A rapid propagation culture medium, culture method and application of arbuscular mycorrhizal fungi

Abstract

The invention discloses a rapid propagation culture medium, a culture method and an application of arbuscular mycorrhizal fungi, and belongs to the technical field of arbuscular fungi culture. The rapid propagation culture medium of the invention comprises the following raw materials in parts by weight: 40 to 60 parts of zeolite, 20 to 30 parts of quartz sand, 20 to 50 parts of peat, 10 to 15 parts of tea stem extract, 8 to 15 parts of xylose, 1 to 2 parts of L-ornithine, 0.5 to 1 part of potassium humate, 2 to 4 parts of ammonium nitrate and 2 to 4 parts of potassium dihydrogen phosphate. The invention prepares a culture medium using ingredients such as tea stem extract, which can significantly increase the propagation speed of arbuscular mycorrhizal fungi, shorten the culture cycle, promote the growth of plants, and provide new technical ideas for arbuscular mycorrhizal fungi culture and plant cultivation.

Description

Technical Field

[0001] The invention relates to the technical field of arbuscular fungi cultivation, and in particular to a rapid propagation culture medium, a cultivation method and an application of arbuscular mycorrhizal fungi. Background Art

[0002] Arbuscular mycorrhizalfungi (AMF) is a type of fungus that is widely found in the soil and can form symbiotic arbuscular mycorrhizae with most terrestrial plants. Fungi obtain necessary carbohydrates and other nutrients from plants, while plants also obtain the nutrients they need from fungi, thus achieving a high degree of unity of mutual benefit and mutual exchange. Current studies have shown that AMF has important physiological and ecological functions, can effectively promote the absorption and utilization of mineral nutrients and water by plants, promote plant growth and development, enhance plant tolerance to environmental stress, and affect plant secondary metabolism.

[0003] At present, the methods for artificially cultivating arbuscular mycorrhizal fungi mainly include pot culture method, glass bead chamber culture method and transformation root modified dual culture method, but due to the strong dependence of AMF on plant roots, the existing artificial culture methods have a long culture cycle, slow AMF reproduction speed, low activity, and limited application prospects. Therefore, it is necessary to provide a culture medium that can quickly cultivate AMF and improve AMF activity. Summary of the invention

[0004] The purpose of the present invention is to provide a rapid propagation medium, a culture method and an application of arbuscular mycorrhizal fungi to solve the problems existing in the above-mentioned prior art. The present invention uses tea stem extract and other ingredients to prepare a culture medium, which can significantly increase the propagation speed of arbuscular mycorrhizal fungi, shorten the culture cycle, promote plant growth, and provide a new technical idea for arbuscular mycorrhizal fungi culture and plant cultivation.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a rapid propagation culture medium for arbuscular mycorrhizal fungi. The rapid propagation culture medium comprises the following raw materials in parts by weight: 40-60 parts of zeolite, 20-30 parts of quartz sand, 20-50 parts of peat, 10-15 parts of tea stem extract, 8-15 parts of xylose, 1-2 parts of L-ornithine, 0.5-1 part of potassium humate, 2-4 parts of ammonium nitrate and 2-4 parts of potassium dihydrogen phosphate.

[0007] Preferably, the method for preparing the tea stem extract comprises the following steps:

[0008] (1) mixing tea stems with water and grinding them into a slurry, inoculating a microbial agent, and fermenting to obtain a tea stem fermentation liquid;

[0009] (2) The tea stem fermentation liquid is concentrated, mixed with an ethanol aqueous solution, subjected to ultrasonic extraction, filtered, and dried to obtain a tea stem extract.

[0010] Preferably, the mass ratio of the tea stems to water is 1:(10-15).

[0011] Preferably, the microbial agent is composed of Bacillus coagulans, Bacillus megaterium and Saccharomyces in a ratio of viable bacteria = (2-4): (2-4): (1-2); the total viable bacteria count of the microbial agent is 5×10 8～12 CFU / g; the inoculation amount of the microbial agent is 3-5%.

[0012] Preferably, the fermentation temperature is 35-45°C and the fermentation time is 48-72 hours.

[0013] Preferably, the tea stem fermented liquid is concentrated to 1 / 4 of its original volume; the volume ratio of the concentrated tea stem fermented liquid to the ethanol aqueous solution is 1:(3-5).

[0014] Preferably, the volume fraction of the ethanol aqueous solution is 60-80%.

[0015] Preferably, the power of the ultrasonic extraction is 400 W, the temperature is 50-55° C., and the time is 30-60 min.

[0016] The present invention also provides an application of the rapid propagation culture medium in culturing arbuscular mycorrhizal fungi.

[0017] The present invention also provides a method for rapid cultivation of arbuscular mycorrhizal fungi, comprising the following steps:

[0018] a) inoculating spores of arbuscular mycorrhizal fungi into the above-mentioned rapid propagation medium, and adjusting the water content of the medium to 30-50%;

[0019] b) Transplanting the tea tree seedlings into the rapid propagation medium of step a), watering them with Hoagland nutrient solution once a week, and after culturing for 3 to 4 months, removing the above-ground parts of the tea trees, and mixing the remaining roots and the rapid propagation medium, crushing and drying them to obtain an arbuscular mycorrhizal fungal agent.

[0020] The present invention discloses the following technical effects:

[0021] The present invention provides a culture medium capable of rapidly propagating arbuscular mycorrhizal fungi. The present invention ferments tea stems by microorganisms, and extracts components in the tea stem fermentation liquid by ethanol ultrasonic extraction, and combines raw materials such as L-ornithine, zeolite, quartz sand, peat, xylose, potassium humate, ammonium nitrate, potassium dihydrogen phosphate, etc., to improve the reproduction speed and infection ability of arbuscular mycorrhizal fungi, and can also improve the growth rate of plants. Compared with other existing technologies, the culture medium of the present invention has easy-to-obtain raw materials, low cost, simple preparation method, and fast propagation speed of arbuscular mycorrhizal fungi, providing new technical ideas for arbuscular mycorrhizal fungi culture and plant cultivation. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The Bacillus coagulans BCN019 used in the examples of the present invention was purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd., Bacillus megaterium was purchased from Guangzhou Zhenwei Microbiology Technology Co., Ltd., and the yeast (Saccharomyces) was Angel Yeast.

[0028] Example 1

[0029] This embodiment provides a rapid propagation culture medium, and the preparation method is as follows:

[0030] 50 parts by weight of zeolite (particle size of 40-60 mesh), 25 parts by weight of quartz sand (particle size of 10-20 mesh), and 40 parts by weight of peat are mixed evenly, and after high-temperature steam sterilization, 12 parts by weight of tea stem extract, 10 parts by weight of xylose, 1.5 parts by weight of L-ornithine, 0.8 parts by weight of potassium humate, 3 parts by weight of ammonium nitrate and 3 parts by weight of potassium dihydrogen phosphate are added, and mixed evenly to obtain a rapid propagation medium.

[0031] Wherein, the preparation process of tea stem extract is:

[0032] The dried tea stems were mixed with water in a mass ratio of 1:12 and crushed into a slurry; Bacillus coagulans, Bacillus megaterium and yeast were mixed in a ratio of viable bacteria of 3:3:2 to obtain a microbial agent (total viable bacteria of 5×10 10 CFU / g); 4% (w / w) microbial agent was added to the obtained slurry, and after fermentation at 42°C for 60 hours, the temperature was raised to 100°C and sterilized for 30 minutes to obtain tea stem fermentation liquid.

[0033] The tea stem fermentation liquid is concentrated to 1 / 4 of the original volume, 4 times the volume of 70% ethanol aqueous solution is added, and ultrasonic extraction is performed at 50°C and 400W for 40 minutes, and then the clarified filtrate is taken and freeze-dried to obtain the tea stem extract.

[0034] Example 2

[0035] This embodiment provides a rapid propagation culture medium, and the preparation method is as follows:

[0036] 40 parts by weight of zeolite (particle size of 40-60 mesh), 30 parts by weight of quartz sand (particle size of 10-20 mesh), and 20 parts by weight of peat are mixed evenly, and after high-temperature steam sterilization, 15 parts by weight of tea stem extract, 8 parts by weight of xylose, 1 part by weight of L-ornithine, 0.5 parts by weight of potassium humate, 4 parts by weight of ammonium nitrate and 4 parts by weight of potassium dihydrogen phosphate are added and mixed evenly to obtain a rapid propagation medium.

[0037] Wherein, the preparation process of tea stem extract is:

[0038] The dried tea stems were mixed with water in a mass ratio of 1:10 and crushed into a slurry; Bacillus coagulans, Bacillus megaterium and yeast were mixed in a ratio of viable bacteria of 2:2:1 to obtain a microbial agent (total viable bacteria of 5×10 10 CFU / g); 3% (w / w) microbial agent was added to the obtained slurry, and after fermentation at 35°C for 72 hours, the temperature was raised to 100°C and sterilized for 30 minutes to obtain tea stem fermentation liquid.

[0039] The tea stem fermentation liquid was concentrated to 1 / 4 of the original volume, 5 times the volume of 80% ethanol aqueous solution was added, and ultrasonic extraction was performed at 50°C and 400W for 30 minutes, and then the clarified filtrate was taken and freeze-dried to obtain the tea stem extract.

[0040] Example 3

[0041] This embodiment provides a rapid propagation culture medium, and the preparation method is as follows:

[0042] 60 parts by weight of zeolite (particle size of 40-60 mesh), 20 parts by weight of quartz sand (particle size of 10-20 mesh), and 45 parts by weight of peat are mixed evenly, and after high-temperature steam sterilization, 10 parts by weight of tea stem extract, 15 parts by weight of xylose, 2 parts by weight of L-ornithine, 1 part by weight of potassium humate, 2 parts by weight of ammonium nitrate and 2 parts by weight of potassium dihydrogen phosphate are added, and mixed evenly to obtain a rapid propagation medium.

[0043] Wherein, the preparation process of tea stem extract is:

[0044] The dried tea stems were mixed with water in a mass ratio of 1:15 and crushed into a slurry; Bacillus coagulans, Bacillus megaterium and yeast were mixed in a ratio of viable bacteria of 2:3:1 to obtain a microbial agent (total viable bacteria of 5×10 10 CFU / g); 5% (w / w) microbial agent was added to the obtained slurry, and after fermentation at 45°C for 48 hours, the temperature was raised to 100°C and sterilized for 30 minutes to obtain tea stem fermentation liquid.

[0045] The tea stem fermentation liquid is concentrated to 1 / 4 of the original volume, 3 times the volume of 60% ethanol aqueous solution is added, and ultrasonic extraction is performed at 50°C and 400W for 60 minutes, and then the clarified filtrate is taken and freeze-dried to obtain the tea stem extract.

[0046] Comparative Example 1

[0047] The difference between this comparative example and Example 1 is that the tea stem extract is replaced by an equal amount of peat.

[0048] Comparative Example 2

[0049] The difference between this comparative example and Example 1 is that L-ornithine is replaced by an equal amount of potassium humate.

[0050] Comparative Example 3

[0051] The difference between this comparative example and Example 1 is that the preparation process of the tea stem extract is adjusted as follows: the tea stems and water are mixed in a mass ratio of 1:12 and crushed into a slurry; the slurry is concentrated to 1 / 4 of the original volume, 4 times the volume of 70% ethanol aqueous solution is added, and ultrasonic extraction is performed at 50°C and 400W for 40 minutes, followed by filtration, and the clarified filtrate is obtained, which is freeze-dried to obtain the tea stem extract.

[0052] Comparative Example 4

[0053] The difference between this comparative example and Example 1 is that the ultrasonic extraction is replaced by reflux extraction, and the reflux temperature is 75° C. and the time is 40 min.

[0054] Comparative Example 5

[0055] The difference between this comparative example and Example 1 is that Bacillus coagulans is replaced by Bacillus subtilis (purchased from Guangzhou True Microbiology Technology Co., Ltd.).

[0056] Test Example 1

[0057] The rapid propagation medium prepared in Examples 1 to 3 and Comparative Examples 1 to 5 was placed in a pot with a length of 0.5 m, a width of 0.5 m, and an inner height of 40 cm, and the thickness of the medium was 35 cm. Glomus radicis was used as the test strain, and spores of Glomus radicis were inoculated in the medium, the inoculation depth was 5 cm, and the inoculation amount was 100 per gram of the medium. Then sterile water was poured until the water content of the medium was 40%.

[0058] The 3-month-old Zijuan tea seedlings were transplanted into the above-mentioned culture medium pots, with one plant transplanted per pot. They were placed at room temperature for normal cultivation, watered with Hoagland nutrient solution once a week, and watered with sterile water to keep the water content of the culture medium at 30% to 50%. After 3 months of cultivation, watering was stopped. Part of the tea tree roots were collected, and the mycorrhizal infection rate was determined and recorded. The determination method was referenced to (Sheng Pingping, Liu Runjin, Li Min. Comparison of arbuscular mycorrhizal observation and infection rate determination methods [J]. Journal of Mycology, 2011, 30(4):7. DOI:CNKI:SUN:JWXT.0.2011-04-004.). The aboveground fresh weight of the tea seedlings was determined before and after cultivation, and the growth rate was calculated. After removing the aboveground part of the tea seedlings, the remaining roots and the rapid propagation culture medium were mixed, crushed and dried, and the spore density of the root Glomus was determined by the wet sieve pouring-sucrose separation method and recorded. The statistical results are shown in Table 1.

[0059] Table 1 Culture results of different culture media

[0060]

[0061] It can be seen from Table 1 that, compared with Comparative Examples 1 to 5, the culture medium prepared in Examples 1 to 3 of the present invention can significantly increase the mycorrhizal infection rate of arbuscular mycorrhizal fungi on tea trees, and the spore propagation effect is obvious, which shows that the culture medium of the present invention can significantly promote the reproduction of arbuscular mycorrhizal fungi and increase the reproduction rate; the culture medium prepared in Examples 1 to 3 can also significantly increase the fresh weight of the aboveground part of tea trees, which shows that the culture medium of the present invention can not only improve the vitality of fungi when propagating arbuscular mycorrhizal fungi, but also promote the growth of plants at the same time.

[0062] According to Comparative Example 1 and Comparative Examples 3-5, it can be seen that the tea stem extract prepared by the present invention contains rich nutrients, which have a significant impact on the reproduction speed and mycorrhizal infection effect of arbuscular mycorrhizal fungi. Omitting the tea stem extract, changing the preparation method of the tea stem extract or omitting the fermentation microorganism strain will affect the content and composition of the nutrients in the extract, resulting in poor reproduction speed, mycorrhizal infection effect and plant growth effect of arbuscular mycorrhizal fungi. According to Comparative Example 2, it can be seen that L-ornithine can not only promote plant growth, but also improve the reproduction speed and mycorrhizal infection effect of arbuscular mycorrhizal fungi. The use of the culture medium of the present invention can achieve the purpose of rapid reproduction of arbuscular mycorrhizal fungi, and can be used in the fields of arbuscular mycorrhizal fungi cultivation and plant cultivation.

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A rapid propagation medium for arbuscular mycorrhizal fungi, characterized in that: The rapid propagation culture medium comprises the following raw materials in parts by weight: 40-60 parts of zeolite, 20-30 parts of quartz sand, 20-50 parts of peat, 10-15 parts of tea stem extract, 8-15 parts of xylose, 1-2 parts of L-ornithine, 0.5-1 part of potassium humate, 2-4 parts of ammonium nitrate and 2-4 parts of potassium dihydrogen phosphate; The preparation method of the tea stem extract comprises the following steps: (1) mixing tea stems with water and crushing them into a slurry, inoculating a microbial agent, and sterilizing after fermentation to obtain a tea stem fermentation liquid; (2) The tea stem fermentation liquid is concentrated, mixed with an ethanol aqueous solution, subjected to ultrasonic extraction, filtered, and freeze-dried to obtain a tea stem extract.

2. The rapid propagation medium according to claim 1, characterized in that The mass ratio of the tea stems to water is 1:(10-15).

3. The rapid propagation medium according to claim 1, characterized in that The microbial agent is composed of Bacillus coagulans ( Bacillus coagulans )、Bacillus megaterium ( Bacillus megaterium ) and yeast ( Saccharomyces ) is composed of a viable bacterial count ratio of (2-4): (2-4): (1-2); the total viable bacterial count of the microbial agent is 5×10 8～ 12 CFU / g; the inoculation amount of the microbial agent is 3-5% of the mass of the slurry.

4. The rapid propagation medium according to claim 1, characterized in that The fermentation temperature is 35-45°C and the fermentation time is 48-72 hours.

5. The rapid propagation medium according to claim 1, characterized in that The tea stem fermentation liquid is concentrated to 1 / 4 of the original volume; the volume ratio of the concentrated tea stem fermentation liquid to the ethanol aqueous solution is 1:(3-5).

6. The rapid propagation medium according to claim 1, characterized in that The volume fraction of the ethanol aqueous solution is 60-80%.

7. The rapid propagation medium according to claim 1, characterized in that The power of the ultrasonic extraction is 400W, the temperature is 50-55°C, and the time is 30-60min.

8. Use of the rapid propagation medium according to any one of claims 1 to 7 in culturing arbuscular mycorrhizal fungi, characterized in that: The arbuscular mycorrhizal fungus is Glomus intraradicus.

9. A method for rapid cultivation of arbuscular mycorrhizal fungi, characterized in that: The steps include: a) inoculating spores of arbuscular mycorrhizal fungi into the rapid propagation medium according to any one of claims 1 to 7, and adjusting the water content of the medium to 30 to 50%; b) transplanting the tea tree seedlings into the rapid propagation medium of step a), watering them with Hoagland nutrient solution once a week, and after culturing for 3 to 4 months, removing the above-ground parts of the tea trees, and mixing and crushing the remaining roots and the rapid propagation medium and drying them to obtain an arbuscular mycorrhizal fungal agent; The arbuscular mycorrhizal fungus is Glomus intraradicus.

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