Novel ralstonia solanacearum phages and compositions and application thereof

A technology of R. solanacearum and bacteriophage, applied in the field of microorganisms

Active Publication Date: 2019-01-04
PHAGELUX (NANJING) BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, the phages of R. solanacearum that have been reported at home and abroad include myocauda ФRSA1, ФRSL1 and ∈RS-1; Brachyceridae ФR

Method used

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  • Novel ralstonia solanacearum phages and compositions and application thereof
  • Novel ralstonia solanacearum phages and compositions and application thereof
  • Novel ralstonia solanacearum phages and compositions and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Isolation and purification of phage

[0060] Sample pretreatment: Take 30g of Jiangtian soil samples from Anqiu City, Shandong Province, Fuyang City, Anhui Province, and Chongzhou City, Sichuan Province respectively, soak in 100ml of sterilized 0.9% NaCl, and let it stand overnight in a refrigerator at 4°C Centrifuge at 4000rpm at room temperature to take the supernatant for later use.

[0061] Isolation of phage: Take 20ml of supernatant from the above 3 materials respectively, filter with 0.22μm filter membrane, add to 20ml of 2×TSB liquid medium. Add 200 μl of R. solanacearum "3-2-1" in the logarithmic phase to the material containing Anqiu City, Shandong Province (R. solanacearum from ginger blast, provided by Sichuan Agricultural University ) bacteria solution, add 200 μl of Ralstia solanacie “5-2-2” in the logarithmic phase to the material containing Fuyang City, Anhui Province (Raleria solanaceae derived from ginger Agricultural University provides) ...

Embodiment 2

[0063] Embodiment 2: Electron microscope observation of phage

[0064] Take the phages prepared in Example 1 for electron microscope observation: take 20 μL of the sample and drop it on the copper grid, wait for it to settle naturally for 15 minutes, use filter paper to absorb the excess liquid from the side, and add 1 drop of 2% phosphotungstic acid (PTA) to the copper grid , stained for 10 minutes, sucked the dye solution from the side with filter paper, and observed with electron microscope after drying: the results are as follows: figure 2 As shown, GP1 bacteriophage has a regular polyhedral head structure and a short tail, the diameter of the head is about 60-70nm, and the length of the tail is about 15-20nm ( figure 2 -A); GP2 bacteriophage has a regular polyhedral head structure and a short tail, the diameter of the head is about 60-70nm, and the length of the tail is about 15-20nm ( figure 2 -B); GP3 bacteriophage has a regular polyhedral head structure and a short...

Embodiment 3

[0065] Example 3: Extraction and sequencing of phage genome

[0066] Take 100 mL of each phage prepared in Example 1, add DNaseI and RNaseA at a final concentration of 1 μg / mL, incubate at 37°C for 60 min, add 5.84 g NaCl (final concentration 1 mol / L), dissolve and place in an ice bath for 1 h. Centrifuge at 11,000 rpm for 10 min at 4°C, and transfer the supernatant to a new centrifuge tube. Add solid PEG8000 (final concentration 10%, that is, add 10g to 100mL), after complete dissolution, ice bath for at least 1h. Centrifuge at 11,000 rpm for 20 min at 4°C, and resuspend the pellet with a small amount of SM solution. Add an equal volume of chloroform and isoamyl alcohol for extraction, shake gently for 30 s, centrifuge at 8000 rpm for 1 min, absorb the supernatant, and repeat the extraction until clarification. Add DNase I and RNase A again to a final concentration of 1 μg / mL, and react at 37°C for 30-60 minutes. Add EDTA to a final concentration of 20mmol / L (that is, add ...

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Abstract

The invention relates to the field of microorganisms, in particular to a ralstonia solanacearum phage GP1 with the preservation number of CCTCC M 2016633, a ralstonia solanacearum phage GP2 with the preservation number of CCTCC M 2016634 and a ralstonia solanacearum phage GP3 with the preservation number of CCTCC M 2016635. The phages have the advantages that the phages are strict virulent phagesand can effectively kill ralstonia solanacearum of different plant host sources, especially the ralstonia solanacearum causing ginger blast; the phages are non-toxic to normal microbial flora, DNAs ofthe phages cannot encode virulence genes, and the stability is high under the pH of 5-9; there is no magnitude change in titer at 4 DEG C after preservation for one year; bacteria can be effectivelyinhibited and killed under low titer. The phages can be used for prevention and treatment of crop bacterial wilt caused by ralstonia solanacearum, especially ginger blast, excellent strain resources are provided for developing novel bio-pesticide resistant to bacterial wilt, and a good application and development prospect is achieved.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to novel R. solanacearum phages, their composition, their preparation method and application. Background technique [0002] Ralstonia solanacearum is a gram-negative bacterium of the subphylum β-proteobacteria that grows in soil. The pathogen can usually invade the intercellular spaces of the cortex from wounds in the roots or stems of plants or from the root caps of uninjured secondary roots. By destroying the intercellular glue layer, the cell walls are separated, deformed, and cavities are formed, thereby infecting The xylem parenchyma stimulates the small cells near the vessel to form the intrusion and migrate into the intrusion. After the invading body ruptures, R. solanacearum will continue to be released into the duct, and multiply and rapidly spread and expand in the duct, thereby causing the plant to wilt and die (He Liyuan, Kang Yaowei. Pseudomonas sola...

Claims

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Application Information

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IPC IPC(8): C12N7/00C07K14/005A01N63/00A01P1/00C12R1/92
CPCA01N63/00C07K14/005C12N7/00C12N2795/10221C12N2795/10222
Inventor 许文建丁良肖逍苏胜兵于浩丛郁徐旭凌赵伟王艳周思翔熊剑胜王卫斌马克·恩格尔沈婵娟
Owner PHAGELUX (NANJING) BIO-TECH CO LTD
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