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A method for isolating the long beaker fungus from soil

A beak shell and soil technology, applied in the field of microbiology, can solve the problems of enrichment and separation of long beak shell fungus, difficulty in controlling the dilution factor of soil extract, and many experimental equipment and equipment. Separation and purification efficiency, low cost, and wide application range

Inactive Publication Date: 2020-06-23
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of this method are: 1) the whole process of bacteria separation needs to be carried out in the laboratory, and strict aseptic conditions are required, and there are many experimental equipment and equipment, which is the direct separation of diseased samples during field investigation and field investigation. It is inconvenient to obtain the target bacteria; 2) the workload is large when using this method to isolate the target bacteria in the soil, and it is difficult to control the dilution factor of the soil extract when different soil samples are separating the target bacteria. The isolation efficiency of some slow-growing target bacteria such as bacteria is extremely low
[0004] At present, researchers mainly use the PDA plate separation method or carrot-induced isolation method when isolating target bacteria on plant tissues infected by C. No studies describe methods for the enrichment and isolation of C. elongatum from soil samples using plant material

Method used

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  • A method for isolating the long beaker fungus from soil
  • A method for isolating the long beaker fungus from soil
  • A method for isolating the long beaker fungus from soil

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Separation and purification of the long beaked shell fungus

[0037] (1) Rinse fresh and healthy carrots with tap water, remove the skin and sterilize them in ethanol with a volume concentration of 75% for 1 to 2 minutes, dry the surface with clean napkins, and then cut them into thicknesses of 3 to 5 mm And uniform slices, and then use a 2.5cm diameter puncher to take a disc of equal diameter in the center of the carrot slices, and set aside;

[0038] (2) Place the carrot discs in step (1) in a petri dish with a diameter of 9 cm, place 12 carrot discs in each petri dish, overlap each other, and use an electronic balance to take it from Mengzi, Yunnan soil sample, 1g / part; place the weighed soil sample between two overlapping carrot discs, and place a ball of cotton soaked in sterile water in the center of the Petri dish to keep it moist (such as figure 1 ); placed in a constant temperature incubator at 26°C for 3 to 10 days, and observing the enrichment ...

Embodiment 2

[0041] Example 2: Identification of isolates

[0042] (1) Sequence identification

[0043]After the pure bacterium colony of the target bacterium that step (3) obtains is cultivated on the MYEA medium for 2 weeks, scrape the mycelia and The mixture of spores was extracted with the CTAB method for total DNA, and the extracted total DNA was amplified by PCR using the following primer pairs:

[0044] Upstream primer ITS1F: 5'-CTTGGTCATTTAGAGGAAGTAA-3' and

[0045] Downstream primer ITS4: 5'-TCCTCCGCTTATTGATATGC-3',

[0046] The amplified sequence is shown in SEQ ID No.1. The PCR products were detected by 1.5% agarose gel electrophoresis, and then sent to Biomed for sequencing, and the sequencing results were compared and analyzed in the NCBI database to further identify the isolates.

[0047] PCR reaction system:

[0048]

[0049]

[0050] PCR reaction program: pre-denaturation at 95°C for 5 min; followed by denaturation at 94°C for 45 s, annealing at 56°C for 40 s, ex...

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Abstract

The invention belongs to the technical field of microbes, and particularly relates to a method for separating ceratocystis spp from soil. According to the method, a carrot is used for concentrating the ceratocystis spp, the ceratocystis spp is separated and purified by means of selecting a single ascospore group for lining. Due to the fact that part of operations does not require a strict aseptic condition, the method is applicable to bacterial strain collection during field studying and field investigation, and working efficiency is increased; by means of selecting the single ascospore group for lining, a target bacterium is separated and purified, separating and purifying efficiency is increased effectively, and time is saved; the method for separating the ceratocystis spp from the soil is low in cost, wide in utilization range, safe and environmental-friendly.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a method for isolating C. elongatus from soil. Background technique [0002] Ceratocystis spp. is a soil-inhabiting fungus that seriously harms crops and commercial trees. It has a wide range of hosts and can harm more than 30 species of woody and herbaceous plants belonging to 14 genera. In my country, C. elongatum mainly harms plants such as sweet potato, pomegranate, taro, loquat, eucalyptus, triangular palm and araceae. In recent years, its harm has spread and intensified. The fungus harms tuber crops, mainly causing symptoms such as rot; harming woody plants, mainly causing symptoms such as ulcers and wilting, and in severe cases, the whole plant will wither and die. my country has a vast territory, rich crop types, and various soil types. There are fungi of the genus Erythrocystis hidden in farmland and natural ecological soils. During the long-term co-evoluti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12N1/02C12R1/645
CPCC12N1/02C12N1/14
Inventor 李健强张治萍李倩罗来鑫蒋娜王宁高淑梅曹永松姜治国
Owner CHINA AGRI UNIV
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