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A rapid method for identifying the survival status of second instar larvae of Meloidogyne incognita in vitro

A technology for root-knot nematodes and second-instar larvae incognita, applied in material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of reporting, coloring effect and death relationship, and achieve high sensitivity, shorten cycle and cost, and simple operation Effect

Active Publication Date: 2018-07-03
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Correlation between coloring effect and death has not been reported in plant parasites

Method used

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  • A rapid method for identifying the survival status of second instar larvae of Meloidogyne incognita in vitro
  • A rapid method for identifying the survival status of second instar larvae of Meloidogyne incognita in vitro
  • A rapid method for identifying the survival status of second instar larvae of Meloidogyne incognita in vitro

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Experimental program
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Embodiment 1

[0023] The method for quickly identifying the second instar larvae (J2) of the present invention in vitro survival state, its steps

[0024] as follows:

[0025] a. After 150 or 180 pieces of J2 were treated with 5% NaClO for 2 minutes, they were washed with sterile water to remove the residual NaClO. The washed J2 larvae were placed in 500ul of sterile water, and placed at a constant temperature of 25°C. in the incubator;

[0026] b. Place the Petri dish containing J2 on ice for 8 hours;

[0027] c. Add 40μl 1N NaOH dropwise to 1ml of 2ml centrifuge tube filled with J2, shake for 4min;

[0028] d. Place the petri dish containing J2 in a constant temperature incubator at 37°C for 12 hours;

[0029] e. Collect J2 into a 1.5ml centrifuge tube and centrifuge at 14000rpm at 4°C for 30min;

[0030] d. The worm bodies after the above treatments were washed 3-5 times with sterile water, and then the worm bodies were placed in a 24-well cell culture plate filled with 1ml of steril...

Embodiment 2

[0032] Basically the same as embodiment one. The difference is: freshly hatched J2 larvae will be collected and added to a 24-well cell culture plate containing 500 μl, and 2 μl of 0.22 μm Remove slag with a filter, dissolve the Nile Red solution in acetone at a concentration of 0.5 mg / ml, and place it at 25°C for 4 hours, then place it under a fluorescence microscope for detection of insect fluorescence. Such as Figure 5 with Image 6 shown.

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Abstract

A method for rapidly identifying in vitro survival state of meloidogyne incognita's second-stage juveniles belongs to the field of agricultural pathogenic pest drug screening. With Nile red staining of J2 fat and according to staining part of polypide, survival state of second-stage juveniles is identified. The method specifically comprises the following steps: adding 150-180 pieces of J2 into a 24-pore cell culture plate containing 500 microliters of sterile water, adding 1 microliter of a 0.5mg / ml Nile red solution which has undergone deslagging by a 0.22-micron filter and has been dissolved in acetone to the 24-pore cell culture plate, standing at 25 DEG C for 4h, exposing with yellow-green exciting light with wavelength of 580-630 mm for 120 milliseconds, repeating the operation every 24 h, and observing and recording fluorescence value; judging that nematode is in death state when obvious orange fluorescent light shows from stylet of nematode to the top of mouthparts; and judging that J2 is in non-death state when there is basically no fluorescent light on nematode heads. The invention has the following beneficial effects: the method is simple to operate and has high sensitivity; multiple detections will not affect activity of J2; and statistical error of pesticides screening due to suspended animation can be reduced, and screening period is shortened and screening cost is lowered.

Description

Technical field: [0001] The invention relates to a method for quickly identifying the living state of second-instar larvae of root-knot nematode incognita in vitro, and belongs to the field of drug screening for agricultural pathogenic pests. Background technique: [0002] Plant parasitic nematodes are important plant diseases that are difficult to control worldwide. The nematode has a wide range of parasites and strong adaptability to the environment, and can infect almost all cultivated crops. The direct economic loss caused by crop pathogenic nematodes is as high as 157 billion U.S. dollars every year in the world, and it is showing an increasing trend year by year. Among them, the root-knot nematode (Meloidogyne spp.) causes tens of billions of U.S. dollars in losses to world agriculture every year. At present, more than 90 species of root-knot nematodes have been found, and the common ones are Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne hapla, and Meloidog...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/64
Inventor 邹成钢陆朝军张克勤卞静
Owner YUNNAN UNIV