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Method for obtaining CLas infected diaphorina citri polypide

A technology of citrus psyllids and worms, which is applied in the field of insect physiology and biochemistry, can solve the problems that plague the citrus industry and the difficulty in the prevention and control of Huanglongbing, and achieve the effect of reducing costs and reducing time costs

Pending Publication Date: 2022-07-08
GANNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, Huanglongbing pathogen (CLas) cannot be cultivated artificially, but it can grow and reproduce in the citrus psyllid for a long time. Once the citrus psyllid is infected by Huanglongbing, it can carry and spread rapidly for life. It has brought great difficulties and is the biggest problem plaguing the citrus industry in recent years

Method used

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  • Method for obtaining CLas infected diaphorina citri polypide
  • Method for obtaining CLas infected diaphorina citri polypide
  • Method for obtaining CLas infected diaphorina citri polypide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Cultivation of isolated citrus shoots:

[0045] The isolated plants were raised with Hoagland nutrient solution of different dilution ratios, and the survival status of the branches was observed. It includes the following steps:

[0046] The virus-free seedlings cultivated in the greenhouse were confirmed to be susceptible to the disease by inoculation with citrus huanglongbing, and the tender branches of the trees infected with citrus huanglongbing were collected. After PCR detection, the branches infected with huanglongbing were selected and divided into 4 groups. Three shoots were repeated, and were cultured in tap water, 0.1× Hoagland nutrient solution, 0.5× Hoagland nutrient solution, 1× Hoagland nutrient solution and 2× Hoagland nutrient solution. Physiological state of isolated shoots. Finally, 0.5 × Hoagland nutrient solution was determined as the optimum nutrient solution concentration, see Table 1.

[0047] Among them, PCR detection primers are:

[0048] F...

Embodiment 2

[0054] Screening of susceptible citrus shoots:

[0055] Use the PCR detection technology in Example 1 to detect the DNA corresponding to the positive material and use ddH2 O was diluted to 150ng / μL for real-time PCR experiments.

[0056] Real-time fluorescent quantitative PCR primers are:

[0057] F: 5'TGGAGGTGTAAAAGTTGCCAAA3'

[0058] R: 5'CCAACGAAAAGATCAGATATTCCTCTA 3'

[0059] The reaction system is: ddH 2 O 3.5 μL; SYBR 5 μL; F / R 0.25 μL; cDNA template 1 μL; total system 10 μL.

[0060] The reaction program was: pre-denaturation at 95°C for 1 min; 45 cycles of 95°C for 15s, 59°C for 15s, and 72°C for 45s. All reactions were performed on a Roche LightCycler 480Ⅱ real-time fluorescence quantitative PCR instrument. After the reaction is complete, choose C t In vitro shoots with similar values ​​were used for subsequent experiments.

[0061] according to figure 1 It can be seen from the results in Table 1 that branches 1-2, 4, 7-10 (Z1-Z2, Z4, Z7-Z10) are positive bran...

Embodiment 3

[0065] Experiments on bacteria acquisition of citrus psyllids in different insect stages:

[0066] The citrus psyllid adults, 4-5 instar nymphs, and 3 instar and below nymphs were subjected to bacteria acquisition experiments, and the most suitable bacteria acquisition period was screened, which specifically included the following steps:

[0067] The citrus psyllid adults, 4th-5th instar nymphs, and 3rd instar nymphs were placed on the isolated plants for bacterial acquisition experiments. The isolated plants were placed in a culture tube, and the position was fixed with a sponge. Placed in a cup-shaped plastic closed environment with an insect-proof net above it, such as figure 2 The effect diagram is shown. Through the screening results, it was found that the 3rd instar and below worms are not easy to survive on the isolated branches, and the adult larvae are less efficient than the 4th-5th instar nymphs. Therefore, the 4th-5th instar nymphs were selected for the bacteria ...

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Abstract

The invention provides a method for obtaining CLas infected diaphorina citri polypide, and relates to the technical field of insect physiology and biochemistry. The method comprises the following steps: firstly, screening out a concentration which is most suitable for survival of in-vitro citrus twigs from Hoagland nutrient solutions with different concentrations; selecting citrus twigs infected with Candidatus Liberibacter asiaticum by using conventional PCR (Polymerase Chain Reaction) and real-time fluorescent quantitative PCR technologies, and performing in-vitro culture; the method comprises the following steps: selecting diaphorina citri adults, nymphs of 4-5 years old and nymphs of 3 years old or below, screening an optimal bacterium obtaining period, and finally obtaining diaphorina citri polypides infected with the Candidatus Liberibacter asiaticum by utilizing a constructed system, so as to provide a basic material for subsequent theoretical research.

Description

technical field [0001] The invention relates to the technical field of insect physiology and biochemistry, in particular to a method for obtaining CLas-infected citrus psyllid parasites. Background technique [0002] Citrus Huanglongbing (HLB) is one of the most devastating diseases in the world's citrus industry. Since there is currently no effective treatment for susceptible trees, Huanglongbing is also known as the "cancer" of citrus. So far, CLas cannot be artificially cultivated, but it can grow and reproduce in the citrus psyllid for a long time. Once infected by CLas, citrus psyllid can be carried for life and spread rapidly, which is helpful for the study of Huanglongbing and its prevention and control. It has brought huge difficulties and is the biggest problem that has plagued the citrus industry in recent years. Nowadays, the prevention and control of citrus huanglongbing mainly focuses on the transmission of the insect-borne citrus psyllid. With the management ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/11
CPCC12Q1/6888C12Q1/6851C12Q2600/158C12Q2561/113C12Q2563/107C12Q2531/113
Inventor 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
Owner GANNAN NORMAL UNIV
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