector heterosporus leafhopper. The above test results show the circulation pathway of WBDp between the vector and the plant: the mouth needle of heterosporus leafhopper pierces the vascular tissue of the infected wheat, and the phytoplasma enters the digestive system of the vector insect through the mouth needle along with the food, passes through the filter chamber and reaches the midgut, infects the midgut epithelial cells and enters the muscle layer, is released into the blood cavity and finally reaches the salivary gland, and releases the phytoplasma into the new plant through the mouth needle, completing the systemic transmission of the phytoplasma. By conducting a virus transmission experiment on infected leafhoppers, PCR was used to detect WBDp in parent female leafhoppers, offspring leafhopper adults and fed wheat, and immunofluorescence was used to label the ovaries and eggs of parent female leafhoppers. It was shown that the infected parent leafhoppers could not transmit WBDp to their offspring leafhoppers through the reproductive system, that is, WBDp was not transmitted through leafhopper eggs.

[0089] As described above, the present invention can be well implemented. The above embodiments are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims

1. A method for establishing the circulation of phytoplasmas between vectors and plants by immunological labeling, characterized in that: The phytoplasma is wheat blue dwarf disease phytoplasma, The vector is the heterosphagnum leafhopper; The method comprises: at different times after the leafhopper is fed with the virus, using antibodies to the surface immune membrane proteins of the wheat blue dwarf phytoplasma to immunolabel the leafhopper tissue infected with the wheat blue dwarf phytoplasma, observing the distribution of the wheat blue dwarf phytoplasma in different tissues of the leafhopper at different time periods, and clarifying the cyclic transmission process; The cyclic transmission process is determined as follows: the mouth needle of the leafhopper pierces and sucks the wheat vascular tissue infected with the wheat blue dwarf phytoplasma, the wheat blue dwarf phytoplasma enters the digestive system of the leafhopper along with the food through the mouth needle of the leafhopper, reaches the midgut after passing through the filter chamber, infects the midgut epithelial cells and enters the muscle layer, is released into the blood cavity and finally reaches the salivary gland, and then the wheat blue dwarf phytoplasma is released into the new wheat plant through the mouth needle of the leafhopper, completing the systemic transmission of the wheat blue dwarf phytoplasma; The tissues are the digestive tract, ovary and salivary gland tissues of the heteroptera leafhopper.

2. The method according to claim 1, characterized in that The method comprises: transferring the starved leafhoppers to wheat with wheat blue dwarf disease phytoplasma to feed poison, and dissecting to obtain the leafhopper tissues after feeding poison.

3. The method according to claim 1 or 2, characterized in that: The heteroptera leafhopper tissue is labeled with a cross-linked product of wheat blue dwarf phytoplasma surface immune membrane protein antibody and fluorescein FITC.

4. The method according to claim 3, characterized in that Immunolabeling was used to determine the distribution of wheat blue dwarf phytoplasma in the digestive tract and salivary gland tissues of the leafhopper at different times after feeding with the leafhopper.

5. Use of the method of claim 1 in revealing the interaction between wheat blue dwarf phytoplasma and heteroptera leafhopper.

6. Use of the method according to claim 1 in the prevention and treatment of phytoplasma diseases.

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