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A kind of separation method of smut and used culture medium

A separation method and technology of smut fungus, applied in the field of smut fungus separation method and the medium used, can solve the problems of inability to focus on the target smut fungus, low overall efficiency, and difficulty in obtaining, so as to improve the efficiency of separation and purification , improved efficiency, and simple sample handling

Active Publication Date: 2019-08-30
INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The shortcoming of this method is: a, the purpose of general fungus isolation process is relatively poor, can only isolate all suspected fungi as much as possible from the material, and then identify which bacterial strain is smut from many fungi one by one; the whole separation Process fails to focus on target smut
It is more difficult to obtain materials, and there is more contamination by other yeast fungi, and the overall efficiency of this method is still low

Method used

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  • A kind of separation method of smut and used culture medium
  • A kind of separation method of smut and used culture medium
  • A kind of separation method of smut and used culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] This embodiment is to isolate smut fungus from Pteris cerevisiae without smut disease.

[0059] Prepare several kinds of solid media, namely ①PDA: 200g peeled potatoes boiled in water and finally filtered to obtain about 800ml of potato juice, add 20g glucose, 17g agar powder, natural pH value, add pure water to make up to 1L. ②YEPSL: Yeast extract 1%, peptone 0.4%, sucrose 0.4% and agar powder 1.7%, natural pH value; In addition, YEPSL liquid medium does not add agar powder. ③YPD: Yeast extract 1%, peptone 2%, glucose 2% and agar powder 1.7%. ④ HFJ separation medium: 0.4% sucrose, 0.5% calcium carbonate, 0.02% magnesium sulfate, 0.02% potassium dihydrogen phosphate, 1.5% agar powder, adjust the pH value to 6.8.

[0060] The above-mentioned several mediums were bottled and sealed, and sterilized under high temperature and high pressure at 115°C and 0.20Mpa for 18 minutes. Streptomycin and cephalosporin were added to the culture medium with agar powder cooled to 60-65°...

Embodiment 2

[0070] This embodiment is to isolate smut fungus from Zizania zizania with smut disease (Zizania stem enlargement is the result of Zizania smut Ustilago esculenta infection).

[0071] The difference between this example and Example 1 is that only the solid and liquid medium of HFJ solid medium and YEPLS are prepared. The effects of these two media on the separation of stem-expanded Zizania stems were compared. Among them, the proportion of solid HFJ separation medium is slightly different from that of Example 1: 1% glucose, 0.2% calcium carbonate, 0.05% magnesium sulfate, 0.03% potassium dihydrogen phosphate, 2% agar powder, pH 6.5.

[0072] 15 colonies were picked from each culture medium for identification, and the results: the yeast colonies isolated from the YEPSL medium were all yeasts; while the smut was isolated from the HFJ medium, respectively. are Ustilagoesculenta and Pseudozyma aphidis.

[0073] The ITS sequence sequencing results of two smut fungi are:

[0074]...

Embodiment 3

[0080] This embodiment is to isolate smut from storage rice without smut disease.

[0081] The difference between this example and Example 2 is that only HFJ solid medium and YEPLS liquid medium are prepared. Among them, the proportion of the solid HFJ separation medium is slightly different from that of Example 2: 1% mannitol, 0.4% calcium carbonate, 0.04% magnesium sulfate, 0.05% potassium dihydrogen phosphate, 2% agar powder, and the pH value is 7.0.

[0082] The stored rice without smut disease was ground and then isolated and cultivated.

[0083] Finally, 10 non-reflective white colonies were picked for culture and identification. ITS sequence alignment results: 8 colonies were Ustilago esculenta (100% similarity in NCBI); the other 2 colonies were similar to Pseudozymaantarctica in NCBI Yes 100%.

[0084] >ITS Ustilago esculenta (rice)

[0085] TTTTCTTGAGGTGTGGCTCGCACCTGTCTAACTAAATCGAGCTACCACATTTTAACACGGTTGCATCGGTTGGCTGTCAAACAGTGCGCGCGGCGAATTCATTTTCGCCCGCGCTCTGCGAGACG...

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Abstract

The invention relates to an ustilago esculenta separation method and a used culture medium. The separation method comprises the steps that vessel coating and culture are conducted on a sample, the used culture medium does not contain any nitrogen source and is prepared from, by mass, a carbon source 0.4%-1%, calcium carbonate 0.2%-0.6%, magnesium sulfate 0.01-0.05%, potassium dihydrogen phosphate 0.01-0.03% and agar powder 1.2-2%, wherein a pH value is 6.5-7.0. The culture medium also contains antibiotics capable of inhibiting bacteria rather than fungi. A white single colony is selected for fungus shaking after culture till turbidity can be seen with naked eyes, and if no sundry fungus pollution exists through microscopic observation, the fungus is a target strain obtained through separation. The two large problems of material limitations and pollution of a large number of sundry fungi are solved to the great degree, the ustilago esculenta separation method has no special requirement for separation materials, and the working efficiency of ustilago esculenta separation and purification is greatly improved.

Description

technical field [0001] The invention relates to a strain separation technology, in particular to a method for separation of smut fungus and the culture medium used. Background technique [0002] Smut disease is an important class of crop diseases, such as wheat dwarf smut, rice smut, sorghum smut and maize tumor smut, etc., which can lead to severe crop yield reduction. On the other hand, some secondary metabolites of smut, such as mannose erythritol lipid and smut acid, are important natural surfactants, which are being researched and developed in recent years and have broad application prospects. Whether it is the study of smut disease or the development and utilization of its secondary metabolites, the separation and purification of various smut fungi are the most basic work in related research. Improving the efficiency of separation and purification of smut can significantly shorten the research time in the initial stage and accelerate the progress of scientific researc...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N1/02C12R1/645
Inventor 李智敏严准严理余永廷高春生曾粮斌陈佳程毅孙向平薛召东
Owner INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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