Method for inducing nematode-trapping fungi to generate trapping organ by using nematode extracellular vesicles

Abstract

The invention discloses a method for inducing nematode-trapping fungi to generate a trapping organ by using nematode extracellular vesicles. The method comprises the following steps of (1) culturing nematodes; (2) obtaining the nematode extracellular vesicles and carrying out characterization analysis on the nematode extracellular vesicles; (3) culturing the Arthrobotrys oligospora which is the nematode trapping fungus; and (4) inducing generation of Arthrobotrys oligospora trapping organs by using the nematode extracellular vesicles. According to the method, Extracellular vesicles (EVs) of caenorhabditis elegans are extracted, and the obtained EVs are utilized to successfully induce Arthrobotrys oligospora to generate a trapping organ, so that the EVs secreted by the nematode contain a substance for inducing nematode-trapping fungi to generate the trapping organ; and the method has important significance for understanding an interaction mechanism of nematodes and nematode-trapping fungi, and has important application value in the field of nematode biological control.

Description

technical field

[0001] The invention relates to the technical field of molecular microbiology, in particular to a method for using nematode extracellular vesicles to induce nematode-preying fungi to produce predatory organs. Background technique

[0002] Plant parasitic nematodes, including root-knot nematodes, cyst nematodes and pine xylophilus, are widely distributed around the world. According to statistics, plant parasitic nematodes cause economic losses of about 157 billion U.S. dollars to various crops around the world every year. In my country, such nematode diseases have become the second largest type of diseases in agricultural production, and are one of the important limiting factors in agricultural production. Plant-parasitic nematodes have the characteristics of diverse hosts, easy transmission, concealment, stubbornness, and rapid population growth. Coupled with the particularity of soil ecology, it is very difficult to control them. For a long time, people ha...

Examples

Embodiment 1

[0047] A method of using extracellular vesicles secreted by nematodes of the present invention to induce nematode-predating fungi to produce predatory organs, comprising the following steps:

[0048] (1) Cultivation of nematodes:

[0049] Medium and buffer of step1 nematodes: NGM solid medium and S Medium liquid medium are used to cultivate nematodes; M9 buffer is used to wash worms and hatch eggs; NGM solid medium 1L: weigh 2.5g of bacto-peptone, chloride Sodium chloride 3g, agar powder 20g, the remaining reagents except agar powder were dissolved in 800mL deionized water, after being completely dissolved, the volume was adjusted to 1L, and 250mL was divided into 500mL conical flasks, and agar powder was added in proportion to each bottle, 121 ℃ autoclave for 20 minutes, when cooled to about 55 ℃, add filter-sterilized 1mL 1M CaCl 2 , 1mL 1M MgSO 4 , 25mL 1M KPO 4 Buffer, 1mL 5mg / mL cholesterol (dissolved in 95% ethanol), mix well, divide into plates, dry and set aside; ...

Embodiment 2

[0076] The difference between embodiment 2 and embodiment 1 is:

[0077] In step (1), the nematodes are synchronized: observe the nematodes cultured for 2 days under a microscope. The eggs in the mother body are arranged in a straight line, and there are fewer larvae in the petri dish. This is the best period for synchronization, and M9 buffer is used. Wash the nematodes off the solid medium. During the cleaning process, repeated vigorous washing should be avoided to cause the agar in the medium to be washed down, which will affect the efficiency of the nematodes in the lysis process. Put the washed nematodes into a 15mL centrifuge tube, add an appropriate amount of M9 buffer to 15mL, let it settle naturally for 3-5min, remove the supernatant, and re-add M9 buffer to 15mL to wash the nematodes; this step needs to be repeated 4-6 times, top Wash the bacteria as far as possible until the clear liquid becomes clear; after removing the excess supernatant, add 2 mL of nematode lysa...

Embodiment 3

[0082] The difference between embodiment 3 and embodiment 1 is: embodiment 2

[0083] The difference between embodiment 2 and embodiment 1 is:

[0084]In step (1), the nematodes are synchronized: put the nematodes cultured for 2.5 days under a microscope, and the eggs in the mother body are arranged in a straight line, and there are fewer larvae in the petri dish. This is the best period for synchronization, and M9 buffer is used. Wash the nematodes off the solid medium. During the cleaning process, repeated vigorous washing should be avoided to cause the agar in the medium to be washed down, which will affect the efficiency of the nematodes in the lysis process. Put the washed nematodes into a 15mL centrifuge tube, add an appropriate amount of M9 buffer to 15mL, let it settle naturally for 3-5min, remove the supernatant, and re-add M9 buffer to 15mL to wash the nematodes; this step needs to be repeated 4-6 times, top Clean the bacteria as much as possible until the liquid tu...

technical field

[0001] The invention relates to the technical field of molecular microbiology, in particular to a method for using nematode extracellular vesicles to induce nematode-preying fungi to produce predatory organs. Background technique

[0002] Plant parasitic nematodes, including root-knot nematodes, cyst nematodes and pine xylophilus, are widely distributed around the world. According to statistics, plant parasitic nematodes cause economic losses of about 157 billion U.S. dollars to various crops around the world every year. In my country, such nematode diseases have become the second largest type of diseases in agricultural production, and are one of the important limiting factors in agricultural production. Plant-parasitic nematodes have the characteristics of diverse hosts, easy transmission, concealment, stubbornness, and rapid population growth. Coupled with the particularity of soil ecology, it is very difficult to control them. For a long time, people ha...

Examples

Embodiment 1

[0047] A method of using extracellular vesicles secreted by nematodes of the present invention to induce nematode-predating fungi to produce predatory organs, comprising the following steps:

[0048] (1) Cultivation of nematodes:

[0049] Medium and buffer of step1 nematodes: NGM solid medium and S Medium liquid medium are used to cultivate nematodes; M9 buffer is used to wash worms and hatch eggs; NGM solid medium 1L: weigh 2.5g of bacto-peptone, chloride Sodium chloride 3g, agar powder 20g, the remaining reagents except agar powder were dissolved in 800mL deionized water, after being completely dissolved, the volume was adjusted to 1L, and 250mL was divided into 500mL conical flasks, and agar powder was added in proportion to each bottle, 121 ℃ autoclave for 20 minutes, when cooled to about 55 ℃, add filter-sterilized 1mL 1M CaCl 2 , 1mL 1M MgSO 4 , 25mL 1M KPO 4 Buffer, 1mL 5mg / mL cholesterol (dissolved in 95% ethanol), mix well, divide into plates, dry and set aside; ...

Embodiment 2

[0076] The difference between embodiment 2 and embodiment 1 is:

[0077] In step (1), the nematodes are synchronized: observe the nematodes cultured for 2 days under a microscope. The eggs in the mother body are arranged in a straight line, and there are fewer larvae in the petri dish. This is the best period for synchronization, and M9 buffer is used. Wash the nematodes off the solid medium. During the cleaning process, repeated vigorous washing should be avoided to cause the agar in the medium to be washed down, which will affect the efficiency of the nematodes in the lysis process. Put the washed nematodes into a 15mL centrifuge tube, add an appropriate amount of M9 buffer to 15mL, let it settle naturally for 3-5min, remove the supernatant, and re-add M9 buffer to 15mL to wash the nematodes; this step needs to be repeated 4-6 times, top Wash the bacteria as far as possible until the clear liquid becomes clear; after removing the excess supernatant, add 2 mL of nematode lysa...

Embodiment 3

[0082] The difference between embodiment 3 and embodiment 1 is: embodiment 2

[0083] The difference between embodiment 2 and embodiment 1 is:

[0084]In step (1), the nematodes are synchronized: put the nematodes cultured for 2.5 days under a microscope, and the eggs in the mother body are arranged in a straight line, and there are fewer larvae in the petri dish. This is the best period for synchronization, and M9 buffer is used. Wash the nematodes off the solid medium. During the cleaning process, repeated vigorous washing should be avoided to cause the agar in the medium to be washed down, which will affect the efficiency of the nematodes in the lysis process. Put the washed nematodes into a 15mL centrifuge tube, add an appropriate amount of M9 buffer to 15mL, let it settle naturally for 3-5min, remove the supernatant, and re-add M9 buffer to 15mL to wash the nematodes; this step needs to be repeated 4-6 times, top Clean the bacteria as much as possible until the liquid tu...

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