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Single-spore isolation method for plasmodiophora brasicae physiological race

A technology for single spore separation and club rhizogenes, applied in the biological field, can solve the problems of low infection rate, imperfect separation technology, limited physiological races, etc., and achieve the effect of shortening the experimental process and low infection rate of single spore separation.

Inactive Publication Date: 2017-10-13
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From these research results, it can be seen that the physiological races isolated by the existing technology are very limited. The main reason is that the separation technology is not perfect, especially the infection rate is too low and the seedling rate is not high.

Method used

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  • Single-spore isolation method for plasmodiophora brasicae physiological race
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  • Single-spore isolation method for plasmodiophora brasicae physiological race

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Infection Rate Survey

[0054] The single spore isolation was carried out by agar method instead of hydroponic cultivation of bacteria liquid ( figure 1 ), the agar method for single spore isolation is to drop a suitable concentration of bacterial solution on a glass slide containing 1.0% agarose, and observe under a microscope. Cut off the agar block where a single spore was observed, and continue to observe until there is only one spore in the agar block. Move the agar block containing single spore into the seedlings cultivated in the freezer box for inoculation, and carry out hydroponic cultivation after a few days until the root system is infected and carry out substrate culture ( figure 2 ). In this step, the original technology is improved, and the ordinary culture medium is replaced by a freezing box, which saves costs, ensures that a single spore has sufficient time to contact the root hair to increase the infection rate, and replaces the previous ...

Embodiment 2

[0056] Embodiment 2: the determination of inoculated seedling age

[0057] The seedlings at the age of 1 day, 2 days, 3 days and 4 days were selected for monospore inoculation, and it was found that the 1-day-old seedlings were infected 32 days after inoculation, and the 2-day-old and 3-day-old seedlings were infected with The time of infection is earlier, 8 days into the early stage of infection, 12 days into the peak of infection. The 4-day-old seedlings were infected 24 days after inoculation. Therefore, 2 days and 3 days are the best inoculation period (see Figure 3-6 ). It was analyzed that the slow infection of 1-day-old seedlings may be due to the displacement of the agar blocks after they germinated and continued to grow.

Embodiment 3

[0058] Embodiment 3: investigation of plant survival rate

[0059] Due to the limited space in the freezer box, the seedlings can only grow in the freezer box for 6 days. Later, hydroponics is used. In the traditional method, the single-spore seedlings are placed in a centrifuge tube for cultivation, and the nutrient solution is poured with a syringe every day to maintain the plants. Growth is time-consuming and laborious, and the survival rate of seedlings is low in this process, and later cultivation is also relatively difficult. Experimental comparison found that one of the problems can be greatly improved by transferring seedlings to sterilized media after infection. The specific experimental method is as follows:

[0060] Experiment with 2-day-old seedlings, and 400 plants were cultivated in two batches. After a few days of inoculation, some of them were hydroponics first, and then turned to soil culture after infection (plan one) (see Figure 7 ); the other part is hyd...

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Abstract

The invention relates to a single-spore isolation method for plasmodiophora brasicae. The method is characterized by including: freezing box filter paper culture: transferring an agar block containing single spore into a freezing box, putting the freezing box on a filter paper cultivated Chinese cabbage seedling root for inoculation; firstly conducting dark culture in the freezing box; letting the agar block contact root hairs to facilitate plasmodiophora infection; transition culture: conducting hydroponic culture after 6 days of growth, transferring the seedling into a centrifugal tube containing distilled water, putting the centrifugal tube into a light-tight centrifugal tube box and conducting culture in an illumination culture box in an environment with a temperature of 23DEG C, a humidity of 60% and illumination of 16h / d for 2 days, then using a Hongland's nutrient solution to conduct hydroponic culture, replenishing the nutrient solution every day until the root hair is infected, and then transferring the seedling into a matrix for culture; when the seedling grows to 4 true leaves, conducting sterile soil culture, and 40 days later, collecting plasmodiophora for physiological race identification. The method provided by the invention can increase the incidence rate to 47.86%, and enhances the plant survival rate from 27% to 67% through inoculation seedling period selection and culture condition optimization.

Description

technical field [0001] The invention relates to a method for isolating single spores of Plasmodium phylloxera of Chinese cabbage, is a technique for separating physiological races of plant pathogenic bacteria, and belongs to the field of biotechnology. Background technique [0002] Clubroot of cruciferous crops, also known as root cancer, is a worldwide soil-borne disease caused by the infection of Plasmodiophora brassicae, which seriously affects the production of Chinese cabbage and other cruciferous crops. Since Plasmodium usually infects in a mixed state of multiple physiological races, its resistance cannot last long. Therefore, it is of great application value to isolate various physiological races of Plasmodium through isolation technology of single spores of Plasmodium. [0003] At present, there have been some reports on the identification of physiological races using the single spore isolation technology of Pluzobacterium spp., for example, in 2008, Xue et al. used...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N1/02C12R1/645
Inventor 冀瑞琴冯辉刘一凡王赛章云李承彧王玉刚刘志勇黄胜楠
Owner SHENYANG AGRI UNIV
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