Method for authenticating and distinguishing fusarium culture causing soybean root rot

A technology for soybean root rot and Fusarium spp. is applied in the measurement/inspection of microorganisms, biochemical equipment and methods, etc., which can solve the problem of high cost of pathogen identification, achieve good application value and reduce costs

Inactive Publication Date: 2018-07-13
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The identification method of ITS sequence is accurate, but the cost for large-scale identification of bacteria is still quite high

Method used

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  • Method for authenticating and distinguishing fusarium culture causing soybean root rot
  • Method for authenticating and distinguishing fusarium culture causing soybean root rot
  • Method for authenticating and distinguishing fusarium culture causing soybean root rot

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: the morphological characteristic of fusarium

[0043] Isolation and purification of pathogenic bacteria

[0044] The wilted diseased plants were collected from the soybean planting field and stored in dry ice for future use. Separation and purification of pathogenic bacteria in the laboratory: rinse the sample with tap water; disinfect with 2% sodium hypochlorite for 10 minutes, rinse with 75% alcohol for 2-3 times, and use sterile water. Rinse several times. Use a sterile knife to cut out the junction of stem disease and health, about 1 cm, inoculate it on a PDA solid medium (containing 100 μg / mL ampicillin) plate, and culture at 25 ° C. After 3 days, with ddH 2 O (containing 100 μ g / mL ampicillin) rinses the colony surface, observes the concentration of the bacterium amount under the electron microscope, and the bacterium amount is diluted to only 3-5 spores in the visual field under the 10-fold microscope, draws an appropriate amount of spore liquid ...

Embodiment 2

[0051] Embodiment 2: the molecular identification of Fusarium

[0052] DNA extraction

[0053] Nearly 1 g of mycelium was suspended in 650 μL of 2×CTAB extract (add 0.2% β-mercaptoethanol before use), then 4 μL of RNase enzyme was added, and ground in a mortar. The ground tissue fluid was incubated in a water bath at 65°C for 30 minutes, and extracted with an equal volume of chloroform:isoamyl alcohol (24:1). Precipitate DNA with two volumes of absolute ethanol, wash with 75% ethanol, ddH 2 O was dissolved and stored at -20°C for later use. DNA quality was detected by spectrophotometer and agarose gel electrophoresis.

[0054] PCR amplification

[0055] Fungal universal primer ITS1F (5'-CTTGGTCATTTTAGAGGAAGTAA-3', Gardes M, Bruns TD. ITSprimers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Molecular Ecology, 1993, 2, 113-118) and ITS4 (5'-TCCTATCCGCTTATTG -3', White TJ, Bruns TD, Lee SB, TaylorJW. Amplification an...

Embodiment 3

[0060] Embodiment 3: CAPS molecular marker development and strain distinction

[0061] Select one Fusarium from each of the four types of Fusarium, use ITS1F and ITS4 primers, and use DNA as a template to carry out PCR amplification, and the PCR products are separated by electrophoresis on 3% agarose gel. From the results of electrophoresis, it can be seen that the amplified band of Fusarium solanifolia runs significantly slower than that of the other three species of bacteria, while the amplified bands of the other three species of bacteria move at the same speed. spp. can be directly distinguished from the other 3 species of Fusarium ( Figure 4 -A). Then, compare the restriction endonuclease restriction endonuclease site difference of the ITS sequence of other 3 kinds of Fusarium, find 3 kinds of specific restriction endonucleases, BspCNI, AvaI and PstI (New England BioLabs, USA), Develop specific CAPS molecular markers. The ITS PCR amplification products of the four Fus...

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Abstract

The invention discloses a method for authenticating and distinguishing fusarium culture causing soybean root rot. The method is characterized in that: fusarium solani and other 3 kinds of fusarium areseparated by using 3 percent agarose gel electrophoresis according to morphology and a genetic evolution tree authentication result built by an ITS sequence, and in terms of an amplified ITS sequencelength; comparing the ITS sequences of the rest 3 kinds of fusarium, finding out a restriction enzyme cutting site BspCNI, AvaI and PstI of a specific-limited incision enzyme; forming different CAPSmolecular markers with ITS sequences obtained by performing enzymatic hydrolysis and amplification on different combinations of the 3 kinds of enzymes, separating the molecular markers with 1.5 percent agarose gel electrophoresis in order to separate the 3 kinds of fusarium. By adopting the method, the authentication and distinguishment of the fusarium culture can be realized by means of the agarose gel electrophoresis and the CAPS molecular markers, the method is suitable for large-scale and high-flux culture authentication and distinguishment, in particular to the rapid authentication and distinguishment of the main fusarium causing soybean root rot in the national soybean production areas.

Description

technical field [0001] The invention belongs to the field of microorganisms and biotechnology, and relates to a method for identifying and distinguishing Fusarium strains that cause soybean root rot. Background technique [0002] Accurately identifying the types of pathogenic bacteria and understanding the distribution and biological characteristics of pathogenic bacteria are the premise and basis for taking effective measures to prevent and control crop diseases. Traditional identification and classification of pathogenic bacteria mainly rely on identification of morphological characteristics and detection of pathogenicity, which is time-consuming and laborious, and may not be accurate. With the development of molecular biology techniques, the ribosome transcribed spacer (ITS) sequence of fungi is widely used in the classification and identification of pathogenic fungi because of its high conservation (Guadet J, Julien J, Lafay JF, Brygoo Y. Phylogeny of some Fusarium spec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04
CPCC12Q1/686C12Q1/6895C12Q2521/301
Inventor 刘世名郑娜张刘萍彭德良
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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