Molecular identification primer and identification method for hyacinth dolichos sclerotinia rot
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An identification method and molecular identification technology are applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection. Accurate results and high sensitivity
Pending Publication Date: 2020-07-24
SHANGHAI JIAO TONG UNIV
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Since the plant disease phenotype can only be observed in the middle and late stages of disease onset, and at this time the pathogen has already invaded the body, the phenotype identification method is of little significance for the prevention and control of sclerotinia sclerotiorum
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Embodiment 1
[0040] With trichoderma, sclerotinia soybean as contrast, identify sclerotinia lentils with molecular identification primer of the present invention and identification method:
[0041] Step 1. Using Trichoderma and Sclerotinia soybean as controls, the experimental strains of Trichoderma, soybean and Sclerotinia lentils were respectively inoculated in the PDA medium, in a constant temperature air bath shaker at a speed of 200rPm, at 28°C Under culture for 24h. The DNA of each strain was extracted using a genomic DNA extraction kit (DP307), and stored at -20°C until use.
[0042] Step 2, using Sclerotinia lentils and control strain DNA as templates, using Sclerotinia sclerotiorum PCR-specific primers LpITS-F and LpITS-R to carry out conventional PCR amplification, the PCR reaction system is as follows: 50 μ l of the reaction system contains 2× TaqPCR MasterMix 25μl, DNA 2μl, LpITS-F 2μl, LpITS-R 2μl, ddH 2 O 19 μl.
[0043] The amplification reaction conditions were: pre-denatu...
Embodiment 2
[0051] With Trichoderma, Sclerotinia sclerotiorum as contrast, identify Sclerotinia lentils with molecular identification primer of the present invention and identification method:
[0052] Step 1. Taking Trichoderma and Sclerotinia sclerotiorum as controls, Trichoderma, rapeseed and Sclerotinia lentils experimental strains were respectively inoculated in PDA medium, in a constant temperature air bath shaker at a speed of 200rPm, at 28°C Under culture for 24h. The DNA of each strain was extracted using a genomic DNA extraction kit (DP307), and stored at -20°C until use.
[0053] Step 2, using Sclerotinia lentils and control strain DNA as templates, using Sclerotinia sclerotiorum PCR-specific primers LpITS-F and LpITS-R to carry out conventional PCR amplification, the PCR reaction system is as follows: 50 μ l of the reaction system contains 2× TaqPCR MasterMix 25μl, DNA 2μl, LpITS-F 2μl, LpITS-R 2μl, ddH 2 O 19 μl.
[0054] The amplification reaction conditions were: pre-den...
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Abstract
The invention discloses a molecular identification primer and identification method for hyacinth dolichos sclerotinia rot, and relates to the technical field of sclerotinia rot. The molecular identification primer comprises an identification sclerotinia species primer pair and an identification sclerotinia microspecies primer pair, wherein the identification sclerotinia species primer pair includes a forward primer LpITS-F and a reverse primer LpITS-R; and the identification sclerotinia microspecies primer pair includes a forward primer Lp18S-F and a reverse primer Lp18S-R. The identificationmethod comprises two identification processes; in the first identification process, the identification sclerotinia species primer pair is utilized to identify whether a to-be-detected strain is sclerotinia or not; for the strain which is the sclerotinia by an identification result, the second identification process is continued; and the identification sclerotinia microspecies primer pair is utilized to identify the sclerotinia microspecies type of the strain. The invention provides a specific primer used for identifying the hyacinth dolichos sclerotinia rot caused by the sclerotinia; the specific primer can quickly identify the hyacinth dolichos sclerotinia rot at the early stage, and thus, a technical basis is provided for integrated control over the hyacinth dolichos sclerotinia rot.
Description
technical field [0001] The invention relates to the technical field of sclerotinia, in particular to a molecular identification primer and an identification method of lentil sclerotinia. Background technique [0002] Sclerotinia sclerotiorum (Lib.) de Bary] can infect more than 400 kinds of plants including rapeseed, soybean, sunflower and other important crops, causing serious diseases and doing great harm to plant production, but lentil sclerotiorum Sclerotinia caused by bacteria has not been reported. Lentils are one of the important vegetable varieties in our country, not only edible but also medicinal. Sclerotinia sclerotiorum damages the base of the stem near the ground or near the branches near the surface. It is water-soaked at first, and then turns grayish white. The cortical tissue becomes dry and fibrous. In severe cases, the plant dies, causing serious losses to lentil production. Sclerotinia spores radiate for more than a month, spread with the wind, and infec...
Claims
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