Method for fast separating, identifying and storing sweet potato sclerotium rolfsii

A technology for leprosy and sweet potato, which is applied in the field of separation, identification and preservation of microorganisms, can solve the problems of large workload of bacterial species preservation, pathogenicity of bacterial species contamination, disordered bacterial species, etc., and achieves small workload and easy management. , reduce the effect of processing

Inactive Publication Date: 2016-07-13
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Abstract
  • Description
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Problems solved by technology

[0004] The most commonly used preservation method for the pathogenic bacteria of white silkworm is to inoculate the white silkworm on the slant of PDA or on the PDA medium plate and store it in a refrigerator at 4°C for low temperature storage. 2-3 months, so it is necessary to continuously re-transfer culture and preserve the white silkworm
However, multiple transfer cultures and preservation can easily lead to contamination and weakened pathogenicity of the strains. When the number of strains is large, the workload of strain preservation is large, which can easily lead to confusion or even loss of strains

Method used

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  • Method for fast separating, identifying and storing sweet potato sclerotium rolfsii

Examples

Experimental program
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Effect test

Embodiment 1

[0049] In March 2011, a typical sample of sweet potato white silk disease was collected from the sweet potato nursery field of Baiyun Base, Guangdong Academy of Agricultural Sciences. The yam and potato pieces are covered with white hyphae and white to brown sclerotia; prepare a PDA medium plate according to the conventional method, cool to 60°C and add streptomycin at 1 / 1000 quality of the PDA medium.

[0050] In the ultra-clean bench, use a sterilized inoculation needle to pick up the mycelia or use a sterilized tweezers to pick up the sclerotia on the PDA medium plate, and place it in a light incubator at 25°C for cultivation. After 2 days, the sclerotia grows white mycelia. like figure 1 As shown, the picked mycelium grows a large amount of new mycelium after two days of cultivation, and picks the medium block with mycelium with an inoculation needle at the edge of the white mycelium, and puts it on a new PDA medium plate. Purification culture was performed, and it was ob...

Embodiment 2

[0053] In December 2011, a typical sweet potato mildew disease sample was collected from the sweet potato quality comparison test field of Baiyun Base, Guangdong Academy of Agricultural Sciences. The harvested sweet potato pieces were covered with white hyphae and white to brown sclerotia; according to conventional methods Prepare a PDA medium plate, add 1 / 1000 streptomycin while cooling to 50°C.

[0054] In the ultra-clean bench, use a sterilized inoculation needle to pick up the hyphae or use a sterilized tweezers to pick up the sclerotia on the PDA medium plate, and place it in a light incubator at 25°C for cultivation. After 3 days, the sclerotia grows white mycelia. On the edge of the white mycelium, use an inoculation needle to pick up the medium block with mycelium, put it on a new PDA medium plate, and purify it. It is observed that 15 bacterial strains can produce rapeseed sclerotia.

[0055] After the purified 15 strains were cultured at 25°C for 3 days, a mycelial b...

Embodiment 3

[0058] Inoculate the 15 pathogenic bacterial strains confirmed in Example 1 onto a new PDA medium plate, and place them at 25°C for 10 days to grow mature tan sclerotia on the plate, such as figure 2 Then, under the condition of ultra-clean bench, use tweezers to pick the tan sclerotium and put it into a sterile preservation tube; twist the preservation tube with sclerotia to a semi-tight state, and place it in a silica gel desiccator to dry After 3 days, tighten the cap of the preservation tube and store it directly at -20°C until August 2011. In October 2015, the strain storage tube stored at -20°C was taken out, and under sterile conditions, the sclerotium was picked up with sterilized forceps and placed on a PDA medium plate, and new colonies could grow within 2 days.

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Abstract

The invention discloses a method for fast separating, identifying and storing sweet potato sclerotium rolfsii.The method comprises the following steps that 1, a PDA medium plate is inoculated with hyphae or sclerotia selected from a disease sample of the sweet potato sclerotium rolfsii and placed at the temperature of 20 DEG C to 30 DEG C for culture; 2, after new hyphae grow out of the PDA medium plate, new hyphae are selected and placed on a new PDA medium plate for purifying culture; 3, after purified strains are cultured for 2 d to 4 d at the temperature of 20 DEG C to 40 DEG C, a healthy sweet potato stem branch is inoculated with hypha blocks as inocula, PDA blocks are adopted as blank control, culture is carried out for 1-3 days under the sealing condition at the temperature of 20 DEG C to 30 DEG C, and when the phenomenon that the stem branch rots occurs, the sweet potato sclerotium rolfsii is confirmed; 4, sclerotium culture is carried out on the sweet potato sclerotium rolfsii, and then sclerotia are dried and stored.According to the method, the process of fast separating, identifying and storing the strains of the sweet potato sclerotium rolfsii can be simple, the workload is small, efficiency is high, and the preserved strains are easy to manage and long in survival time and can survive for 4 years or longer.

Description

technical field [0001] The invention relates to the technical field of isolation, identification and preservation of microorganisms, in particular to a method for rapidly isolating, identifying and preserving sweet potato spp. Background technique [0002] Sweet potato canker sore is caused by Sclerotium rolfsii Sacc., which may occur from the sweet potato seedling stage to the harvest stage. The sweet potato yield loss of the diseased field reaches more than 20%, and even the harvest is severe. During the storage or sale of sweet potatoes, the rot of the stems and tails of sweet potatoes is often caused by Sepia spp., which affects the quality and commerciality of sweet potatoes. At present, the harm of sweet potato canker sore has become an important limiting factor of the sweet potato industry. The occurrence regularity of sweet potato canker sore, the biological characteristics of the pathogen, the sensitivity to fungicides and the pathogenicity to sweet potato are studi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N1/04C12N1/02C12R1/645
CPCC12N1/14C12N1/02C12N1/04
Inventor 黄立飞房伯平陈景益叶芍君黄实辉
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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