Method for separating single oospore of phytophthora infestans

A potato late blight and oospore technology, applied in the biological field, can solve the problems of difficult operation, time-consuming, low success rate, etc., and achieve the effect of reducing operation difficulty, saving operation time and high success rate

Pending Publication Date: 2018-11-23
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The object of the present invention is to provide a method for separating single oospores of potato infestans, which solves the problem of existing potato

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  • Method for separating single oospore of phytophthora infestans
  • Method for separating single oospore of phytophthora infestans
  • Method for separating single oospore of phytophthora infestans

Examples

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Example Embodiment

[0018] Example 1

[0019] (1) Activate the potato Phytophthora infestans strain 902-3 (collected from Yunnan in 2015), insert the strain into a rye medium, and cultivate it under dark conditions at 19°C for 16 days to collect hyphae;

[0020] (2) Weigh 80 mg of mycelium into a 1.5 mL centrifuge tube, add 1 mL of sterile water and place it in a refrigerator at 4°C for 3 hours to stimulate the sporangia to release zoospores;

[0021] (3) Then, centrifuge at 1000r / min for 2min, remove the supernatant, and add 30mg / mL of breakdown enzymatic hyphae, sporangia and zoospores. The breakdown enzyme is configured with pH=7.0 PBS buffer , And filter with 0.22μm bacterial filter;

[0022] (4) Centrifuge at 1000r / min for 1min, remove the supernatant, add 1mL of sterile distilled water; filter through nylon mesh with pore diameters of 76μm, 40μm and 20μm in turn, wash the 20μm nylon mesh with sterile water in a small beaker, and collect Egg spore suspension;

[0023] (5) Adjust the concentration of...

Example Embodiment

[0027] Example 2

[0028] (1) Activate the potato infestation bacteria strain 902-3 (collected from Yunnan in 2015), insert the strain into a rye medium, and cultivate it under dark conditions at 19°C for 15 days to collect hyphae;

[0029] (2) Weigh 100 mg of mycelium into a 1.5 mL centrifuge tube, add 1 mL of sterile water and place it in a refrigerator at 4°C for 2-3 hours to stimulate the sporangia to release zoospores;

[0030] (3) Then, centrifuge at 1000r / min for 2min, remove the supernatant, and add 30mg / mL of breakdown enzymatic hyphae, sporangia and zoospores. The breakdown enzyme is configured with pH=7.0 PBS buffer , And filter with 0.22μm bacterial filter;

[0031] (4) Centrifuge at 1000r / min for 1min, remove the supernatant, add 1mL of sterile distilled water; filter through nylon mesh with pore diameters of 76μm, 40μm and 20μm in turn, wash the 20μm nylon mesh with sterile water in a small beaker, and collect Egg spore suspension;

[0032] (5) Adjust the concentration o...

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Abstract

The invention provides a method for separating a single oospore of phytophthora infestans. The method comprises the following steps: separating hyphae from the oospore at a suitable temperature by using a certain concentration of driselase, and then removing excess hyphae and sporangiospores by a multiple low-speed centrifugation method, and finally, coating an oospore suspension on water agar andobserving under a light microscope and separating the single oospore using a blade. The method is also applicable to strains generating less oospore, the success rate is higher, the operation time isgreatly shortened, and meanwhile the operation difficulty is also greatly reduced. The method is an effective method for rapidly separating the single oospore on a large scale in a pathogen population research experiment. The method is successfully applied to the genetic test study of phytophthora infestans population.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for isolating a single oospore of potato infestans. Background technique [0002] Potato Late Blight (Potato Late Blight) is caused by Phytophthora infestans, a devastating oomycete disease that causes potato stem and leaf death and tuber rot. Potato-producing areas all over the world have occurred, and the epidemic year generally reduces production by 30%. In the 1840s, a large number of Irish potatoes died, and the production was cut in half, causing more than 1 million people to starve to death and 2 million people to emigrate overseas. At that time, there were various speculations about the cause of potato death. In 1842, von Martius first believed that it was caused by pathogens. In 1857, Speerschneider proved that mold on leaves could cause tuber rot. From 1861 to 1863, DeBary (deBary) determined that the lesions on the leaves and tuber rot were caused by...

Claims

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Application Information

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IPC IPC(8): C12N3/00C12R1/645
CPCC12N3/00
Inventor 沈林林詹家绥祝雯周世豪蔡铭铭
Owner FUJIAN AGRI & FORESTRY UNIV
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