A method for inducing the formation of a predatory organ of a nematode-trapping fungus

Abstract

The present invention discloses a method for inducing the formation of feeding organs of nematode-trapping fungi, belonging to the field of applied microorganisms. The specific steps are as follows: culturing the activated nematode-trapping fungi until spores are produced; washing the cultured spores and diluting them to obtain a spore suspension with a concentration of 30,000 to 60,000 spores / mL; applying the spore suspension to an agar medium covered with cellophane, evenly applying the spore suspension, and then culturing at 25 to 28°C for 28 to 36 hours. After adding a mixture of a feeding organ inducer and water, the mixture is further cultured at 25 to 28°C for 36 to 60 hours. The nematode-trapping fungi will produce a large number of feeding organs; the feeding organ inducer is one of oleic acid, linoleic acid, or pregnenolone. The present invention is the first to discover that using pregnenolone, oleic acid, or linoleic acid to induce nematode-trapping fungi to produce a large number of feeding organs.

Description

Technical Field

[0001] The invention relates to a method for inducing the formation of a predatory organ of a nematode-trapping fungus, and belongs to the field of applied microorganisms. Background Art

[0002] Compared with pests, agricultural parasitic nematodes cause much greater losses each year. Agricultural producers usually attribute crop damage to pests or other reasons, but often ignore parasitic nematodes. Due to the hidden and destructive nature of agricultural parasitic nematodes, and the fact that they are often ignored, they cause damage to agriculture that is difficult to control.

[0003] Plant-parasitic nematodes are widely distributed, diverse, and rapidly reproducing. They parasitize various plant organs, absorbing nutrients and causing irreversible damage to crops. They also pollute soil, rendering it unsuitable for agricultural cultivation and resulting in even greater economic losses.

[0004] Parasitic nematode control has evolved from long-term, ineffective composting, flooding, and soil solarization to highly effective chemical pesticides. However, due to the high cost of pesticides and the damage to soil and food safety caused by their extensive use, highly toxic pesticides have been banned. With the increasing demand for healthier lifestyles and a preference for pesticide-free, green foods, there is an urgent need to find new, environmentally friendly, and effective nematode control agents. The development of biological nematode control is attracting considerable attention, with nematode-predating fungi playing a key role.

[0005] Nematode-trapping fungi are important members of soil ecology. Nearly 380 species have been reported worldwide, belonging to the genus Arthrospora ( Arthrobotrys ), Septimia ( Monacrosporium ), Monocromosporum ( Dactylella ) etc. As the main natural enemy of nematodes, nematode-predating fungi generally lead a saprophytic life. When nematodes appear in a nutrient-poor environment, they change their growth stage to become pathogenic fungi, forming predatory organs to capture and digest nematodes to obtain nutrition (the infection process includes: attraction, capture, piercing and digestion).

[0006] The predatory organs of nematode-trapping fungi, such as insect-catching rings and sticky mesh structures, are usually generated under suitable environmental conditions. Temperature and humidity will affect the generation of predatory organs. The supply of nutrients in the environment, such as the concentration of carbon and nitrogen sources, also has a great influence on the formation of traps. Generally, in a barren environment lacking sufficient available nutrients, and under the activation of inducing substances, nematode-trapping fungi will activate the predatory mechanism to produce predatory organs. The present invention uses a mixture of oleic acid, linoleic acid, pregnenolone and water at appropriate concentrations to induce nematode-trapping fungi to produce predatory organs. This method provides good experimental materials for further studying the molecular mechanism of predatory organ formation, and also provides new ideas for highly effective nematode biocontrol agents.

[0007] After literature search, no public reports of documents with the same content as the present invention were found. Summary of the Invention

[0008] In order to enable nematode-trapping fungi to produce more predatory organs, the present invention provides a method for inducing the formation of predatory organs of nematode-trapping fungi, which specifically comprises the following steps:

[0009] (1) Cultivate the activated nematode-trapping fungus until spores are produced.

[0010] (2) Wash the spores cultured in step (1) and dilute them to obtain a spore suspension.

[0011] (3) Spreading the diluted spore suspension in step (2) onto an agar medium covered with cellophane, spreading the spore suspension evenly and incubating at a constant temperature, adding a mixture of a predatory organ inducer and water, and continuing to incubate at a constant temperature. After the incubation is completed, the nematode-predatory fungus produces a large number of predatory organs.

[0012] The predatory organ inducer is one of oleic acid, linoleic acid or pregnenolone.

[0013] Preferably, the nematode-trapping fungus is Arthrosporum oligosporum.

[0014] Preferably, the culture medium used for culturing the nematode-trapping fungus in step (1) is corn starch culture medium.

[0015] Preferably, the amount of spores in the spore suspension in step (2) is 30,000 to 60,000 spores / mL.

[0016] Preferably, the amount of the predatory organ inducer in step (3) is 1 mL of a mixture of the predatory organ inducer and water added to every 100 uL of spore suspension, and the concentration of the predatory organ inducer in the mixture of the predatory organ inducer and water is 100 pmol / L~100 umol / L.

[0017] More preferably, the concentration of the predatory organ inducer in the mixed solution of the predatory organ inducer and water in step (3) is 100 nmol / L.

[0018] Preferably, in step (3), the spore suspension is evenly spread and then cultured at a constant temperature of 25-28° C. for 28-36 hours.

[0019] Preferably, the constant temperature culture condition in step (3) is 25-28° C. for 36-60 h.

[0020] Technical effects of the present invention:

[0021] (1) The present invention first discovered that pregnenolone, oleic acid or linoleic acid can induce nematode-trapping fungi to produce a large number of predatory organs. The amount of predatory organs induced by pregnenolone, oleic acid or linoleic acid increased by 2000%, 1200% and 1600% respectively compared with those not induced by these three reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Pregnenolone induces the nematode-predating fungus Arthrospora oligosporida to produce predatory organs.

[0023] Figure 2 Oleic acid induces the nematode-predating fungus Arthrospora oligosporida to produce predatory organs.

[0024] Figure 3 Linoleic acid induces the nematode-predating fungus Arthrospora oligosporida to produce predatory organs. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the contents described above.

[0026] The preparations of the reagents and culture media used in the examples are as follows. Unless otherwise specified, all reagents used can be purchased through conventional commercial channels.

[0027] (1) Preparation of a mixed solution of oleic acid, linoleic acid, pregnenolone and water: Accurately weigh 0.031 g of pregnenolone, 0.028 g of oleic acid and 0.028 g of linoleic acid into a 1.5 mL sterilized centrifuge tube, add sterile water to each tube to a volume of 1 mL, and prepare a 100 mmol / L stock solution. Store at 4 °C and dilute to obtain a mixed solution of the predatory organ inducer and water with the corresponding final concentration when used.

[0028] (2) Agar medium: Add 20 g of agar to every 1000 mL of purified water and sterilize at 121°C for 20 minutes.

[0029] (3) Corn starch medium: Weigh 30 g corn kernels, add 1000 mL pure water, boil (100°C) for 20 minutes, filter the liquid through 6 layers of gauze, add 5 g yeast extract, add 20 g agar, sterilize at 121°C for 20 minutes, pour into a plate with a diameter of 6 cm, wait for complete solidification and set aside.

[0030] (4) PDA culture medium: Peel 200 g of potatoes, cut into pieces, and boil for 30 minutes. Filter through 6 layers of gauze to collect the supernatant. Add 20 g of glucose and 20 g of agar. Add water to make the volume 1000 mL. Sterilize at 121°C for 20 minutes, then pour into a 6 cm diameter plate and wait for complete solidification before use. Example 1

[0031] A method for inducing the growth of Arthrosporum oligosporum using pregnenolone Arthrobotrys oligospora ) A method for producing a predatory organ, the specific steps of which are as follows:

[0032] (1) Activation of Arthrospora oligosporida: Activate the preserved Arthrospora oligosporida on PDA medium;

[0033] (2) Spore culture of Arthrospora oligosporida: The Arthrospora oligosporida block activated in step (1) was inoculated onto a corn starch culture medium and cultured at 28°C for 10 days to obtain a large number of spores.

[0034] (3) Wash the spores on the plate with 3 mL of sterile water and dilute with sterile water to obtain a spore suspension with a concentration of 40,000 spores / mL.

[0035] (4) Take 100uL of the diluted spore suspension in step (3) and spread it on a 6cm agar medium covered with cellophane, and place it in a 28℃ constant temperature incubator to germinate for 28h. Then, inject 1mL of a mixture of pregnenolone and water (the concentration of pregnenolone in the mixture is 100nmol / L) into each plate from the side wall, slowly tilt the plate to allow the mixture of pregnenolone and water to soak the plate, and continue to place it in a 28℃ constant temperature incubator for induction for 48h, when a large number of predatory organs are produced.

[0036] For comparison, a blank control was also set up in this example, i.e., the mixture without pregnenolone was added. The results are as follows: Figure 1 As shown, from Figure 1 It can be seen that adding pregnenolone to the mixture of water can effectively increase the number of predatory organs of the spore-forming fungus. Example 2

[0037] A method for inducing the growth of Arthrosporum oligosporum using oleic acid Arthrobotrys oligospora ) A method for producing a predatory organ, the specific steps of which are as follows:

[0038] (1) Activation of Arthrospora oligosporida: Activate the preserved Arthrospora oligosporida on PDA medium;

[0039] (2) Spore culture of Arthrodiosporium oligosporum: The Arthrodiosporium blocks activated in step (1) were inoculated onto corn starch culture medium and cultured at 28°C for 10 days to obtain a large number of spores.

[0040] (3) Wash the spores on the plate with 3 mL of sterile water and dilute with sterile water to obtain a spore suspension with a concentration of 30,000 spores / mL.

[0041] (4) Take 100uL of the diluted spore suspension in step (3) and spread it on a 6cm agar medium covered with cellophane, and place it in a constant temperature incubator at 25℃ to germinate for 36h. Then, inject 1mL of a mixture of oleic acid and water (the concentration of oleic acid in the mixture is 100nmol / L) into each plate from the side wall, slowly tilt the plate to allow the mixture of oleic acid and water to soak the plate, and continue to place it in a constant temperature incubator at 28℃ for 36h to induce, and a large number of predatory organs will be produced.

[0042] For comparison, a blank control was also set up in this example, that is, no mixture of oleic acid and water was added. The results are as follows: Figure 2 As shown, from Figure 2 It can be seen that adding a mixture of oleic acid and water can effectively increase the number of predatory organs of the spore-forming fungus. Example 3

[0043] A method for inducing the growth of Arthrosporum oligosporum ( Arthrobotrys oligospora ) A method for producing a predatory organ, the specific steps of which are as follows:

[0044] (1) Activation of Arthrospora oligosporida: Activate the preserved Arthrospora oligosporida on PDA medium;

[0045] (2) Spore culture of Arthrodiosporium oligosporum: The Arthrodiosporium block activated in step (1) was inoculated onto corn starch culture medium and cultured at 28°C for 10 days to obtain a large number of spores.

[0046] (3) Wash the spores on the plate with 3 mL of sterile water and dilute with sterile water to obtain a spore suspension with a concentration of 60,000 spores / mL.

[0047] (4) Take 100uL of the diluted spore suspension in step (3) and spread it on a 6cm agar medium covered with cellophane, and place it in a constant temperature incubator at 28℃ to germinate for 30h. Then, inject 1mL of a mixture of linoleic acid and water (the concentration of linoleic acid in the mixture is 100nmol / L) into each plate from the side wall, slowly tilt the plate to allow the mixture of linoleic acid and water to soak the plate, and continue to place it in a constant temperature incubator at 25℃ for induction for 60h, when a large number of predatory organs are produced.

[0048] For comparison, a blank control was also set up in this example, that is, no mixture of linoleic acid and water was added. The results are as follows: Figure 3 As shown, from Figure 3It can be seen that adding a mixture of linoleic acid and water can effectively increase the number of predatory organs of the spore-forming fungus.

Claims

1. A method for inducing the formation of a nematode-trapping fungus predatory organ, characterized in that: The specific steps include: (1) Cultivating the activated nematode-trapping fungus until spores are produced; (2) washing the spores cultured in step (1) and diluting them to obtain a spore suspension; (3) Spreading the diluted spore suspension in step (2) onto an agar medium covered with cellophane, spreading the spore suspension evenly and incubating at a constant temperature, adding a mixture of a predatory organ inducer and water, and continuing to incubate at a constant temperature, until the nematode-predating fungus produces a large number of predatory organs after the incubation is completed; The predatory organ inducer is one of oleic acid, linoleic acid or pregnenolone; The nematode-trapping fungus is Arthrosporum oligosporum ( Arthrobotrys oligospora ).

2. The method for inducing the formation of a nematode-trapping fungus predatory organ according to claim 1, characterized in that: The culture medium used for culturing the nematode-trapping fungus in step (1) is corn starch culture medium.

3. The method for inducing the formation of a nematode-trapping fungus predatory organ according to claim 1, characterized in that: The amount of spores in the spore suspension of step (2) is 30,000 to 60,000 spores / mL.

4. The method for inducing the formation of a nematode-trapping fungus predatory organ according to claim 1, characterized in that: The amount of the predatory organ inducer used in step (3) is 1 mL of a mixture of the predatory organ inducer and water added to every 100 uL of the spore suspension, and the concentration of the predatory organ inducer in the mixture of the predatory organ inducer and water is 100 pmol / L~100 umol / L.

5. The method for inducing the formation of a nematode-trapping fungus predatory organ according to claim 1, characterized in that: In step (3), the spore suspension is evenly spread and then cultured at a constant temperature of 25-28°C for 28-36 hours.

6. The method for inducing the formation of a nematode-trapping fungus predatory organ according to claim 1, characterized in that: The constant temperature culture condition in step (3) is 25-28°C for 36-60 hours.

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