A method for amplifying and propagating Ralstonia solanacearum phage

A R. solanacearum and phage technology, which is applied in the field of phage expansion and reproduction of R. solanacearum, can solve the problems of preventing and controlling the re-infestation of objects and restricting the development of phages, and achieve the effect of avoiding re-infestation

Active Publication Date: 2020-08-07
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, in the above-mentioned process, it is necessary to expand the production of phages. The phages of plant pathogenic bacteria in the prior art usually adopt their host bacteria to reproduce, and the host bacteria are plant pathogenic bacteria. However, when carrying out phage propagation, the host bacteria It is not completely lysed by phage, and it is easy to cause damage to the control object again when it is applied in the field, thus seriously restricting the development of phage application

Method used

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  • A method for amplifying and propagating Ralstonia solanacearum phage
  • A method for amplifying and propagating Ralstonia solanacearum phage
  • A method for amplifying and propagating Ralstonia solanacearum phage

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Embodiment 1

[0021] 1. Acquisition of R. solanacearum: From naturally occurring tobacco plants, the dilution coating separation method is used to isolate, and a single colony is picked for 3-5 times of purification to obtain wild R. solanacearum.

[0022] 2. Screening of non-toxic strains: wild R. solanacearum isolated from naturally occurring tobacco plants, after more than 20 generations of continuous culture, pick colonies with poor fluidity and carry out pathogenicity measurement. The measurement results show that the strain loses its pathogenicity to tobacco. The pathogenicity is a mutant strain of the virulent R. solanacearum, named RSsw326-2.

[0023] 3. Determination of pathogenicity: Pick a single colony of the mutant strain obtained by subculture and shake it in NB liquid medium (28°C, 180rpm) to a concentration of about 10 8 CFU / mL, using root irrigation to inoculate 5 true leaves of tobacco seedlings, 30 days after the inoculation treatment, the inoculated tobacco was the same ...

Embodiment 2

[0030] 1. Screening of non-toxic strains: R. solanacearum isolated from naturally occurring tobacco plants, after more than 20 generations of continuous culture, picked colonies with poor fluidity, and carried out pathogenicity determination. The measurement results showed that the strain lost its pathogenicity to tobacco. sex, which is a mutant strain of virulent R. solanacearum, named RSsw326-2.

[0031] 2. Determination of pathogenicity of non-toxic strains: Tobacco seedlings with 5 true leaves, variety: Cuibi No. 1, inoculation concentration of R. solanacearum: 10 8 CFU / mL; inoculation method: root irrigation inoculation;

[0032] 3. Determination of the host range of non-toxic strains:

[0033] The host range of avirulent strains was determined using the spot method. The specific operation is:

[0034] Take 1mL log phase R. solani ( Ralstonia solanacearium ) culture into the CPG semi-solid medium, mix well and pour it onto the pre-prepared CPG solid agar plate, after ...

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Abstract

The invention discloses a method for expanding reproduction of ralstonia solanacearium bacteriophage. An avirulent ralstonia solanacearium strain RSsw326-2 disclosed by the invention is preserved in CCTCC (China Center for Type Culture Collection) and has the preservation number of CCTCC NO. M 2018197. The avirulent ralstonia solanacearium disclosed by the invention is ralstonia solanacearium obtained by separating from naturally infected tobacco plants; by subculture, dissociant is obtained; by pathogenicity determination, the dissociant losing pathogenicity for tobacco seedlings is the ralstonia solanacearium avirulent strain; when the avirulent ralstonia solanacearium avirulent is utilized to reproduce the ralstonia solanacearium bacteriophage, the ralstonia solanacearium bacteriophagewith a high titer can be obtained. The method is expected to be used as a carrier for greatly expanding reproduction of the bacteriophage in the ralstonia solanacearium bacteriophage biological pesticide development so as to avoid secondary damage to prevention and control objects due to the toxicity of a host strain.

Description

technical field [0001] The invention relates to the fields of biotechnology and microbial products, in particular to a method for expanding and propagating bacteriophage of R. solanacearum. Background technique [0002] Phages are viruses that attack bacteria and also genetic material that confers biological traits on host bacteria. The phenomenon that phages can kill bacteria was discovered by Frederick W.Twort in 1915, so the research on using phages to control bacteria has been going on for nearly a hundred years. Phages must parasitize in living bacteria and have strict host specificity, which depends on the molecular structure and complementarity of phage adsorption organs and surface receptors of recipient bacteria. Phages are the most prevalent and widespread group of viruses. Bacteriophages are usually found in places full of bacterial communities, such as soil and animal guts. With the development of molecular biology technology, phage has been widely used in man...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N1/20C12R1/01
CPCC12N1/20C12N7/00C12N2795/00051
Inventor 蔡学清林志坚夏志辉胡方平
Owner FUJIAN AGRI & FORESTRY UNIV
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