Method for inducing formation of nematode-trapping fungus predatory organ

Abstract

The invention discloses a method for inducing nematode-trapping fungus predatory organ formation, and belongs to the field of applied microorganisms. The method comprises the following specific steps: culturing activated nematode-trapping fungi until spores are generated; washing off the cultured spores, and diluting to obtain a spore suspension with the concentration of 30000-60000 spores / mL; coating the spore suspension into an agar culture medium paved with glass paper, uniformly coating the spore suspension, culturing for 28-36 hours at the temperature of 25-28 DEG C, adding a mixed solution of a predatory organ inducer and water, and continuously culturing for 36-60 hours at the temperature of 25-28 DEG C, so that the nematode-trapping fungi can generate a large number of predatory organs; the predatory organ inducer is one of oleic acid, linoleic acid or pregnenolone. It is found for the first time that a large number of predatory organs can be generated when pregnenolone, oleic acid or linoleic acid is used for inducing nematode-trapping fungi.

Description

Technical Field

[0001] The present invention relates to a method for inducing the formation of predatory organs of nematode-trapping fungi, belonging to the field of applied microbiology. Background Art

[0002] Compared with pests, agricultural parasitic nematodes cause much greater losses every year. Agricultural producers usually attribute crop damage to pests or other reasons, often neglecting parasitic nematodes. Due to the concealment and destructiveness of agricultural parasitic nematodes, and the fact that they are often overlooked, they cause uncontrollable damage to agriculture.

[0003] Plant parasitic nematodes are widely distributed, have many varieties, and reproduce quickly. They parasitize on various plant organs to absorb nutrients, causing irreversible damage to crops. Moreover, plant parasitic nematodes can also cause soil pollution, making the soil no longer suitable for agricultural planting, thus causing greater economic losses.

[0004] For the control of parasitic nematodes, it has changed from composting, flooding, and soil solarization with long cycles and insignificant effects to highly efficient chemical pesticide control. However, due to the high cost of pesticides and the harm to soil and food safety after a large amount of highly toxic pesticides are applied, highly toxic pesticides have been banned. With people's pursuit of a healthy life and their preference for green foods without pesticides, there is an urgent need to find a new type of environmentally friendly and effective nematode control agent. The development direction of the biological control of nematodes has attracted much attention, and nematode-trapping fungi play an important role in this.

[0005] Nematode-trapping fungi are important members of the soil ecosystem. Nearly 380 species have been reported globally and belong to Arthrobotrys ( Arthrobotrys ), Dactylella ( Monacrosporium ), Monacrosporium ( Dactylella ), etc. As the main natural enemies of nematodes, nematode-trapping fungi generally live a saprophytic life. When nematodes appear in a nutrient-poor environment, they change their growth stage into pathogenic fungi, forming predatory organs to capture and digest nematodes to obtain nutrients (the infection process includes: attraction, capture, piercing, and digestion).

[0006] The predatory organs of nematode-trapping fungi, such as trapping rings and sticky reticulate structures, are usually generated under suitable environmental conditions. Temperature and humidity will affect the formation of predatory organs, and the supply of nutrients in the environment, such as the concentrations of carbon sources and nitrogen sources, also has a great impact on the formation of traps. Generally, in a nutrient-poor 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 mixed solution of oleic acid, linoleic acid, pregnenolone and water at an appropriate concentration 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 efficient nematode biocontrol agents.

[0007] Through literature search, no public report of literature identical to the content of the present invention was found. Summary of the Invention

[0008] In order to enable nematode-trapping fungi to produce more trapping organs, the present invention provides a method for inducing the formation of trapping organs of nematode-trapping fungi, which specifically includes the following steps: (1) Cultivate the activated nematode-trapping fungi until spores are produced.

[0009] (2) Wash down the spores obtained by culturing in step (1), and dilute to obtain a spore suspension.

[0010] (3) Spread the diluted spore suspension obtained in step (2) on an agar medium covered with cellophane. After spreading the spore suspension evenly, incubate at a constant temperature, then add a mixed solution of a trapping organ inducer and water and continue to incubate at a constant temperature. After the cultivation is completed, the nematode-trapping fungi produce a large number of trapping organs.

[0011] The trapping organ inducer is one of oleic acid, linoleic acid or pregnenolone.

[0012] Preferably, the nematode-trapping fungus is Arthrobotrys oligospora.

[0013] Preferably, the medium used for culturing the nematode-trapping fungi in step (1) is a corn starch medium.

[0014] Preferably, the amount of spores in the spore suspension in step (2) is 30,000 - 60,000 spores / mL.

[0015] Preferably, the dosage of the trapping organ inducer in step (3) is to add 1 mL of the mixed solution of the trapping organ inducer and water to every 100 μL of the spore suspension, and the concentration of the trapping organ inducer in the mixed solution of the trapping organ inducer and water is 100 pmol / L - 100 μmol / L.

[0016] More preferably, the concentration of the trapping organ inducer in the mixed solution of the trapping organ inducer and water in step (3) is 100 nmol / L.

[0017] Preferably, the conditions for incubating at a constant temperature after spreading the spore suspension evenly in step (3) are: incubate at 25 - 28 °C for 28 - 36 h.

[0018] Preferably, the conditions for continuing to incubate at a constant temperature in step (3) are to incubate at 25 - 28 °C for 36 - 60 h.

[0019] Technical effects of the present invention: (1) The present invention for the first time discovers that pregnenolone, oleic acid or linoleic acid can be used to induce a large number of predatory organs in nematode-trapping fungi. After induction with pregnenolone, oleic acid or linoleic acid, the amounts of predatory organs are increased by 2000%, 1200% and 1600% respectively compared with those without using these three reagents. Description of the Drawings

[0020] Figure 1 Figure shows that pregnenolone induces the production of predatory organs in the nematode-trapping fungus Arthrobotrys oligospora.

[0021] Figure 2 Figure shows that oleic acid induces the production of predatory organs in the nematode-trapping fungus Arthrobotrys oligospora.

[0022] Figure 3 Figure shows that linoleic acid induces the production of predatory organs in the nematode-trapping fungus Arthrobotrys oligospora. Detailed Embodiments

[0023] The present invention will be further described in detail below with reference to the drawings and specific embodiments, but the protection scope of the present invention is not limited to the content described.

[0024] The reagents and the preparation of the culture medium used in the embodiments are as described below. Unless otherwise specified, the reagents used can be obtained through conventional commercial channels.

[0025] (1) Preparation of the 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 respectively and place them in 1.5 mL sterilized centrifuge tubes. Add sterile water to a volume of 1 mL respectively to prepare stock solutions of 100 mmol / L, store them at 4 °C, and dilute them to obtain the corresponding mixed solutions of predatory organ inducer and water at the final concentration when in use.

[0026] (2) Agar medium: Add 20 g of agar to every 1000 mL of pure water and sterilize at 121 °C for 20 minutes.

[0027] (3) Corn starch medium: Weigh 30 g of corn kernels, add 1000 mL of pure water, boil (100 °C) for 20 minutes, filter the liquid with 6 layers of gauze to collect it, add 5 g of yeast extract, add 20 g of agar, sterilize at 121 °C for 20 minutes and then pour it into a petri dish with a diameter of 6 cm, and wait for it to solidify completely before use.

[0028] (4)PDA medium: 200 g of peeled potatoes, cut into pieces, boiled for 30 minutes, filtered through six layers of gauze to collect the supernatant, added with 20 g of glucose and 20 g of agar. Add water to make up the volume to 1000 mL, sterilize at 121 °C for 20 minutes, then pour into a petri dish with a diameter of 6 cm, and wait for it to solidify completely before use. Example 1

[0029] A method for inducing the production of predatory organs in Arthrobotrys oligospora ([[]] Arthrobotrys oligospora ) by pregnenolone, the specific steps are as follows: (1)Activation of Arthrobotrys oligospora: Activate the preserved Arthrobotrys oligospora strain on PDA medium; (2)Spore culture of Arthrobotrys oligospora: Inoculate the activated Arthrobotrys oligospora fungal block in step (1) onto corn starch medium and culture at 28 °C for 10 days to obtain a large number of spores.

[0030] (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.

[0031] (4)Take 100 μL of the diluted spore suspension in step (3) and spread it on a 6 cm agar medium covered with cellophane, place it in a constant temperature incubator at 28 °C for 28 h to germinate, then inject 1 mL of the mixed solution of pregnenolone and water (the concentration of pregnenolone in the mixed solution is 100 nmol / L) into each plate from the side wall, slowly tilt the plate to make the mixed solution of pregnenolone and water soak the plate, and continue to place it in a constant temperature incubator at 28 °C for induction for 48 h, and a large number of predatory organs will be produced.

[0032] For comparison, a blank control was also set up in this example, that is, the mixed solution without pregnenolone was added. The results are as Figure 1 shown. It can be seen from Figure 1 that the mixed solution of pregnenolone and water can effectively increase the number of predatory organs of Arthrobotrys oligospora. Example 2

[0033] A method for inducing the production of predatory organs in Arthrobotrys oligospora ([[]] Arthrobotrys oligospora ) by oleic acid, the specific steps are as follows: (1)Activation of Arthrobotrys oligospora: Activate the preserved Arthrobotrys oligospora strain on PDA medium; (2)Spore culture of Arthrobotrys oligospora: Inoculate the activated Arthrobotrys oligospora fungal block in step (1) onto corn starch medium and culture 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 30,000 spores / mL.

[0035] (4) Take 100 μL of the diluted spore suspension in step (3) and spread it on a 6-cm agar medium covered with cellophane. Place it in a constant temperature incubator at 25 °C for 36 h to germinate. Then, inject 1 mL of the mixed solution of oleic acid and water (the concentration of oleic acid in the mixed solution is 100 nmol / L) into each plate from the side wall. Slowly tilt the plate to make the mixed solution of oleic acid and water infiltrate the plate and continue to place it in a constant temperature incubator at 28 °C for 36 h to induce a large number of predatory organs to be produced.

[0036] For comparison, this example also sets a blank control, that is, without adding the mixed solution of oleic acid and water. The results are as Figure 2 shown. It can be seen from Figure 2 that adding the mixed solution of oleic acid and water can effectively increase the number of predatory organs of Example 3

[0037] A method for inducing predatory organs in Arthrobotrys oligospora Arthrobotrys oligospora with pregnenolone, the specific steps are as follows: (1) Activation of Arthrobotrys oligospora: Activate the preserved Arthrobotrys oligospora strain on PDA medium; (2) Spore culture of Arthrobotrys oligospora: Inoculate the activated Arthrobotrys oligospora fungal block in step (1) onto corn starch medium and culture it at 28 °C for 10 days to obtain a large number of spores.

[0038] (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.

[0039] (4) Take 100 μL of the diluted spore suspension in step (3) and spread it on a 6-cm agar medium covered with cellophane. Place it in a constant temperature incubator at 28 °C for 30 h to germinate. Then, inject 1 mL of the mixed solution of pregnenolone and water (the concentration of pregnenolone in the mixed solution is 100 nmol / L) into each plate from the side wall. Slowly tilt the plate to make the mixed solution of pregnenolone and water infiltrate the plate and continue to place it in a constant temperature incubator at 25 °C for 60 h to induce a large number of predatory organs to be produced.

[0040] For comparison, this example also sets a blank control, that is, without adding the mixed solution of pregnenolone and water. The results are as Figure 3 shown. It can be seen from Figure 3 that adding the mixed solution of pregnenolone and water can effectively increase the number of predatory organs of Arthrobotrys oligospora.

Claims

1. A method for inducing the formation of predatory organs of nematode-trapping fungi, characterized in that: Specifically, it includes the following steps: (1) Cultivate the activated nematode-trapping fungus until spores are produced; (2) Wash down the spores obtained from step (1) and dilute them to obtain a spore suspension; (3) Spread the diluted spore suspension obtained in step (2) onto an agar medium covered with cellophane. After spreading the spore suspension evenly, incubate it at a constant temperature. Then add a mixed solution of a nematode-trapping organ inducer and water and continue to incubate at a constant temperature. After the cultivation is completed, the nematode-trapping fungus produces a large number of nematode-trapping organs; The nematode-trapping organ inducer is one of oleic acid, linoleic acid or pregnenolone.

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

3. The method for inducing the formation of the predatory organs of nematode-trapping fungi according to claim 1, wherein: The amount of spores in the spore suspension described in step (2) is 30,000 - 60,000 spores / mL.

4. The method for inducing the formation of predatory organs of nematode-trapping fungi according to claim 1, wherein: In step (3), the dosage of the nematode-trapping organ inducer is to add 1 mL of the mixed solution of the nematode-trapping organ inducer and water to every 100 μL of the spore suspension. The concentration of the nematode-trapping organ inducer in the mixed solution of the nematode-trapping organ inducer and water is 100 pmol / L - 100 μmol / L.

5. The method for inducing the formation of the predatory organs of nematode-trapping fungi according to claim 1, wherein: The conditions for incubating at a constant temperature after spreading the spore suspension evenly in step (3) are: incubate at 25 - 28 °C for 28 - 36 h.

6. The method for inducing the formation of the predatory organs of nematode-trapping fungi according to claim 1, wherein: The conditions for continuing to incubate at a constant temperature in step (3) are 25 - 28 °C for 36 - 60 h.

Images