A fully synthetic mushroom bed substrate for Morchella

Abstract

The invention discloses a fully synthetic mushroom bed substrate for Morchella. It belongs to the technical field of basic scientific research and teaching tools for edible fungi physiology, biochemistry and molecular biology. It includes the following raw materials: silicon dioxide particles, potassium nitrate, anhydrous calcium sulfate, anhydrous potassium hydrogen phosphate, anhydrous magnesium sulfate, sodium tetraborate, anhydrous zinc sulfate, anhydrous copper sulfate, anhydrous ferric chloride, anhydrous manganese sulfate, and anhydrous sodium molybdate. The invention solves the problem that there is no carrier with a simple formula, completely known chemical composition, high controllability and high repeatability in the current scientific research on Morchella, and the preparation steps are simple, so the invention is suitable for scientific research universities and research institutes to carry out basic research experiments and teaching on the physiology, biochemistry and molecular biology of Morchella growth and fruiting.

Description

Technical Field

[0001] The invention relates to the technical field of basic scientific research and teaching tools for edible fungi physiology, biochemistry and molecular biology, and more specifically to a fully synthetic mushroom bed matrix for Morchella oleracea. Background Art

[0002] Since the successful artificial domestication of Morchella, the cultivation area in China has expanded rapidly, leading to a trend of basic research on functional genomics, gene expression, physiology and biochemistry related to the growth and fruiting of Morchella in the field of scientific research. Since most edible fungi belong to the phylum Basidiomycetes, Morchella is one of the few Ascomycetes that can be cultivated artificially, and it is delicious and has high economic value. In addition, its genetic background is clear and its whole genome has been sequenced and published, making Morchella suitable as a model organism for studying the growth and fruiting of large Ascomycetes. The depth of its research has reached the level of molecular biology.

[0003] However, all current experiments related to the growth and fruiting of morels have to cultivate morels on ordinary soil. The composition of soil is extremely complex, and the specific structure of macromolecules such as humic acid in soil organic matter has not been fully resolved to this day. The composition of soil varies from place to place around the world, and its composition will continue to change with specific conditions such as dryness and wetness, season, temperature, and fertilization. Therefore, as a carrier, soil has a high degree of unknown composition, uncontrollability, and low repeatability, resulting in the mycelial network and mushroom primordium of morels growing in this carrier being in different molecular biological states each time, causing uncertainty, low repeatability, and low reliability in basic research. In addition, the extremely complex composition of known and unknown components in the soil adds unnecessary complexity to the research.

[0004] Therefore, in order to make Morchella enter the laboratory from the fields and become a model organism suitable for basic research, especially physiological, biochemical and molecular biology, it is necessary to learn from the research growth carriers used by species with relatively mature scientific research. For example, the more precise molecular biology experiments on rice require the use of vermiculite mixed with Hoagland culture medium as the cultivation matrix, Arabidopsis and tobacco are planted on agar-containing culture medium, and actinomycetes are inoculated on Gao's No. 1 culture medium. All the components of these culture media are known. This provides sufficient certainty, controllability and repeatability for scientific research.

[0005] Therefore, scientific research on morels must also find a new cultivation carrier to replace the traditional natural soil. The simpler the composition of this new matrix, the better, and all the ingredients need to be known and easy to purchase, and preferably do not contain any organic compounds, because organic compounds are prone to various condensations, cyclizations, Maillard reactions, etc. when sterilized at 121°C, generating unknown substances, which have unpredictable interference with scientific research. The formula containing only inorganic substances is simple and clear, and more certain, controllable and repeatable. This will be conducive to the study of the physiological, biochemical and molecular biological mechanisms of the growth and fruiting of morels in the laboratory.

[0006] Therefore, how to provide a fully synthetic mushroom bed substrate for Morchella oleracea is a problem that those skilled in the art urgently need to solve. Summary of the invention

[0007] In view of this, the present invention provides a fully synthetic Morchella fruiting bed substrate, which is suitable for research universities and scientific research institutes to conduct basic research on the physiological and biochemical mechanisms of Morchella cultivation and fruiting and is a cultivation carrier for teaching tools.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A fully synthetic mushroom bed substrate for morels comprises the following raw materials in parts by mass: 99.8500-99.9500 parts of silicon dioxide particles, 0.0250-0.0350 parts of potassium nitrate, 0.0490-0.0590 parts of anhydrous calcium sulfate, 0.0050-0.0150 parts of anhydrous dipotassium hydrogen phosphate, 0.0003-0.0005 parts of anhydrous magnesium sulfate, 0.00005-0.00015 parts of sodium tetraborate, 0.00005-0.00015 parts of anhydrous zinc sulfate, 0.00005-0.00015 parts of anhydrous copper sulfate, 0.00005-0.00015 parts of anhydrous ferric chloride, 0.00005-0.00015 parts of anhydrous manganese sulfate, and 0.00005-0.00015 parts of anhydrous sodium molybdate.

[0010] Preferably: the raw materials include the following parts by mass: 99.9000 parts of silicon dioxide particles, 0.0300 parts of potassium nitrate, 0.0540 parts of anhydrous calcium sulfate, 0.0150 parts of anhydrous dipotassium hydrogen phosphate, 0.0004 parts of anhydrous magnesium sulfate, 0.0001 parts of sodium tetraborate, 0.0001 parts of anhydrous zinc sulfate, 0.0001 parts of anhydrous copper sulfate, 0.0001 parts of anhydrous ferric chloride, 0.0001 parts of anhydrous manganese sulfate, and 0.0001 parts of anhydrous sodium molybdate.

[0011] Preferably, the particle size of the silicon dioxide particles is 150 to 200 meshes.

[0012] The present invention also provides a method for preparing any of the above-mentioned matrices, comprising the following steps: mixing the raw materials uniformly, adding deionized water, then sealing and sterilizing, and cooling for standby use.

[0013] Preferably, the mass ratio of the raw material to deionized water is 100:25.

[0014] Preferred: Sterilization: 121°C, sterilization time 120 min; cooling to below 20°C.

[0015] The present invention provides application of the above-mentioned matrix in agricultural and industrial production.

[0016] Preferred: For basic scientific research and experiments.

[0017] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a fully synthetic mushroom bed substrate for morels, and the technical effect achieved is: the present invention solves the problem that there is no carrier with a simple formula, completely known chemical composition, high controllability and high repeatability in the current scientific research on morels, and the preparation steps are simple, which is suitable for scientific research universities and research institutes to carry out basic research experiments and teaching on the physiology, biochemistry and molecular biology of morel growth and fruiting.

[0018] In addition, compared with the research of the prior art, for example, the invention of ZL202210150334.1, which has been published, adopts a structure in which the outer layer is mainly calcium carbonate (doped with various nanoscale inorganic compound crystals) and the center is silicon dioxide. The preparation is complicated and requires the process of nanoscale material engineering to prepare. It is not suitable for cheap, stable and easy acquisition by laboratories of biological disciplines in universities and scientific research institutes. The present invention does not have a complex microstructure and can be used by directly mixing the materials and adding water for sterilization. It is suitable for cheap, stable and easy acquisition and use by laboratories of biological disciplines in universities and scientific research institutes. Moreover, the invention of ZL202210150334.1, which has been published, mainly uses calcium carbonate as the main raw material other than silicon dioxide, while the present invention mainly uses calcium sulfate. The invention of ZL202210150334.1, which has been published, is calculated by volume, and the amount of silicon dioxide used is similar to that of calcium carbonate. The present invention uses silicon dioxide as the absolute main component of more than 99.9%, and other inorganic compounds account for only a very small part. Since the proportion of calcium carbonate in the invention of ZL202210150334.1 is relatively high, and according to the principle of the invention, calcium carbonate will be continuously dissolved by the organic acid secreted by the morel hyphae, this causes the proportion of calcium carbonate in the entire carrier to become lower and lower over time, and the entire component ratio is constantly changing, which is not suitable for the requirements of relatively stable conditions in physiology, biochemistry and molecular biology. The silicon dioxide of the present invention exceeds about 99.9%, and the silicon dioxide will not be decomposed and metabolized by the morel hyphae and environmental microorganisms, nor will it be dissolved by carbonic acid formed by organic acids and carbon dioxide dissolved in water. It is an inert substance and plays a role of pure background in the carrier, forming a super stable system in which the components of about 99.9% or more remain unchanged throughout the whole process, which is very suitable for the requirements of conditional stability in the fields of physiology, biochemistry and molecular biology in basic scientific research.

[0019] At the same time, compared with various published Morchella cultivation formulas for agricultural production practices retrieved in the past, they all contain complex organic matter such as soil humus, straw, livestock manure, etc., resulting in complex ingredients and containing many organic components that have not yet been resolved by current scientific research. During high-temperature sterilization, organic matter will further undergo various organic reactions and side reactions with uncertainty and complexity, resulting in more complex ingredients. Although the cost is low, it is only suitable for production practice and not suitable for precise scientific research. The present invention is composed entirely of inorganic compounds, does not contain any organic matter, can be sterilized at high temperature for an ultra-long time (121°C, sterilization time 120min, sterilization time longer than 121°C and sterilization time 30min used by most common culture media) without decomposition and side reactions, and is very stable.

[0020] The present invention avoids these problems and is suitable for precise scientific research. DETAILED DESCRIPTION

[0021] The following will be described clearly and completely in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The embodiment of the invention discloses a fully synthetic mushroom bed substrate for Morchella oleracea.

[0023] The raw materials required in the examples were all commercially available, all chemicals were analytically pure or above, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0024] Example 1

[0025] A fully synthetic mushroom bed substrate for morels comprises the following raw materials in parts by mass: 99.8500 parts of silicon dioxide particles, 0.0250 parts of potassium nitrate, 0.0490 parts of anhydrous calcium sulfate, 0.0050 parts of anhydrous dipotassium hydrogen phosphate, 0.0003 parts of anhydrous magnesium sulfate, 0.00005 parts of sodium tetraborate, 0.00005 parts of anhydrous zinc sulfate, 0.00005 parts of anhydrous copper sulfate, 0.00005 parts of anhydrous ferric chloride, 0.00005 parts of anhydrous manganese sulfate and 0.00005 parts of anhydrous sodium molybdate.

[0026] To further optimize the technical solution: the particle size of the silica particles is 150 mesh.

[0027] Example 2

[0028] A fully synthetic mushroom bed substrate for morels comprises the following raw materials in parts by mass: 99.9000 parts of silicon dioxide particles, 0.0300 parts of potassium nitrate, 0.0540 parts of anhydrous calcium sulfate, 0.0150 parts of anhydrous dipotassium hydrogen phosphate, 0.0004 parts of anhydrous magnesium sulfate, 0.0001 parts of sodium tetraborate, 0.0001 parts of anhydrous zinc sulfate, 0.0001 parts of anhydrous copper sulfate, 0.0001 parts of anhydrous ferric chloride, 0.0001 parts of anhydrous manganese sulfate and 0.0001 parts of anhydrous sodium molybdate.

[0029] To further optimize the technical solution: the particle size of the silica particles is 180 mesh.

[0030] Example 3

[0031] A fully synthetic mushroom bed substrate for morels comprises the following raw materials in parts by mass: 99.9500 parts of silicon dioxide particles, 0.0350 parts of potassium nitrate, 0.0590 parts of anhydrous calcium sulfate, 0.0150 parts of anhydrous dipotassium hydrogen phosphate, 0.0005 parts of anhydrous magnesium sulfate, 0.00015 parts of sodium tetraborate, 0.00015 parts of anhydrous zinc sulfate, 0.00015 parts of anhydrous copper sulfate, 0.00015 parts of anhydrous ferric chloride, 0.00015 parts of anhydrous manganese sulfate and 0.00015 parts of anhydrous sodium molybdate.

[0032] To further optimize the technical solution: the particle size of the silica particles is 200 mesh.

[0033] Example 4

[0034] The preparation method of the matrix comprises the following steps:

[0035] Mix the raw materials evenly, add deionized water, then seal and sterilize (pack in polypropylene bags), cool and set aside.

[0036] To further optimize the technical solution: the mass ratio of raw materials to deionized water is 100:25.

[0037] To further optimize the technical solution: Sterilization: 121℃, sterilization time 120min; cool down to below 20℃.

[0038] Technical Effects

[0039] Specific applications:

[0040] The matrix prepared in Example 1 was used for transcriptome research on the growth and fruiting process of Morchella mycelium

[0041] 1) Add 300 grams of Morchella sclerotia (this embodiment: the sclerotia in the "Chuan Morchella No. 6" strain produced by Sichuan Jindi Tianlingjian Company) to every 300 kg of sterilized wet carrier, mix well, and pack in 3 trays with drainage holes at the bottom, each tray contains 100 kg, and a total of 3 trays are used as 3 parallel repetitions of the basic scientific research routine setting. When the Morchella mycelium germinates and covers the entire carrier, refer to the conventional cultivation technology of Morchella published in Invention 201510532171.3 to make an exogenous nutrient bag, and place the exogenous nutrient bag on the surface of the carrier of the present invention according to the usage of the invention, so that the mycelium drills into the nutrient bag to absorb nutrients. At the same time, the carrier is sampled once every 10 days (until the mushrooms are fully grown and no new fruiting bodies are produced) with a micro soil sampler.

[0042] 2) After quick freezing with liquid nitrogen, the total RNA of the Morchella hyphae densely distributed in the carrier was extracted (the reagents included: Shanghai Bioengineering Fungal Total RNA Rapid Extraction Kit (Product No. B518629), followed the steps in the instructions), and the transcriptome was determined by conventional analytical methods (conventional Illumina platform transcriptome sequencing).

[0043] The experimental results show that: due to the strong stability of the carrier of the present invention and the small amount of interfering substances, the total RNA sample of Morchella preserved therein is less damaged, OD260 / 280=1.8-2.2, OD260 / 230≥2.0; while natural soil contains a large amount of RNA decomposing enzymes, and it is difficult to extract high-quality total RNA of Morchella in an experiment of cultivating Morchella in natural soil, OD260 / 280<1.8, OD260 / 230<2.0, and the bands are blurred and severely degraded, resulting in unsatisfactory experimental results.

[0044] The matrix prepared in Example 2 was used for proteomic research on the mycelial growth and fruiting process of Morchella

[0045] Step 1) is the same as the transcriptome study, except that the Morchella sclerotia are derived from the sclerotia of the "G10" strain produced by Yibin Zhengde Biotechnology Co., Ltd.;

[0046] 2) After quick freezing with liquid nitrogen, the total protein of the densely distributed Morchella hyphae in the carrier was extracted, and the proteome was determined by conventional analytical methods (conventional proteome sequencing based on chromatography and mass spectrometry).

[0047] The experimental results show that: due to the strong stability of the carrier of the present invention and the small amount of interfering substances, the total protein sample of Morchella preserved therein is less damaged, and the total amount of protein extracted is ≥1 microgram. However, natural soil not only has a large amount of protein combined with humus, but also contains proteases. In the experiment of cultivating Morchella in natural soil, it is not only difficult to extract high-quality total protein of Morchella, but also it will be degraded and destroyed by soil proteases, and the total amount of protein extracted is <1 microgram, resulting in unsatisfactory experimental results.

[0048] The matrix prepared in Example 3 was used for metabolomic study of the mycelial growth and fruiting process of Morchella edulis. Step 1) The same as the transcriptomic study;

[0049] 2) After quick freezing with liquid nitrogen, the total metabolites of the densely distributed Morchella mycelium in the carrier were extracted, and the metabolome was determined by conventional analytical methods (conventional metabolome testing based on chromatography and mass spectrometry).

[0050] The experimental results show that: due to the strong stability of the carrier of the present invention and the small amount of interfering substances, the metabolome samples of Morchella preserved therein are less damaged, and the product peaks are clearly distinguishable. However, natural soil itself contains a variety of organic acids and other substances that are easily confused with Morchella metabolites. The metabolome of Morchella extracted from the experiment of cultivating Morchella in natural soil will be seriously interfered by the small molecular organic matter contained in the soil itself, and the product peaks are blurred and difficult to distinguish, resulting in unsatisfactory experimental results.

[0051] The matrix prepared in Example 2 was used to evaluate the effect of the herbicide "glyphosate" on the fruiting of Morchella

[0052] 1500 kg of sterilized wet carrier was added with 1500 g of Morchella sclerotia (sclerotia of the "G10" strain produced by Yibin Zhengde Biotechnology Co., Ltd.) and mixed. Then the mixture with a total weight of about 1500 kg was divided into 15 trays with drainage holes at the bottom, divided into 5 groups, 3 in each group as 3 parallel replicates of the basic scientific research routine setting. 0 dose, 1 dose, 2 doses, 3 doses, and 4 doses of the herbicide "glyphosate" were added to the 5 groups respectively. After the mycelium of the morel mushrooms germinates and covers the entire carrier, an exogenous nutrient bag is made by referring to the conventional cultivation technology of morels disclosed in Invention 201510532171.3. The exogenous nutrient bag is placed on the surface of the carrier of the present invention according to the usage of the invention, and the mycelium is allowed to drill into the nutrient bag to absorb nutrients until the mushrooms are fully grown and no new fruiting bodies are produced. The yield of fruiting bodies of morels is counted, and it is found that the higher the application rate of the herbicide "glyphosate", the lower the yield of morels, which proves that "glyphosate" has an inhibitory effect on the fruiting of morels.

[0053] Since the carrier of the present invention is mostly composed of silicon dioxide, with only a small amount of inorganic minerals, the known components will not react with "glyphosate" to generate other substances (for example, various condensations, cyclizations, Maillard reactions, etc. will not easily occur to generate unknown substances), nor will it adsorb "glyphosate". However, natural soil contains some chemical substances that can react with "glyphosate" to reduce its effect, and macromolecules such as humic acid can also adsorb "glyphosate" to reduce its effect, which interferes with the experimental results, has poor repeatability, and causes great errors in the experimental results.

[0054] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0055] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fully synthetic mushroom bed substrate for Morchella edulis, characterized in that: The invention comprises the following raw materials in parts by weight: 99.8500~99.9500 parts of silicon dioxide particles, 0.0250~0.0350 parts of potassium nitrate, 0.0490~0.0590 parts of anhydrous calcium sulfate, 0.0050~0.0150 parts of anhydrous dipotassium hydrogen phosphate, 0.0003~0.0005 parts of anhydrous magnesium sulfate, 0.00005~0.00015 parts of sodium tetraborate, 0.00005~0.00015 parts of anhydrous zinc sulfate, 0.00005~0.00015 parts of anhydrous copper sulfate, 0.00005~0.00015 parts of anhydrous ferric chloride, 0.00005~0.00015 parts of anhydrous manganese sulfate, and 0.00005~0.00015 parts of anhydrous sodium molybdate.

2. The matrix according to claim 1, characterized in that It includes the following raw materials in parts by mass: 99.9000 parts of silicon dioxide particles, 0.0300 parts of potassium nitrate, 0.0540 parts of anhydrous calcium sulfate, 0.0150 parts of anhydrous dipotassium hydrogen phosphate, 0.0004 parts of anhydrous magnesium sulfate, 0.0001 parts of sodium tetraborate, 0.0001 parts of anhydrous zinc sulfate, 0.0001 parts of anhydrous copper sulfate, 0.0001 parts of anhydrous ferric chloride, 0.0001 parts of anhydrous manganese sulfate, and 0.0001 parts of anhydrous sodium molybdate.

3. The matrix according to claim 2, characterized in that The particle size of the silicon dioxide particles is 150-200 meshes.

4. The method for preparing a matrix according to any one of claims 1 to 3, characterized in that: The following steps are involved: Mix the raw materials evenly, add deionized water, then seal and sterilize, cool and set aside; The mass ratio of the raw material to deionized water is 100:25; The sterilization is carried out at 121°C for 120 min; the temperature is lowered to below 20°C.

5. Use of the matrix according to claim 3 in agricultural and industrial production.

6. The use according to claim 5, characterized in that For basic scientific research and experiments.