CN114717335A - Method for obtaining CLas infected diaphorina citri polypide body - Google Patents

Method for obtaining CLas infected diaphorina citri polypide body Download PDF

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CN114717335A
CN114717335A CN202210504089.XA CN202210504089A CN114717335A CN 114717335 A CN114717335 A CN 114717335A CN 202210504089 A CN202210504089 A CN 202210504089A CN 114717335 A CN114717335 A CN 114717335A
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diaphorina citri
infected
obtaining
real
screening
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余海中
张金波
方瑜婕
段硕
陈玮
卢占军
胡威
朱博
姚锋先
黄爱军
苏华楠
彭婷
袁小勇
汪和贵
欧阳智刚
舒佳旺
刘佳阳
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Gannan Normal University
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Abstract

The invention provides a method for obtaining a CLas infestation diaphorina citri insect body, and relates to the technical field of insect physiological biochemistry. Firstly, screening out the concentration which is most suitable for survival of the in vitro citrus tender branches from Hoagland nutrient solutions with different concentrations; selecting and in vitro culturing the citrus twig infected with the yellow dragon germ by using the conventional PCR and real-time fluorescent quantitative PCR technology; selecting adult diaphorina citri, nymphs of 4-5 years and nymphs of 3 years and below, screening the most suitable nymph obtaining period, and finally obtaining diaphorina citri bodies infected with yellow dragon bacteria by utilizing the constructed system, thereby providing a basic material for the subsequent theoretical research.

Description

Method for obtaining CLas infected diaphorina citri polypide body
Technical Field
The invention relates to the technical field of insect physiological biochemistry, in particular to a method for obtaining a CLas infected diaphorina citri insect body.
Background
Citrus greening disease (HLB) is one of the most devastating diseases in the citrus industry worldwide. Since no effective treatment method for the susceptible tree body of the yellow shoot disease exists at present, the yellow shoot disease is also called cancer of citrus. The yellow dragon disease germs (CLas) can not be artificially cultured yet, but can grow and propagate in the diaphorina citri bodies for a long time, and the diaphorina citri can be carried for life and rapidly spread once being infected by the yellow dragon germs, which brings great difficulty to the research of the yellow dragon disease and the prevention and control thereof, and is the biggest problem which troubles the citrus industry in recent years. At present, the prevention and control of citrus greening diseases mainly focuses on the spread of insect vectors of citrus psyllids, and with the management and prevention of governments and fruit growers, the stock of field citrus psyllids is relatively small, and the infected citrus greening diseases are rare, so that a lot of resistance is brought to the development of bacteria obtaining experiments and corresponding functional researches of the citrus psyllids by taking the citrus psyllids as objects. Therefore, a system for quickly and efficiently obtaining the materials infected with the yellow dragon germs is urgently established in a laboratory.
Disclosure of Invention
The invention aims to provide a method for quickly obtaining diaphorina citri infected with liberobacter asiaticum, which directly utilizes isolated plants to quickly obtain a large amount of susceptible materials and greatly reduces the cost of experimental materials.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for obtaining a CLas infected diaphorina citri polypide, which comprises the following steps: screening the infected shoots and culturing in vitro, screening C using real-time fluorescent quantitative PCRtAnd (4) placing the diaphorina citri on the susceptible branches with similar values to infect the susceptible branches to obtain susceptible psylla polyporus frondosus bodies.
In the invention, the infected branches are obtained by PCR detection and screening.
In the invention, the PCR detection amplification primers are as follows:
F：5’GCGCGTATGCAATACGAGCGGCA 3’
R：5’GCGTCGCGACTTCGCAACCCAT 3’。
in the invention, the PCR detection reaction program is as follows: pre-denaturation at 95 ℃ for 3 min; 30s at 95 ℃, 30s at 62 ℃, 1min at 72 ℃ for 10s, and 34 cycles; 5min at 72 ℃; 5min at 25 ℃.
In the invention, the condition of the in vitro culture is to culture by using Hoagland nutrient solution with 0.1-2 times of concentration.
In the invention, the real-time fluorescent quantitative PCR amplification primer is as follows:
F：5’TGGAGGTGTAAAAGTTGCCAAA 3’
R：5’CCAACGAAAAGATCAGATATTCCTCTA 3’。
in the invention, the real-time fluorescent quantitative PCR reaction system is as follows: ddH2O3.5 μ L; SYBR 5. mu.L; F/R is 0.25 mu L; 1 mu L of cDNA template; total system 10. mu.L.
In the invention, the real-time fluorescent quantitative PCR reaction program is as follows: pre-denaturation at 95 ℃ for 1 min; 95 ℃ for 15s, 59 ℃ for 15s, 72 ℃ for 45s, 45 cycles.
In the invention, the pest period of the diaphorina citri is 4-5 years nymphs.
In the invention, the infection days are 2-8 days.
Compared with the prior art, the invention has the following beneficial effects:
the invention directly utilizes the isolated plant, thereby greatly reducing the cost of experimental materials; in addition, the rate of the diaphorina citri infection is 100% in the 5 th day by the high-efficiency bacterium obtaining system constructed by the invention, so that a large amount of infection materials can be quickly obtained, and the time cost is reduced.
Drawings
FIG. 1 is a diagram of PCR detection of citrus excised shoot susceptibility, where the reference numerals denote the excised citrus shoots, M: takara DL 2000maker, yang: susceptible shoots, yin: non-infected branches;
FIG. 2 is a graph showing the effect of in vitro plant breeding;
FIG. 3 is a partially enlarged schematic view of a cultivated isolated plant;
FIG. 4 is a graph of adult vs. nymph harvest, where the upper curve represents the nymph of diaphorina citri at 4-5 years old and the lower curve represents the adult diaphorina citri;
FIG. 5 is a diagram of the dissection of the midgut and diaphorina citri nymphs;
fig. 6 is a graph showing the germ-carrying rate of diaphorina citri nymphs after 5 days of feeding, M: takara DL 2000maker, yang: susceptible shoots, yin: non-infected branches;
FIG. 7 is a graph showing the results of the disease rate in different days.
Detailed Description
The invention provides a method for obtaining a CLas infestation diaphorina citri polypide body, which comprises the following steps: screening the infected shoots and culturing in vitro, screening C using real-time fluorescent quantitative PCRtSusceptible branches of similar valueAnd putting the diaphorina citri on the susceptible branches for infection to obtain susceptible diaphorina citri polypore bodies.
In the present invention, susceptible shoots are obtained using PCR assay screening. The invention uses PCR detection technology to screen positive infected branches, and ensures the reliability of experimental results.
In the invention, the PCR detection amplification primers are:
F：5’GCGCGTATGCAATACGAGCGGCA 3’
R：5’GCGTCGCGACTTCGCAACCCAT 3’。
in the present invention, the PCR detection reaction procedure is: pre-denaturation at 95 ℃ for 3 min; 30s at 95 ℃, 30s at 62 ℃, 1min at 72 ℃ for 10s, and 34 cycles; 5min at 72 ℃; 5min at 25 ℃.
In the invention, the condition of the in vitro culture is to culture by using 0.1-2 times concentration Hoagland nutrient solution. Preferably, the condition of the in vitro culture is to culture by using 0.5-1 fold concentration of Hoagland nutrient solution. In the invention, the concentration of Hoagland nutrient solution is screened, and the infected branches obtained by culture can survive in vitro for 8-10 days. The Hoagland nutrient solution is prepared by using a Hoagland nutrient solution mother solution according to the required concentration. The Hoagland nutrient stock solution used in the present invention is commercially available from Coolaber as 500 × modified Hoagland nutrient solution (NS10205-250L), but this should not be construed as limiting the scope of the present invention.
In the invention, the real-time fluorescent quantitative PCR amplification primers are as follows:
F：5’TGGAGGTGTAAAAGTTGCCAAA 3’
R：5’CCAACGAAAAGATCAGATATTCCTCTA3’。
in the invention, the real-time fluorescent quantitative PCR reaction system is as follows: ddH2O3.5 μ L; SYBR 5. mu.L; F/R is 0.25 mu L; 1 mu L of cDNA template; total system 10. mu.L. The invention further screens the screened positive susceptible material by using real-time fluorescence PCR, and then selects CtSubsequent experiments are carried out on the isolated branches with similar values, so that the germ carrying capacity of the citrus branches can be kept basically consistent, and the accuracy of the experiment effect is improved.
In the invention, the real-time fluorescent quantitative PCR reaction program is as follows: pre-denaturation at 95 ℃ for 1 min; 95 ℃ for 15s, 59 ℃ for 15s, 72 ℃ for 45s, 45 cycles.
In the invention, the pest period of the diaphorina citri is 4-5 years nymph. The method can improve the bacteria-obtaining efficiency by screening the insect period of the diaphorina citri.
In the invention, the infection days are 2-8 days. Preferably, the infection days are 4-6 days.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Culturing in-vitro citrus twigs:
in-vitro plants are fed by adopting Hoagland nutrient solutions with different dilution times, the survival state of branches is observed, and Hoagland nutrient solutions with the best growth vigor and the longest survival time and corresponding to the dilution times are selected for carrying out subsequent experiments. The method comprises the following steps:
the method comprises the steps of inoculating citrus yellow dragon germs to virus-free nursery stocks cultured in a greenhouse to confirm infected trees, collecting tender branches of the citrus yellow dragon disease infected trees, selecting branches infected with the citrus yellow dragon disease after PCR detection, dividing the branches into 4 groups, repeating 3 branches in each group, culturing the branches in tap water, 0.1 x Hoagland nutrient solution, 0.5 x Hoagland nutrient solution, 1 x Hoagland nutrient solution and 2 x Hoagland nutrient solution respectively, replacing the culture solution periodically, and observing the physiological state of the isolated branches. Finally, the optimal nutrient solution concentration of 0.5 XHoagland nutrient solution is determined, and the optimal nutrient solution concentration is shown in table 1.
Wherein, the PCR detection primer is:
F：5’GCGCGTATGCAATACGAGCGGCA 3’
R：5’GCGTCGCGACTTCGCAACCCAT 3’
PCR reaction procedure: pre-denaturation at 95 ℃ for 3 min; 30s at 95 ℃, 30s at 62 ℃, 1min at 72 ℃ for 10s, and 34 cycles; 5min at 72 ℃; 5min at 25 ℃.
TABLE 1 survival time of shoots treated with different Hoagland nutrient solutions
Treatment concentration 0.1× 0.5× 1× 2× Tap water
Survival time/d 4 8 6 4 3
Example 2
Screening the susceptible citrus twig material:
detection of DNA corresponding to the material positively obtained by the PCR detection technique in example 1 Using ddH2O is diluted to 150 ng/. mu.L and is used for real-time fluorescence quantitative PCR experiment.
The real-time fluorescent quantitative PCR primer is as follows:
F：5’TGGAGGTGTAAAAGTTGCCAAA 3’
R：5’CCAACGAAAAGATCAGATATTCCTCTA 3’
the reaction system is as follows: ddH2O 3.5μL；SYBR 5μL；F/R is 0.25 mu L; 1 mu L of cDNA template; total system 10. mu.L.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 1 min; 95 ℃ for 15s, 59 ℃ for 15s, 72 ℃ for 45s, 45 cycles. The reactions were all performed on a Roche LightCycler 480 II real-time fluorescent quantitative PCR instrument. Selecting C after the reaction is finishedtSubsequent experiments were performed on isolated shoots of similar values.
According to the results shown in FIG. 1 and Table 1, branches 1-2, 4 and 7-10 (Z1-Z2, Z4 and Z7-Z10) are positive branches, and C is selectedtZ1 and Z9 with close values are a group; z8 and Z10 are a group for subsequent experiments.
TABLE 2 real-time fluorescent quantitative PCR detection of citrus in-vitro shoot infection
Z1 Z2 Z3 Z4 Z5 Z6 Z7 Z8 Z9 Z10 7-3 7-4 Yin body Yang (Yang)
Ct 25.3 28.4 - 29.2 - - 26.8 27.3 25.9 27.2 - - - 29.4
Example 3
The experiment for obtaining the bacteria of the diaphorina citri in different insect stages comprises the following steps:
respectively carrying out a bacteria-obtaining experiment on the diaphorina citri imago, the nymphs of 4-5 years and the nymphs of 3 years and the following nymphs, and screening out the most suitable bacteria-obtaining period, wherein the method specifically comprises the following steps:
respectively placing adult diaphorina citri, nymphs of 4-5 years and nymphs of 3 years and below on isolated plants for bacteria obtaining experiments, placing the isolated plants in a culture tube, fixing the positions of the isolated plants by using sponges, placing the isolated plants on a foam platform, adding a cup-shaped plastic closed environment, and arranging an insect-proof net above the closed environment, as shown in an effect diagram of fig. 2. Screening results show that 3-year-old and below nymphs are not easy to survive on isolated branches, and the adult nymphs have lower bacterium harvesting efficiency than 4-5-year-old nymphs, so 4-5-year-old nymphs are selected for bacterium harvesting experiments, as shown in fig. 4.
Picking 4-5-year old diaphorina citri nymphs to citrus twigs infected with yellow dragon germs, picking 10-15 surviving diaphorina citri at intervals of 3 days, dissecting and separating the oral needle, the midgut and the residual tissues, and detecting the susceptibility by using the PCR technology in the embodiment 1.
During the process of acquiring the psylla, the yellow dragon pathogen is inhaled from the psylla oral needle, reaches the midgut, and finally passes through the barrier of the midgut to enter the hemolymph. Generally, psyllid infection is considered as a purposeful strip for whole insect detection, and in the invention, in order to eliminate the interference of possible residual germs in the oral needle, anatomical separation is carried out, so that the test result is more accurate. The amplification results are shown in FIG. 6: the amplification results of the head and the stylet of the psylla are 1-24 of the first row and 25-30 of the third row; the amplification results of the residual tissues of the psyllids are 1-24 in the second row and 25-30 in the third row; and the third row 1-8 is the amplification result of the whole psyllid.
As can be seen from fig. 7, the rate of psyllid infestation at day 5 using the method of the present invention was 100%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A method for obtaining a CLas body infecting diaphorina citri is characterized by comprising the following steps: screening the infected shoots and culturing in vitro, screening C using real-time fluorescent quantitative PCRtAnd (4) placing the diaphorina citri on the susceptible branches with similar values to infect the susceptible branches to obtain susceptible psylla polyporus frondosus bodies.
2. The method according to claim 1, wherein the diseased shoots are obtained using PCR assay screening.
3. The method of claim 2, wherein the PCR detection amplification primers are:
F：5’GCGCGTATGCAATACGAGCGGCA 3’
R：5’GCGTCGCGACTTCGCAACCCAT 3’。
4. the method of claim 2, wherein the PCR detection reaction program is: pre-denaturation at 95 ℃ for 3 min; 30s at 95 ℃, 30s at 62 ℃, 1min at 72 ℃ for 10s, and 34 cycles; 5min at 72 ℃; 5min at 25 ℃.
5. The method according to claim 1, wherein the ex vivo culture is performed under conditions in which 0.1-to 2-fold concentration of Hoagland nutrient solution is used.
6. The method of claim 1, wherein the real-time fluorescent quantitative PCR amplification primers are:
F：5’TGGAGGTGTAAAAGTTGCCAAA 3’
R：5’CCAACGAAAAGATCAGATATTCCTCTA 3’。
7. the method of claim 6, wherein the real-time fluorescent quantitative PCR reaction system is: ddH2O3.5 μ L; SYBR 5. mu.L; F/R is 0.25 mu L; 1 mu L of cDNA template; total system 10. mu.L.
8. The method of claim 6, wherein the real-time fluorescent quantitative PCR reaction program is: pre-denaturation at 95 ℃ for 1 min; 95 ℃ for 15s, 59 ℃ for 15s, 72 ℃ for 45s, 45 cycles.
9. The method according to claim 1, wherein the diaphorina citri has a nymph of 4-5 years.
10. The method of claim 1, wherein the number of infected days is 2 to 8 days.
CN202210504089.XA 2022-05-10 2022-05-10 Method for obtaining CLas infected diaphorina citri polypide body Pending CN114717335A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116769819A (en) * 2023-05-11 2023-09-19 广东省农业科学院果树研究所 Application of citrus CsWRKY40 gene in repelling diaphorina citri

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116769819A (en) * 2023-05-11 2023-09-19 广东省农业科学院果树研究所 Application of citrus CsWRKY40 gene in repelling diaphorina citri
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