Method for converting pathogenic bacteria of pokkah boeng disease of sugarcane

A technology of sugarcane tip rot and pathogenic bacteria, applied in the field of fungal genetic transformation, to achieve the effect of reducing the analysis of gene insertion sites, small insertion fragments, and rapid methods

Pending Publication Date: 2018-03-02
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, a large-scale and efficient method for obtaining mutants of sugarcane tip rot has not been established so far. Therefore, establishing a set of rapid and effective methods for making mutants resistant to sugarcane tip rot is of great significance for the prevention, control and research of this disease.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] A method for transforming sugarcane tip rot pathogenic bacteria, comprising the following steps:

[0033] 1) Preparation of sugarcane tip rot pathogen spore suspension

[0034] Inoculate the pathogenic bacteria of sugarcane tip rot (strain CNO~1) on the first PDA medium for activation, pick the hyphae at the edge of the colony and inoculate them in the sterilized PDB medium for shaking culture, collect the bacteria by centrifugation and filtration, and then use Sterile water made to a concentration of 1 x 10 6 ~1×10 7 The sugarcane tip rot pathogenic spore suspension of CFU / ml is for subsequent use;

[0035] 2) Hypertonic treatment

[0036] Spread a 20-25 μm sterile filter membrane on a hypertonic culture medium (petri dish with a diameter of 9 cm) on an ultra-clean workbench, and then take 250-350 μl of sugarcane tip rot pathogenic spore suspension and evenly spread it on the On a 20-25 μm sterile filter membrane, the hypertonic treatment time is 6-12 hours, and dr...

Embodiment 2

[0052] A method for transforming sugarcane tip rot pathogenic bacteria, comprising the following steps:

[0053] 1) Preparation of sugarcane tip rot pathogen spore suspension

[0054] Inoculate the pathogenic bacteria of sugarcane tip rot (bacterial strain YN41) on the first PDA medium for activation, pick the edge hyphae of the colony and inoculate them in the sterilized PDB medium for shaking culture, centrifuge and filter to collect the thalline, and then use sterile Water made with a concentration of 1×10 6 The sugarcane tip rot pathogenic spore suspension of CFU / ml is for subsequent use;

[0055] 2) Hypertonic treatment

[0056] On the ultra-clean workbench, spread the 20 μm sterile filter membrane on the hyperosmotic medium (diameter 9 cm culture dish), and then take 250 μl of sugarcane tip rot pathogenic spore suspension and evenly spread it on the 20 μm sterile filter. On the membrane, the hyperosmotic treatment time is 6 hours, and the filter membrane containing fung...

Embodiment 3

[0072] A method for transforming sugarcane tip rot pathogenic bacteria, comprising the following steps:

[0073] 1) Preparation of sugarcane tip rot pathogen spore suspension

[0074] Inoculate the pathogenic bacteria of sugarcane tip rot (strain CNO~1) on the first PDA medium for activation, pick the hyphae at the edge of the colony and inoculate them in the sterilized PDB medium for shaking culture, collect the bacteria by centrifugation and filtration, and then use Sterile water made to a concentration of 1 x 10 7 The sugarcane tip rot pathogenic spore suspension of CFU / ml is for subsequent use;

[0075] 2) Hypertonic treatment

[0076] On the ultra-clean workbench, spread the 25 μm sterile filter membrane on the hyperosmotic medium (diameter 9 cm culture dish), and then take 350 μl of sugarcane tip rot pathogenic spore suspension and evenly spread it on the 25 μm sterile filter. On the membrane, the hyperosmotic treatment time was 12 hours, dried to obtain a filter memb...

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Abstract

The invention discloses a method for transforming sugarcane tip rot pathogenic bacteria. The prepared spore suspension of sugarcane tip rot pathogen is evenly coated on a sterile filter membrane, and the expression cassette containing the fusion of tryptophan operon and hygromycin resistance gene is mixed with gold powder to form microprojectiles; Insertion mutants were obtained through resistance screening culture and molecular detection. The expression cassette used in the present invention is directly inserted to obtain mutants, and this method is easier to transform and obtain mutants.

Description

technical field [0001] The invention relates to a method for transforming sugarcane tip rot pathogenic bacteria, and belongs to the technical field of fungal genetic transformation. Background technique [0002] The pathogen of sugarcane tip rot (Pokkah Boeng Disease) is Fusarium moniliformae (Fusarium moniliformae), which can produce beaded conidia or pseudocephalic sporulation in the asexual stage, and its sexual form is Gibberella moniliformis. Sugarcane tip rot is a worldwide disease that seriously threatens the sugarcane industry and has a great impact on the yield and quality of sugarcane. In my country, the incidence of tip rot is increasing gradually, and the incidence of tip rot in some areas is above 25%, up to 40%. It has become the main disease of sugarcane in the early and middle stages of growth. The research on this pathogen is still relatively weak, especially the research on gene function has not been reported yet. Using the phenotype and pathogenicity of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89C12R1/77
CPCC12N15/895
Inventor 张木清王继华徐世强
Owner GUANGXI UNIV
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