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Method for high-throughput screening of pine wood nematode inhibitor

A pine wood nematode and inhibitor technology, applied in the field of high-throughput screening of pine wood nematode inhibitors, can solve the problem of high cost

Active Publication Date: 2021-02-19
ZHEJIANG FORESTRY ACAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this kind of trunk injection is still expensive, and it can only be applied to ancient and famous trees.

Method used

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  • Method for high-throughput screening of pine wood nematode inhibitor
  • Method for high-throughput screening of pine wood nematode inhibitor
  • Method for high-throughput screening of pine wood nematode inhibitor

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0029] Example 1. Nematode Isolation and Culture

[0030] (1) Prepare PDA medium: take 200 grams of peeled potatoes, cut them into small pieces, add 1000 ml of water and boil for 30 minutes, filter the potato pieces with 4 layers of gauze, make up the filtrate to 1000 ml, add 20 grams of glucose, 15 grams of agar grams, after melting, subpackage, 15 pounds of sterilization (ie 121 degrees) for 15 minutes. Prepare a PDA plate on a 9cm sterile petri dish, inoculate Botrytis cinerea with a 0.5cm hole puncher, culture it to cover the entire plate at 25°C, and place it upside down at 4°C for later use;

[0031] (2) Cut the large piece of pine wood into small pieces of about 0.5*3cm, wrap it with gauze, put it in a sealed plastic, add sterile water, soak it in a water bath at 20-25°C overnight, and filter the soaking solution with 2-4 layers of gauze , the filtrate was centrifuged at 1000g for 1-5min, the supernatant was removed, and 1-5ml of worm liquid was left;

[0032] (3) Add...

example 2

[0033] Example 2. Nematode Screening

[0034] (1) Use a 1mL pipette to draw 1mL of sterile water and add it to a 9cm plate with nematodes, so many times, wash the nematode plate, and then collect the cleaned worm liquid into a sterile beaker;

[0035] (2) After mixing the collected insect liquid, filter it through a 10-20 μm nylon filter membrane. The filtrate is confirmed to be a larvae liquid by a 10*40 times microscope. Take out from the filter, rinse in sterile water, so that the required adult liquid is obtained, and the imago liquid is used here for chemical testing;

[0036] (3) Centrifuge the worm solution obtained above at 1000g for 5 minutes at low speed, remove part of the supernatant, adjust the solution to 0.5 agarose concentration with 1 / 1000 agarose solution, so that the nematodes are evenly dispersed, and observe with a 40 times microscope, Adjust the number of nematodes per mL to 2000-4000;

[0037] (4) Take a 96-well plate, add 25 μL of worm solution to eac...

example 3

[0038] Example 3. Preparation and data processing of several biopesticide test panels

[0039] (1) Prepare the reagents in a 96-well plate according to the formula in Table 1 below:

[0040] Table 1. Test Plate Reagent Recipe

[0041] Group Name 1, 2, 3, 4 5, 6, 7, 8 9, 10, 11, 12

[0042] A I 25μL+25μL water J 25μL+25μL water K 25μL+25μL water

[0043] B L 25μL + 25μL water M 25μL + 25μL water N 25μL + 25μL water

[0044] C L 25μL + 25μL water M 25μL + 25μL water N 25μL + 25μL water

[0045] D L 25μL + 25μL water M 25μL + 25μL water N 25μL + 25μL water

[0046] E L 25μL + 25μL water M 25μL + 25μL water N 25μL + 25μL water

[0047] F L 25μL + 25μL water M 25μL + 25μL water N 25μL + 25μL water

[0048] G L 25μL + 25μL water M 25μL + 25μL water N 25μL + 25μL water

[0049] H 50 μL water

[0050] in FIG. 1:

[0051] A. positive control; B. test sample 1; C. test sample 2; D. test sample 3;

[0052] E. Test sample 4; F. Test sample 5; G. Test sample 6; H. Negative co...

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Abstract

The invention relates to the field of biological pest control, in particular to a method for high-throughput screening of a pine wood nematode inhibitor. Pine trees are the most important afforestation tree species in China, but pine wood nematode diseases are always first forest diseases and insect pests in nearly thirty years, the harm is very serious, but the prevention and control drugs are limited, so that more prevention and control drugs need to be supplemented and screened urgently for more effective prevention and control. The invention aims to provide technical support for screeningmore pine wood nematode control agents. The method for high-throughput screening of the pine wood nematode inhibitor comprises the following steps: 1) collecting pine wood nematode, namely separatingpine wood nematode from epidemic wood, and carrying out enlarged culture; (2) pine wood nematode screening, namely distinguishing and counting cultured nematodes according to adults and larvae; (3) test board manufacturing, namely setting a contrast and test area in a microwell plate and preparing a reagent with a certain concentration; and (4) data recording and processing, namely recording deadand alive changes of the nematodes along with time, counting drug effects and screening.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for high-throughput screening of pine wood nematode inhibitors. Background technique [0002] Pine xylophilus disease is a major international quarantine disease caused by pine wilting and death caused by pine xylophilus. Since the first discovery of pine wood nematode disease in Zijinshan black pine in Nanjing in 1982, the disease has occurred in more than 300 cities and counties in 18 provinces. Over the past 30 years, pine wood nematode has been the number one forest disease and insect pest in my country. Pine trees are the most important tree species for afforestation in my country. The national pine forest covers an area of ​​more than 500 million mu. The main tree species are Masson pine, black pine, red pine, Ryukyu pine, Huashan pine, Yunnan pine, Simao pine, Huangshan pine, Huashan pine, red pine, etc. All are susceptible species. In terms of controlling pine wood...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/15
CPCG01N33/15
Inventor 彭华正金群英朱汤军张飞英叶华琳戚全英
Owner ZHEJIANG FORESTRY ACAD
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