A kind of preservation method of solanacearum phage

A preservation method and technology of R. solanacearum, applied in the direction of microorganism-based methods, biochemical equipment and methods, preservation of microorganisms, etc., can solve the problems of short preservation time, large titer drop, unsatisfactory effect, etc., and achieve reasonable operation Effect

Active Publication Date: 2021-10-22
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, in the above-mentioned process, the phage strains need to be preserved. The existing phytopathogenic bacteria, especially the preservation methods of R. solanacearum phages, have a lot of titer reduction and short storage time, and the effect is not ideal, which seriously restricts the application of phages. develop

Method used

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  • A kind of preservation method of solanacearum phage
  • A kind of preservation method of solanacearum phage
  • A kind of preservation method of solanacearum phage

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0015] 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.

[0016] 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.

[0017] 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

[0023] Example 2 Determination of the host range of avirulent strains:

[0024] The host range of avirulent strains was determined using the spot method. The specific operation is:

[0025] 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 solidification, take 0.5 μL phage stock solution dropwise onto the surface of the plate, dry it, and incubate it upside down at 28°C overnight. 2d Observe the formation of plaques. The results showed that the strain could be parasitized by R. solanacearum phage ( figure 2 ).

Embodiment 3

[0027] (1) Inoculate the strain RSsw326-2 on a CPG plate, turn it upside down, and culture it at 28°C for 24-48 hours to make it grow a single colony.

[0028] (2) Pick a single colony of the strain RSsw326-2 in a Erlenmeyer flask filled with 100mL NB medium, and culture it at 28°C with shaking at 180rpm until the concentration is about 10 8 cfu / ml.

[0029] (3) Add 1mL of phage stock solution, mix well, let stand for 15min, shake overnight at 28°C and 130rpm, and culture to completely lyse the bacteria.

[0030] (4) Centrifuge the co-cultivation solution at 12,000 rpm for 5 minutes, and filter the supernatant with a 0.45 μm filter membrane to obtain the phage stock solution.

[0031] (5) Take the cultured phage stock solution, measure its titer and record it for future use.

[0032] (6) The phage stock solution (2.29*10 10 PFU / mL) do the following: ①Store the stock solution at room temperature; ②Store the stock solution at 4°C; ③Store the stock solution at -20°C; ④Store t...

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Abstract

The invention discloses a storage method for Ralstonia solanacearum phage. The non-toxic R. solanacearum RSsw326‑2 disclosed by the invention is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2018197. The non-toxic R. solanacearum in the present invention is the R. solanacearum isolated from naturally occurring tobacco plants, and the avirulent R. solanacearum obtained through subculture, adopts phage stock solution mixed with an equal amount of R. solanacearum in this proposal After adding a final concentration of 40% glycerol and storing at -75°C for 12 months, the titer decreased less, and the storage time for R. solanacearum phage was long and high activity could be maintained.

Description

technical field [0001] The invention relates to the field of biotechnology and the field of microbial products, in particular to a method for preserving bacteriophage of Ralstonia 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/04C12R1/92
CPCC12N1/04
Inventor 林志坚蔡学清胡方平
Owner FUJIAN AGRI & FORESTRY UNIV
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