Plant pathogen purification and isolation method

A plant pathogenic bacteria, purification and separation technology, applied in the field of plant disease purification and separation, can solve the problems of low purity, difficult target, thick needle tip, etc. to achieve the effect of wide applicability, flexible selection and adjustment, and various separation methods.

Inactive Publication Date: 2018-08-03
SICHUAN AGRI UNIV
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AI Technical Summary

Problems solved by technology

At present, although there are many methods for unit cell separation, there are still some imperfections. For example, in the direct picking method of unit cells, it is difficult to guarantee the success rate of picking the unit cells with the picking needle. The needle tip is thick and it is difficult to accurately locate the target; the direct cutting method of the agar block has the problem of misalignment when cutting the agar block containing single spores, because it needs to be separated from the microscope for freehand cutting; the capillary separation method has the same problem as the direct cutting method of the agar block , and there are also problems of missing points or multiple points when spotting samples; the tissue mashing rule is relatively extensive, and the probability of obtaining pure culture bacteria is low

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Experimental program
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Effect test

Embodiment 1

[0059] Embodiment 1 Purification and separation method of plant pathogenic bacteria based on pure culture bacteria

[0060] The method adopts a miniature spore-picking needle, comprising the following steps: Step 1, preparing a single spore of pure cultured bacteria: get the pure cultured bacteria, use the inoculation needle to pick the bacterial block and inoculate it in the PDA medium (depending on the situation, other cultures can also be used) base), inoculate 1-3 dishes, transfer to a constant temperature incubator, incubate at a constant temperature of 25°C for 1-7 days, and wait for sporulation; during the cultivation process of pure culture bacteria, regular microscopic observation, if sporulation has occurred, that is The next step can be carried out. If no spores are produced, continue to cultivate. If no spores are produced after long-term cultivation, it is necessary to consider changing the medium and repeat the above steps until spores are produced;

[0061] Step...

Embodiment 2

[0066] Embodiment 2 Purification and isolation method of plant pathogenic bacteria based on pure culture bacteria

[0067] The method adopts a micropipette and comprises the following steps:

[0068]Step 1. Cultivate purely cultured bacteria monospores: take purely cultured bacteria, use an inoculation needle to pick out bacterial blocks and inoculate them in PDA medium, inoculate 1-3 dishes, transfer to a constant temperature incubator, and cultivate at a constant temperature of 25 °C for 1 -7d, wait for sporulation; during the culture process of pure culture fungus, regular microscopic observation, if sporulation has been produced, the next step can be carried out, if no sporulation, continue to cultivate, if no sporulation after long-term cultivation, need to Consider replacing the medium and repeat the above steps until sporulation;

[0069] Step 2. Preliminary preparation for absorbing single spores. Take out a sterile glass slide, wipe it clean with lens tissue and set...

Embodiment 3

[0074] Embodiment 3 Purification and isolation method of plant pathogenic bacteria based on plant tissue samples

[0075] The method adopts micro sporulation needles and comprises the following steps:

[0076] Step 1. Plant tissue sample acquisition and pre-treatment: Plant tissue samples are usually collected in the field, so before the unit cell isolation begins, it needs to be microscopically tested to check whether it has mature spores. If there is no and Ascocarp is immature, it needs to induce sporulation, if there is, the surface of the sample needs to be disinfected, that is, sterilized with 75% ethanol solution for 30sec., if necessary, use mercuric chloride for secondary disinfection, and then clean it with sterile floating solution Several times, then wipe and blot dry with sterile filter paper, and set aside.

[0077] Step 2. Tissue sectioning and single spore acquisition: Before tissue sectioning, it is necessary to determine the exact position of the pycnidium o...

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Abstract

The invention discloses a plant pathogen purification and isolation method. Mainly two separation materials, namely a pure culture and a plant tissue sample, are obtained; a micro suction tube or a micro spore picking needle is adopted; and purification and isolation are implemented based on a PDA medium, a WA medium and sterile suspension liquid. The purification and isolation method provided bythe invention is improvement and optimization on the basis of an original single spore isolation method; and the method is higher in practicability, and the method is simple and easily available in appliance, low in cost, convenient to operate and relatively high in success rate.

Description

technical field [0001] The invention belongs to the field of purification and separation of plant diseases, and in particular relates to a method for purification and separation of plant pathogenic bacteria. Background technique [0002] The purification and separation technology of plant pathogenic bacteria is the basic technology of plant disease research. The existing technologies mainly include: single cell direct picking method, direct cutting method of agar block, capillary separation method, tissue smashing method, etc. The single cell direct picking method is to use a self-made sporulation needle (made of suture needles, insect needles, thin steel wire, eyebrows, etc.) The method in the culture medium; the direct cutting method of the agar block is the method of cutting out the agar block containing only single spores and transferring it to the medium under a low power microscope (10× or 20×) with a micro-knife; the capillary separation method Under a low-power micr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/02
CPCC12N1/02
Inventor 杨春琳许秀兰刘应高
Owner SICHUAN AGRI UNIV
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