Combination of a pair of maize smut fungus and its application
A kind of smut fungus, a pair of technology, applied in the direction of application, fungi, instruments, etc., can solve the problems of poor test accuracy, consistency and repeatability, uncertain results of artificial inoculation, corn plant rot, etc. Achieve the effects of strong pathogenicity, easy harvesting and transportation, and high success rate of inoculation
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Embodiment 1
[0038] Example 1 Separation and Screening and Identification of White Polygezide Straisage of Obusic Black Fungus
[0039] Perform as follows:
[0040](1) Collecting winter spores and preparing a Winter Spore suspension: In December 2016, the natural air-dried corn tumor flavor is collected in Hainan, directly picking a small amount of winter spores directly from the cornoma black powder. Place in sterilization. In the 2ml centrifuge tube, then 1 ml of sodium hypochlorite solution was added in weight percent, and centrifuged at 2 min, 12000 rpm, 1 min, to the supernatant, add sterilization water to clean the precipitation 3, and finally use sterile water to dilute the precipitate. That is, the petrospore suspension, its final concentration is 10 3 Spore / ml.
[0041] (2) Winter aprotic culture: Take 200 μL of the Winter Spore suspension obtained from step (1) to PDA medium, cultured for 2 days at 25 ° C, which can be observed to observe the fine colony visible to the naked eye.
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Embodiment 2
[0050] Example 2: Screening assay for combination of different maize black powder
[0051] (1) The propagation of the monocrete strain: 5 of the monocretetic strains obtained in Example 1 were evenly applied to different PDA plate media, respectively. 3 days were cultured at 25 ° C, dark conditions to obtain a haploid.
[0052] (2) Preparation of the inoculus of the monocrete strain: Steps (1) from the PDA plate culture medium were scraped from the PDA plate culture medium with a sterilized slide, and in accordance with 1 petri dish (9 cm in diameter of the culture dish). The bacteria were diluted with 1 L of sterilization of water, respectively, with a peripheral strain of 3, 6, 7, 8 or 11, respectively.
[0053] (3) According to the mating type of each haploid strain in Example 1, two two-two combinations of different interstitial monoclonal strains, that is, the second type of loftter strain prepared by step (2). The mother liquor was mixed according to the mass ratio of 1: 1, ...
Embodiment 3
[0060] Example 3 Classification and identification of haplot strains UM01 and UM02 of the present invention
[0061] The genomic DNA of UM01 or UM02 was extracted, the genomic DNA of UM01 or UM02 strain was a template, and the fungi germic primer ITS1 and ITS4 were used for PCR amplification, the primer sequence as:
[0062] ITS1: 5'-tccgtaggtgaacctgcgg-3 ';
[0063] ITS4: 5'-tcctccgcttttgatatgc-3 '.
[0064] The PCR reaction system (25 μl), wherein: ITS1 (10 μmol / L) 0.5 μL, ITS4 (10 μmol / L) 0.5 μL, ES TAQ MASTERMIX 12.5μL, Template 2μL, DDH 2 O-25 μL. The PCR reaction procedure: 95 ° C pre-degeneration is 5 min, 95 ° C denaturation 30s, 56 ° C for 1 min, 35 cycles at 72 ° C, 35 cycles; final 72 ° C for 10 min, 4 ° C. 5 μl of the amplified product was taken for 1% agarose gel electrophoresis, and the PCR product size was detected under the UV transmitter, and the PCR amplification product was directly sent to Biological Engineering (Shanghai) Co., Ltd. to obtain the ITS sequen...
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