A spore production inducer of Pythium oligandrum, preparation method and application thereof

Abstract

The present invention relates to a spore production inducer of Pythium oligandrum, a spore production medium for inducing Pythium oligandrum comprising the inducer, and a preparation method and application thereof. By adding the spore production inducer of the present application, a large number of spores can be rapidly produced by the mycelium of Pythium oligandrum, and the spore production rate thereof can be increased, and the spore production amount can reach more than 5 times the spore production amount without induction. At the same time, the biological control and growth promotion ability of the Pythium oligandrum spores prepared by the present invention is not reduced.

Description

Technical Field

[0001] The invention relates to the technical field of life sciences, and in particular to a spore production inducer of Pythium oligandrum, a preparation method and application thereof. Background Art

[0002] Pythium oligandrum belongs to the phylum Oomycetes. It is a highly aggressive parasitic microorganism widely found in the soil and plant rhizosphere, and has broad-spectrum resistance to pathogenic microorganisms. Pythium oligandrum colonizes in the rhizosphere of crops and can promote plant rooting and growth. It is a microorganism that has both disease prevention and growth-promoting functions. Pythium oligandrum spores are its main active ingredients that can inhibit pathogen infection and promote plant growth. The currently commercialized product "Dolly Vitality" is a microbial preparation jointly developed by ImageTrade International Co., Ltd. and Czech Biological Preparation Co., Ltd. that uses active spores of Pythium oligandrum for broad-spectrum sterilization. It is known as the "Light of Agriculture in the New Century" in Europe and is a recommended product designated by the European Union. It is widely used in fields, cash crops, fruit trees, vegetables, golf courses, and garden flowers.

[0003] The processing of biological pesticides usually involves the preparation of fungal spores. The spores of Pythium oligandrum are generally prepared by directly inoculating pure cultured Pythium oligandrum discs into liquid culture medium to obtain mycelium, and then the spores are separated from the mycelium and released into the water by beating or shaking. The number and efficiency of the spore suspension of Pythium oligandrum obtained by this method need to be improved, and it is difficult to meet the needs of mass production. Therefore, optimizing the spore production conditions of Pythium oligandrum and inducing its efficient spore production are of great significance for the in-depth research and development and utilization of Pythium oligandrum.

[0004] Spore inducers are highly effective promoters that induce fungi to produce spores and increase spore production by adding one or more trace substances. Adding spore inducers to the culture medium can increase the number of mycelium of Pythium oligandrum and induce its efficient spore production, which can provide sufficient experimental materials for various studies on Pythium oligandrum and is a technical guarantee for promoting the development of biological control technology for Pythium oligandrum. Summary of the invention

[0005] Problem that the invention aims to solve

[0006] In response to the above technical problems, the present application provides a spore production inducer of Pythium oligandrum that can induce Pythium oligandrum to produce spores efficiently, as well as a preparation method and application thereof.

[0007] Solutions for solving problems

[0008] On the one hand, the present application provides a spore production inducer for Pythium oligandrum, comprising sodium molybdate, ammonium sulfate and sodium iron EDTA.

[0009] On the other hand, the present application provides a culture medium for inducing spore production of Pythium oligandrum, comprising a basal culture medium and a spore production inducer of Pythium oligandrum.

[0010] On the other hand, the present application provides a method for preparing the aforementioned Pythium oligandrum spore production inducer and a culture medium for inducing Pythium oligandrum spore production.

[0011] On the other hand, the present application provides the use of the aforementioned Pythium oligandrum spore production inducer and / or the culture medium for inducing Pythium oligandrum spore production in inducing rapid and massive spore production by Pythium oligandrum mycelium.

[0012] On the other hand, the present application provides a culture method for inducing rapid and massive spore production of Pythium oligandrum.

[0013] Effects of the Invention

[0014] By adding the spore production inducer of the present application, the mycelium of Pythium oligandrum can quickly produce a large number of spores, improve its spore production rate, and the spore production amount reaches more than 5 times the uninduced spore production amount. At the same time, the biological control and growth promotion ability of the Pythium oligandrum spores prepared by the present invention is not reduced, thus laying a good foundation for the later research on biological control technology of Pythium oligandrum, effectively saving manpower and material resources and a lot of precious time of scientific researchers, promoting the replacement of biological control agents with or reducing the use of chemical pesticides, improving food and ecological environment safety, and having good economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Effects of spore production inducers of Pythium oligandrum on the antibacterial effect of Pythium oligandrum plates

[0016] Figure 2 Effects of spore production inducers of Pythium oligandrum on the antibacterial effect of Pythium oligandrum in vivo DETAILED DESCRIPTION

[0017] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0018] On the one hand, the present application provides a spore production inducer for Pythium oligandrum, comprising sodium molybdate, ammonium sulfate and sodium iron EDTA.

[0019] In some embodiments, the oligandrum spore production inducer includes 1-50g of sodium molybdate, 1-100g of ammonium sulfate and 1-100g of sodium iron EDTA, with a total volume of 1000ml, and the balance is water; preferably, the oligandrum spore production inducer includes 1-25g of sodium molybdate, 1-50g of ammonium sulfate and 1-50g of sodium iron EDTA, with a total volume of 1000ml, and the balance is water; more preferably, the oligandrum spore production inducer includes 1-10g of sodium molybdate, 1-25g of ammonium sulfate and 1-25g of sodium iron EDTA, with a total volume of 1000ml, and the balance is water.

[0020] On the other hand, the present application provides a method for preparing any of the aforementioned Pythium oligandrum spore production inducers, comprising the following steps:

[0021] Weigh 1-50 g of sodium molybdate, 1-100 g of ammonium sulfate, and 1-100 g of sodium iron EDTA, add water to 1000 ml, mix well, and sterilize to obtain a spore production inducer.

[0022] On the other hand, the present application provides a culture medium for inducing spore production of Pythium oligandrum, comprising a basal culture medium and any one of the aforementioned spore production inducers of Pythium oligandrum.

[0023] In some embodiments, in the medium for inducing spore production of Pythium oligandrum, the final concentration of any of the aforementioned spore production inducers of Pythium oligandrum is 0.01-10%, preferably 0.1-5%, and more preferably 0.1-2%.

[0024] In some embodiments, the basal medium is V8 medium.

[0025] In some embodiments, the V8 medium includes V8 vegetable juice, calcium carbonate, and water.

[0026] In some embodiments, the basic culture medium includes 100 ml of V8 vegetable juice, 1 g of calcium carbonate, and the balance is water, based on a total volume of 1000 ml.

[0027] On the other hand, the present application provides a method for preparing a culture medium for inducing spore production of Pythium oligandrum, comprising the following steps:

[0028] (1) Preparation of any one of the above-mentioned Pythium oligandrum spore production inducers;

[0029] (2) A certain amount of a spore production inducer of Pythium oligandrum is added to any of the above-mentioned basal culture media to make the final concentration of the spore production inducer of Pythium oligandrum be 0.01-10%, thereby obtaining a medium for inducing spore production of Pythium oligandrum.

[0030] In some embodiments, the method for preparing a medium for inducing spore production of Pythium oligandrum comprises the following steps:

[0031] (1) Weigh 1-50 g of sodium molybdate, 1-100 g of ammonium sulfate, and 1-100 g of sodium iron EDTA, add water to 1000 ml, mix well, and sterilize to obtain a spore production inducer;

[0032] (2) A certain amount of a spore production inducer of Pythium oligandrum is added to any of the above-mentioned basal culture media to make the final concentration of the spore production inducer of Pythium oligandrum be 0.01-10%, thereby obtaining a medium for inducing spore production of Pythium oligandrum.

[0033] On the other hand, the present application provides the use of any of the aforementioned Pythium oligandrum spore production inducers and / or any of the culture media for inducing Pythium oligandrum spore production in inducing rapid and massive spore production by Pythium oligandrum mycelium.

[0034] On the other hand, the present application provides a culture method for inducing rapid and massive spore production by Pythium oligandrum, comprising using Pythium oligandrum mycelium as a basis and a liquid culture medium to which any of the aforementioned Pythium oligandrum spore production inducers is added as a culture medium to induce rapid and massive spore production by Pythium oligandrum mycelium.

[0035] In some embodiments, the culture method for inducing rapid and massive spore production of Pythium oligandrum comprises the following steps:

[0036] (1) activating Pythium oligandrum on a solid culture medium;

[0037] (2) The cultured mycelium is inoculated into a liquid culture medium containing any of the aforementioned Pythium oligandrum spore production inducers, and cultured in a constant temperature incubator at 25-30° C. in the dark for 5-10 days.

[0038] In some embodiments, the liquid culture medium containing any one of the aforementioned Pythium oligandrum spore production inducers refers to any one of the aforementioned culture media for inducing Pythium oligandrum spore production.

[0039] The terms are explained as follows:

[0040] The water defined in each embodiment of the present application refers to water that can be used for biological cell culture, including but not limited to distilled water, deionized water, pure water, high-purity water, ultrapure water, etc.; preferably ultrapure water.

[0041] The V8 medium of the present application refers to a medium containing V8 vegetable juice, which is a universal medium that can be used to culture fungi such as Phytophthora and Pythium, wherein the V8 vegetable juice includes a mixture of various vegetable juices, such as tomatoes, carrots, watercress, lettuce, celery, spinach, beetroot, celery, etc., which provides essential nutrients for fungal growth; calcium carbonate is used to neutralize the acid generated during the culture process. The V8 medium and V8 vegetable juice described in the present application can be commercially available, or prepared according to any preparation method described in the present application.

[0042] The Pythium oligandrum strain used in this application was purchased from the General Microbiological Center of China Microbiological Culture Collection Administration, with the collection number: CGMCC No.3.17808.

[0043] Example 1: Preparation of spore-inducing culture medium

[0044] (1) Take an appropriate amount of V8 vegetable juice (Zinpro, USA, product number: Y6GpRQmQ), centrifuge at 4000 r / min for 10 min, take 100 ml of the supernatant, add 1 g of calcium carbonate thereto and mix and dissolve, make the obtained V8 vegetable juice calcium carbonate solution dilute to 1000 mL with ultrapure water, sterilize it at 121°C for 20 min, and obtain V8 culture medium.

[0045] (2) Weigh 1 g of sodium molybdate, 1 g of ammonium sulfate, and 10 g of sodium iron EDTA, add ultrapure water to 1000 mL, mix well, and sterilize at 121° C. for 20 min to obtain a spore production inducer for Pythium oligandrum.

[0046] (3) When using, add the Pythium oligandrum spore production inducer to the V8 medium to make the final concentration of the Pythium oligandrum spore production inducer be 0.1%, and mix well before use.

[0047] Example 2: Preparation of spore-inducing culture medium

[0048] (1) Take an appropriate amount of V8 vegetable juice, centrifuge it at 4000 r / min for 10 min, take 100 ml of the supernatant, add 1 g of calcium carbonate to it and mix and dissolve it, and make the obtained V8 vegetable juice calcium carbonate solution up to 1000 mL with ultrapure water, and sterilize it at 121°C for 20 min.

[0049] (2) Weigh 5 g of sodium molybdate, 5 g of ammonium sulfate, and 50 g of sodium iron EDTA, add ultrapure water to 1000 mL, mix well, and sterilize at 121° C. for 20 min to obtain a spore production inducer for Pythium oligandrum.

[0050] (3) When using, add the Pythium oligandrum spore production inducer to the V8 medium to make the final concentration of the Pythium oligandrum spore production inducer be 5%, and mix well before use.

[0051] Example 3: Preparation of spore-inducing culture medium

[0052] (1) Take an appropriate amount of V8 vegetable juice, centrifuge it at 4000 r / min for 10 min, take 100 ml of the supernatant, add 1 g of calcium carbonate to it and mix and dissolve it, and make the obtained V8 vegetable juice calcium carbonate solution up to 1000 mL with ultrapure water, and sterilize it at 121°C for 20 min.

[0053] (2) Weigh 5 g of sodium molybdate, 5 g of ammonium sulfate, and 25 g of sodium iron EDTA, add ultrapure water to 1000 mL, mix well, and sterilize at 121° C. for 20 min to obtain a spore production inducer for Pythium oligandrum.

[0054] (3) When using, add the spore production inducer of Pythium oligandrum to the V8 medium to make the final concentration of the spore production inducer of Pythium oligandrum to be 0.5%, and mix well before use.

[0055] Example 4: Preparation of spore-inducing culture medium

[0056] (1) Take an appropriate amount of V8 vegetable juice, centrifuge it at 4000 r / min for 10 min, take 100 ml of the supernatant, add 1 g of calcium carbonate to it and mix and dissolve it, and make the obtained V8 vegetable juice calcium carbonate solution up to 1000 mL with ultrapure water, and sterilize it at 121°C for 20 min.

[0057] (2) Weigh 10 g of sodium molybdate, 25 g of ammonium sulfate, and 25 g of sodium iron EDTA, add ultrapure water to 1000 mL, mix well, and sterilize at 121° C. for 20 min to obtain a spore production inducer for Pythium oligandrum.

[0058] (3) When using, add the Pythium oligandrum spore production inducer to the V8 medium to make the final concentration of the Pythium oligandrum spore production inducer be 10%, and mix well before use.

[0059] Example 5: Induction of Oligandro Pythium Spores

[0060] (1) Inoculate the Pythium oligandrum strain onto a solid culture medium plate and culture it in a constant temperature incubator at 22° C. for 5-7 days. The solid culture medium formula used is: 1000 mL of the liquid culture medium without the inducer in Example 1, 20 g of agar, and sterilize at 121° C. for 20 min after mixing.

[0061] (2) The activated Pythium oligandrum was transferred back to the solid culture medium plate and placed in a constant temperature incubator at 22°C for 72 hours. When the colony grew to a diameter of 3 cm, a piece of about 1 cm was taken with an inoculation loop. 2 Fresh mycelium at the edge of the colony was transplanted into a 13*13 cm square culture dish containing 50 mL of spore induction medium, sealed with a sealing film and placed in a constant temperature incubator at 22°C for culture.

[0062] (3) After 120 h of culture, the mycelium and culture medium were taken out and blended in a juicer to prepare a spore suspension of Pythium oligandrum. The volume was fixed to 50 mL, and 5 μL was taken on a hemocytometer to observe and calculate the spore production per unit volume.

[0063] The results showed that the spore production of Pythium oligandrum in the culture medium without spore production inducer was 1.26×10 5In contrast, the spore production of Pythium oligandrum cultured in induced spore production medium can reach 8.18×10 5 / ml, an increase of more than 5 times (Table 1).

[0064] Table 1: Effects of spore inducers on spore production of Pythium oligandrum

[0065]

[0066] Example 6: In vitro antibacterial activity test of Pythium oligandrum spore liquid

[0067] (1) The spore solution prepared in Example 5 was diluted and spread on a plate, and then placed in a constant temperature incubator at 22° C. for 72 h. After mycelium grew, a single colony was picked and transferred to a new plate, and the culture was continued for 120 h.

[0068] (2) A 5 mm diameter Pythium oligandrum cake and a 5 mm diameter wheat fusarium cake were punched out by a hole punch and inoculated on a 9 cm diameter PDA plate. The plate inoculated with only the plant pathogen cake was used as the control. Each treatment was repeated 3 times and cultured in a 25°C incubator for 5 days. The inhibition rate of each treatment group was observed ( Figure 1 CK refers to a plate inoculated with only plant pathogen cakes; A1 refers to a strain cultured by traditional spore production technology; A1 (adding inducer) refers to a strain cultured by adding an inducing spore production agent). The results show that the induced spores of Pythium oligandrum produced by the present invention have no significant difference in the inhibition rate of the in vitro plate compared with the uninduced spores, that is, the inducing spore production agent does not affect the biocontrol effect of Pythium oligandrum.

[0069] Example 7: In vivo antibacterial activity test of Pythium oligandrum spore liquid

[0070] (1) Wheat seeds were sterilized with 5% sodium hypochlorite and 75% ethanol and then soaked in sterile water for 24 h until they turned white. They were then transferred to a plate with 8 layers of gauze and 1 layer of filter paper for light culture.

[0071] (2) When the leaf tip slightly exposed the coleoptile, cut off the top 3 mm and drip 2 μL of 1×10 5 / mL of Fusarium graminearum spore solution. After 2 hours, 5mL / plate of A: sterile water, B: Pythium oligandrum spore solution prepared according to the method of Example 5 above, and C: uninduced Pythium oligandrum spore solution were sprayed respectively. Subsequently, the plates were sprayed twice a day, 5mL / plate each time, and the lesion length was compared after 7 days of 16 / 8h light and dark alternation. Figure 2 CK refers to the plate inoculated only with plant pathogen cake; A1 refers to the strain cultured by traditional spore production technology; A1 (with inducer) refers to the strain cultured with the addition of spore production inducer).

[0072] The results showed that the wheat in the control group was infected by pathogens, and the lesions expanded to both ends, while the induced oligandrum spores and the uninduced spores of the present invention could inhibit the infection of pathogens, and there was no significant difference in the living control effect of the two on wheat, that is, the induced spore-producing agent did not affect the biocontrol effect of oligandrum spores.

[0073] Example 8: Growth-promoting effect of Pythium oligandrum spore liquid on wheat

[0074] (1) Wheat seeds were sterilized with 5% sodium hypochlorite and 75% ethanol and then soaked in water for 24 h until they turned white. They were then transplanted into a plug tray filled with vermiculite.

[0075] (2) The spore solution of Pythium oligandrum was prepared according to the method of Example 5, diluted at a ratio of 10% (v / v) and added to the tray to completely soak the vermiculite for the first time, and then 10 mL / hole was added every two days. After culturing for 7 days with 16 / 8 h light and dark alternation, the plant height, stem diameter, fresh weight, dry weight and root activity were measured (Table 2).

[0076] The results show that: the induced Pythium oligandrum spores of the present invention have no significant difference in various indicators of promoting wheat growth compared with the uninduced spores, that is, the spore-producing inducing agent does not affect the growth-promoting effect of Pythium oligandrum.

[0077] Table 2 Effects of spore inducers on the growth-promoting ability of Pythium oligandrum

[0078]

[0079] Note: Because the weight of each wheat plant varies greatly, the samples are measured in groups, with 10 samples in each group;

[0080] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A medium for inducing spore production of Pythium oligandrum, comprising a basal medium and a spore production inducer of Pythium oligandrum, wherein: The spore production inducer of Pythium oligandrum includes 1-10 g of sodium molybdate, 1-25 g of ammonium sulfate, and 1-25 g of sodium iron EDTA based on a total volume of 1000 ml; The basic culture medium is V8 culture medium; In the medium for inducing spore production of Pythium oligandrum, the final concentration of the spore production inducer of Pythium oligandrum is 0.1-10%.

2. The medium for inducing spore production of Pythium oligandrum according to claim 1, characterized in that The final concentration of the oligandrum spore production inducer is 0.1-5%.

3. The medium for inducing spore production of Pythium oligandrum according to claim 2, characterized in that The final concentration of the Pythium oligandrum spore production inducer is 0.1-2%.

4. The medium for inducing spore production of Pythium oligandrum according to claim 1, characterized in that The V8 medium includes V8 vegetable juice, calcium carbonate and water.

5. A method for preparing the spore production inducer of Pythium oligandrum according to claim 1, characterized in that: The following steps are involved: According to the mass of claim 1, sodium molybdate, ammonium sulfate and sodium iron EDTA are weighed respectively, water is added to make up to 1000 ml, and the mixture is sterilized after mixing to obtain the spore production inducer of Pythium oligandrum.

6. A method for preparing a medium for inducing spore production of Pythium oligandrum according to claim 1, comprising the following steps: (1) preparing a spore production inducer of Pythium oligandrum according to the method for preparing a spore production inducer of Pythium oligandrum according to claim 5; (2) A certain amount of a spore production inducer of Pythium oligandrum is added to the basal culture medium so that the final concentration of the spore production inducer of Pythium oligandrum is 0.1-10%, thereby obtaining a culture medium for inducing spore production of Pythium oligandrum.

7. The preparation method according to claim 6, characterized in that: The final concentration of the Pythium oligandrum spore production inducer is 0.1-5%.

8. The preparation method according to claim 7, characterized in that: The final concentration of the Pythium oligandrum spore production inducer is 0.1-2%.

9. Use of the medium for inducing spore production of Pythium oligandrum according to any one of claims 1 to 4 in inducing rapid spore production in large quantities by mycelium of Pythium oligandrum.

10. A culture method for inducing rapid and massive spore production by Pythium oligandrum, comprising using Pythium oligandrum mycelium as a basis and the Pythium oligandrum spore production medium according to any one of claims 1 to 4 as a culture medium to induce rapid and massive spore production by Pythium oligandrum mycelium.

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