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Root-knot nematode biocontrol fungus acremonium implicatum protoplast preparation and regeneration method

A technology for biocontrol of fungi and protoplasts, applied in biochemical equipment and methods, methods using spores, methods based on microorganisms, etc., can solve the problems of large differences in protoplast preparation conditions, affecting enzymatic reactions, etc., and achieve high activity , good repeatability and simple operation

Active Publication Date: 2020-09-25
天津市农业科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of protoplast preparation, the bacterial age, osmotic pressure stabilizer, lyase type, enzymatic hydrolysis temperature and other conditions will affect the enzymatic hydrolysis reaction, resulting in large differences in protoplast preparation conditions.

Method used

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  • Root-knot nematode biocontrol fungus acremonium implicatum protoplast preparation and regeneration method
  • Root-knot nematode biocontrol fungus acremonium implicatum protoplast preparation and regeneration method
  • Root-knot nematode biocontrol fungus acremonium implicatum protoplast preparation and regeneration method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] (1) Strain activation: Inoculate the Acremonium acremonium strain stored at -80°C on PDA medium, and culture it in the dark at 28°C for 5 days to activate the strain;

[0041] (2) Preparation of spore liquid: use a 5mm hole puncher to punch a hole at the edge of the colony cultivated in step (1), take a piece of bacteria and place it in PDB medium at 25°C, cultivate it at 150r / min for 2 days, collect the spores and prepare them into a uniform Spore suspension at a concentration of 1 × 10 8 cfu / mL;

[0042] (3) Mycelia preparation: Take 100 μL of the spore suspension in step (2) in 100 mL of PDB medium at 25°C and 150 r / min for 24, 36, 48, 60, and 72 hours, and filter it with sterilized three-layer lens-cleaning paper. Then use 0.7mol / L NaCl solution with a pH of 6.5 to repeatedly wash and collect the mycelia. After the mycelium is washed to a lump, weigh 0.5g into a sterilized 50mL centrifuge tube. The entire operation process is carried out on a clean bench;

[0043]...

Embodiment 2

[0050] (1) Strain activation: Inoculate the Acremonium acremonium strain stored at -80°C on PDA medium, and culture it in the dark at 28°C for 5 days to activate the strain;

[0051] (2) Preparation of spore liquid: use a 5mm hole puncher to punch a hole at the edge of the colony cultivated in step (1), take a piece of bacteria and place it in PDB medium at 25°C, cultivate it at 150r / min for 2 days, collect the spores and prepare them into a uniform Spore suspension at a concentration of 1 × 10 8 cfu / mL;

[0052] (3) Mycelia preparation: Take 100 μL of the spore suspension in step (2) and place it in 100 mL of PDB medium at 25°C and culture it at 150 r / min for 36 hours, filter it with sterilized three-layer lens-cleaning paper, and then filter it with 0.7 pH of 6.5. mol / L NaCl solution was washed repeatedly to collect the mycelium, and after the mycelium was washed to a lump, weigh 0.5g into a sterilized 50mL centrifuge tube, and the whole operation process was carried out on...

Embodiment 3

[0061] (1) Strain activation: Inoculate the Acremonium acremonium strain stored at -80°C on PDA medium, and culture it in the dark at 28°C for 5 days to activate the strain;

[0062] (2) Preparation of spore liquid: use a 5mm hole puncher to punch a hole at the edge of the colony cultivated in step (1), take a piece of bacteria and place it in PDB medium at 25°C, cultivate it at 150r / min for 2 days, collect the spores and prepare them into a uniform Spore suspension at a concentration of 1 × 10 8 cfu / mL;

[0063] (3) Mycelia preparation: Take 100 μL of the spore suspension in step (2) and put it in 100 mL of PDB medium at 25°C, culture it at 150 r / min for 36 hours, filter it with sterilized three-layer lens-cleaning paper, and then filter it with a concentration of pH 6.5. 0.7mol / L NaCl solution was washed repeatedly to collect mycelia, and after the mycelium was washed to a lump, weighed 0.5g into a sterilized 50mL centrifuge tube, and the whole operation process was carried...

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Abstract

The invention discloses a root-knot nematode biocontrol fungus acremonium implicatum protoplast preparation and regeneration method, and belongs to the technical field of agricultural plant protection. The method comprises the following steps of activating a strain; preparing a spore suspension; preparing clumpy mycelia; preparing a protoplast; and regenerating the protoplast. Compared with the prior art, the method has the beneficial effects that the root-knot nematode biocontrol fungus acremonium implicatum protoplast preparation and regeneration method is simple to operate, good in repeatability and high in activity of the prepared protoplast, the regeneration rate of the protoplast reaches 17.78%. and a good foundation is laid for the establishment of an A.implicatum genetic transformation system and the research of a biocontrol mechanism of the strain.

Description

technical field [0001] The invention relates to the technical field of agricultural plant protection, in particular to a preparation and regeneration method of root-knot nematode biocontrol fungus Acremonium acremonium protoplast. Background technique [0002] The root-knot nematode Meloidogyne spp. is an important plant pathogen with worldwide distribution, which has brought huge economic losses to global agricultural production. Root-knot nematodes are diverse, widely distributed, highly pathogenic, have a wide range of hosts, and have strong environmental adaptability. They can infect almost all vegetable crops. Among vegetable crops, Solanaceae, Cruciferae and Cucurbitaceae are the most harmful. Infestation of vegetable crops by root-knot nematodes will result in reduced yield and quality. In addition, root-knot nematodes will also form compound infections with other pathogens, causing more serious economic losses. [0003] Root-knot nematodes are mainly controlled by...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N3/00C12R1/645
CPCC12N1/14C12N3/00Y02A50/30
Inventor 姚玉荣郝永娟霍建飞贲海燕刘晓琳高苇王万立
Owner 天津市农业科学院
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