Method for preparing and preserving high-purity culture of ralstonia solanacearum causing tobacco bacterial wilt

Abstract

The invention discloses a method for preparing and preserving a high-purity culture of ralstonia solanacearum causing tobacco bacterial wilt. The method provided by the invention comprises the following steps of: dealing with stalks of diseased or infected plants with tobacco bacterial wilt typical symptoms and then keeping the humidity at 27-30 degrees centigrade at a sterile state for 12-24 hours to obtain a culture containing ralstonia solanacearum; identifying and sieving a high-purity culture with the bacteria-containing amount of more than 80% by using a TTC (Triphenyl-tetrazolium chloride) method; and preserving the obtained high-purity culture at the constant temperature from minus 20 degrees centigrade to minus 40 degrees centigrade by utilizing a preserving fluid A or preservingthe obtained high-purity culture at the constant temperature of 20 degrees centigrade by utilizing a preserving fluid B, wherein the preserving fluid A is composed of 100-200 mL of glycerol and 20-40g of skim milk powder and is prepared by the following steps of: adding distilled water to dissolve and determine the volume to 500 mL; shaking up and regularly sterilizing to prepare the preserving fluid A, and the preserving fluid B is prepared by the following steps of: adding 1-2 g of agar into 1000 mL of distilled water to be melted; shaking up and regularly sterilizing to prepare the preserving fluid B. The method for preparing and preserving the high-purity culture of ralstonia solanacearum causing tobacco bacterial wilt, provided by the invention, is simple and feasible and has the advantages of high purity of the target bacteria, stable performance and good preserving effect.

Description

technical field [0001] The invention belongs to the technical field of microorganism purification and preservation, and in particular relates to a preparation method of a high-purity culture of Ralstia solanacearum that causes tobacco bacterial wilt and two preservation methods. Background technique [0002] Tobacco bacterial wilt (Tobacco bacterial wilt) is one of the main bacterial diseases in tobacco production. It occurs in most of the tobacco-producing areas in China, and it tends to increase year by year. Its pathogenic bacterium is Ralstonia solanacearum (Ralstonia solanacearum), and the host of this bacterium is extensive, and existing technology can realize the separation, cultivation and preservation of this bacterium, see " Chinese tobacco disease " (Zhu Xianchao, Wang Yanting, Wang Zhifa, China Agriculture Publishing House, 2002, pp. 152-163), the culture method adopted is as follows: after the surface of the separated material is sterilized, the diseased tissue ...

AI Technical Summary

Problems solved by technology

The disadvantages of this method are: multiple transfers of germs on the artificial medium will cause the mutation of the germs, and some inherent characteristics will be lost; and it may not be possible to isolate the germs due to the small number of germs

[0003] In order to prevent the continuous passage of R. solanacearum on the artificial culture medium, the problem of rapid decline in pathogenicity or the change of other genetic traits, as well as the indeterminate effect of the existing technology to preserve the culture, to develop an economical and effective, And keep the separation, cultivation and preservation method of tobacco bacterial wilt pathogenic bacteria inherent characteristics, it seems particularly necessary