CN110591923A - Method for quickly obtaining mass DSE mycelium inoculant - Google Patents
Method for quickly obtaining mass DSE mycelium inoculant Download PDFInfo
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Abstract
The invention discloses a method for quickly obtaining a large amount of DSE mycelium inoculants, which comprises the steps of inoculating DSE mycelium into a culture medium of wheat bran-vermiculite for amplification culture, and establishing a non-infectious and non-interfering quick culture system under a dark condition.
Description
Technical Field
The invention relates to a method for quickly obtaining a large amount of DSE mycelium inoculants, belonging to the fields of microbiology, biotechnology and environmental management.
Background
Dark septate endophytic fungi (DSE) are the main group of plant root endophytic fungi, and are mainly characterized in that hyphae are darker in color and have obvious membranes, and the hyphae are colonized in the epidermis and cortex of healthy plant roots and even in cells or intercellular spaces of vascular bundle tissues and form symbionts with plants without causing plant diseases. DSEs are widely distributed and encompass different habitats: from coastal beaches to inland plateau mountains, from tropical and temperate zones to frozen regions and north and south regions. Colonization among these different habitat plants suggests that DSEs have little or no host specificity and that DSEs promote uptake and utilization of organic nutrients by the host and thus further promote plant growth. After the DSE is colonized on the plant root system, the stress resistance of a host can be improved, and the slime hyphae extending from the plant root of the DSE can help the plant to maintain the transportation of water and nutrition in a drought environment; melanin in the fungal cell wall can enhance the mechanical strength of the cell wall, endow the cell with heat resistance and radiation resistance, and play a certain role in the change of the enhancement of the stress resistance of host plants. Because DSE has stronger tolerance to adverse environment, its bioremediation effect in heavy metal contaminated soil also becomes a hotspot of research in recent years. Therefore, it is important to explore DSEs for improving the plant resistance to drought and stress such as heavy metal pollution, inducing plants to produce systemic resistance, and resisting pathogenic organism stress, and it is important to obtain a large amount of pure mycelia rapidly. Most of the existing methods are plate or liquid culture, which is easy to pollute, long in time consumption and low in yield. Therefore, it is urgent to establish a DSE mycelium system which can rapidly obtain a large amount of stable and efficient culture medium with definite and uniform components and no pollution.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method which is stable, efficient and pollution-free, has simple culture medium components, can obtain a large amount of mycelium inoculants through simple solid fermentation, is easy to obtain and has low cost.
The invention provides a method for quickly obtaining a large amount of DSE mycelium inoculants, which mainly takes DSE strains and wheat bran-vermiculite as materials, inoculates the DSE mycelium to a culture medium containing wheat bran-vermiculite for expanded culture, establishes a quick culture system without mixed bacteria interference under dark conditions, and realizes the following concrete steps:
(1) activating the DSE strain, inoculating hyphae into a culture dish containing a PDA culture medium, carrying out dark inverted culture at the temperature of 27-29 ℃ for 14-16 days, and punching a plurality of hypha blocks along the edges of the mycelium by using a puncher for later use;
(2) mixing wheat bran and garden vermiculite in equal volume to obtain a mixture, adding tap water, transferring the uniformly mixed wheat bran-vermiculite mixed matrix into a triangular flask, and sealing the opening with an aseptic PVA (polyvinyl alcohol) membrane for sterilization;
(3) inoculating the hypha blocks in the step (1) into triangular flasks filled with the culture medium in the step (2) in an aseptic workbench, inoculating 3-5 hypha blocks in each triangular flask, crushing and uniformly mixing the inoculated hypha blocks by using an inoculating needle, sealing by using an aseptic PVA film, placing the triangular flasks in a dark environment at 27-29 ℃, culturing without shaking for the first 10 days, shaking the culture medium in the triangular flasks to mix the culture medium in the bottles every 2 days from the 11 th day, and growing hyphae in the triangular flasks after 20 days to obtain a large amount of DSE mycelia.
The DSE strain in the step (1) is DSE fungus Exophiala piscicola (Exophiala, H93) which is preserved in China center for agricultural biological strain preservation, and the strain number is as follows: ACCC 32496.
The preparation method of the PDA culture medium in the step (1) is as follows: 200g of potato, 20g of glucose, 20g of agar and 1L of tap water in constant volume.
The mycelium blocks in the step (1) are round blocks with the diameter of 5-7 mm.
The wheat bran in the step (2) is wheat bran or coarse bran.
The mass ratio of the mixture in the step (2) to tap water is 1: 1.4-1.6.
And (2) sterilizing, namely treating at 121 ℃ for 20-25 min, and naturally cooling to room temperature after sterilizing.
The invention has the beneficial effects that: a solid culture system for rapidly obtaining a large amount of DSE mycelium inoculants is innovatively established, the method is simple, stable and efficient, the matrix component is single, the precondition is provided for further researching the interaction of DSE and plants in a large amount and repairing heavy metal environmental pollution by using DSE, and meanwhile, the reference is provided for the large amount of other fungus mycelium inoculants.
Drawings
FIG. 1 shows the state of the culture in a triangular flask of example 1 after 20 days;
FIG. 2 is a diagram showing the state after 30 days of culture in a petri dish and a test tube in comparative example 1;
FIG. 3 is a photograph of the growth of Zea mays Hull No. 4;
FIG. 4 is a micrograph of DSE Exophiala piscivora H93 after colonization of maize roots;
FIG. 5 is a photomicrograph of DSE Exophiala piscicola H93 after colonization of maize roots.
Detailed Description
The invention will be further illustrated with reference to specific examples, but the scope of the invention is not limited thereto. The DSE strain used in the embodiment of the invention is DSE fungus Exophiala piscicola (Exophiala piscicola, H93), is stored in China center for agricultural biological culture Collection, and has the strain number: ACCC 32496.
Example 1
A method for rapidly obtaining a large amount of DSE mycelium inoculants comprises the following specific steps:
(1) preparation of DSE pinophyta piscivora H93 inoculant: after the strain DSE Exophiala piscicola H93 is activated, hyphae are inoculated into a culture dish containing a PDA culture medium, and the preparation method of the PDA culture medium is as follows: preparing 200g of potato, 20g of glucose, 20g of agar and tap water to a constant volume for 1L, performing dark inverted culture at 27 ℃ for 16 days, and punching a plurality of hypha blocks along the edge of a mycelium by using a puncher to serve as an inoculant for later use, wherein the hypha blocks are round blocks with the diameter of 5 mm;
(2) preparation of solid medium: mixing wheat bran for feed and garden vermiculite in equal volume to obtain a mixture, adding tap water, uniformly mixing, wherein the mass ratio of the mixture to the tap water is 1:1.4, transferring the uniformly mixed wheat bran-vermiculite mixed matrix into 250mL triangular bottles, filling 150mL of the uniformly mixed wheat bran-vermiculite mixed matrix into each triangular bottle, sealing the triangular bottles by using a sterile PVA film, sterilizing at 121 ℃ for 20min, and naturally cooling at room temperature;
(3) mass fermentation of mycelia: inoculating the prepared DSE Exophiala pisciophila H93 mycelium blocks in the step (1) into the culture medium cooled in the step (2) in an aseptic workbench, inoculating 5 mycelium blocks with the diameter of 5mm into each triangular bottle, smashing and uniformly mixing the inoculated mycelium blocks by using an inoculating needle, sealing by using an aseptic PVA film, placing the triangular bottles in a dark environment at 27 ℃ for 20 days, carrying out no shaking for the first 10 days so as not to damage the overall structure of the mycelium, shaking the triangular bottles every 2 days from the 11 th day after a small amount of mycelium is grown so as to uniformly distribute the mycelium and absorb sufficient nutrition, and basically fully growing the triangular bottles (as shown in figure 1) after 20 days when the mycelium and wheat bran are mixed into a whole, wherein the fresh weight of the mycelium in each triangular bottle is 46.248g, thus obtaining a large amount of DSE Exophiala pisciophila H93 mycelium.
The DNA of the mycelium cultured by the wheat bran is extracted by adopting a urea extraction method, the DNA is sent to a sequencing company for sequence determination after detection, the obtained sequence is subjected to Blast comparison with the sequence on NCBI, the similarity is 100 percent, and the strain is proved to have no mutation in the rapid expanding culture process, short time and large quantity.
Example 2
A method for rapidly obtaining a large amount of DSE mycelium inoculants comprises the following specific steps:
(1) preparation of DSE pinophyta piscivora H93 inoculant: after the strain DSE Exophiala piscicola H93 is activated, hyphae are inoculated into a culture dish containing a PDA culture medium, and the preparation method of the PDA culture medium is as follows: preparing 200g of potato, 20g of glucose, 20g of agar and tap water to a constant volume for 1L, carrying out dark inverted culture at 28 ℃ for 15 days, and punching a plurality of hypha blocks along the edge of a mycelium by using a puncher for later use as an inoculant, wherein the hypha blocks are round blocks with the diameter of 6 mm;
(2) preparation of solid medium: mixing wheat bran for feed and garden vermiculite in equal volume to obtain a mixture, adding tap water, uniformly mixing, wherein the mass ratio of the mixture to the tap water is 1:1.5, transferring the uniformly mixed wheat bran-vermiculite mixed matrix into 250mL triangular bottles, filling 160mL of the uniformly mixed wheat bran-vermiculite mixed matrix into each triangular bottle, sealing the triangular bottles by using a sterile PVA film, sterilizing at 121 ℃ for 22min, and naturally cooling at room temperature;
(3) mass fermentation of mycelia: inoculating the prepared DSE Exophiala pisciophila H93 hypha blocks in the step (1) into the culture medium cooled in the step (2), inoculating 4 hypha blocks with the diameter of 6mm into each triangular bottle, smashing and uniformly mixing the inoculated hypha blocks by using an inoculating needle, sealing by using an aseptic PVA film, placing the triangular bottles in dark at 28 ℃ for 20 days, culturing for the first 10 days without shaking to avoid damaging the integral structure of the hypha, after a small amount of hypha grows out, shaking the triangular bottles every 2 days from the 11 th day to uniformly mix the culture medium in the bottles so that the hypha is uniformly distributed and absorb sufficient nutrition, and after 20 days, mixing the hypha and wheat bran into a whole, basically fully growing the triangular bottles, wherein the fresh weight of the hypha in each triangular bottle is 46.248g, so that a large amount of DSE Exophiala H93 hypha is obtained.
The DNA of the mycelium cultured by the wheat bran is extracted by adopting a urea extraction method, the DNA is sent to a sequencing company for sequence determination after detection, the obtained sequence is subjected to Blast comparison with the sequence on NCBI, the similarity is 100 percent, and the strain is proved to have no mutation in the rapid expanding culture process, short time and large quantity.
Example 3
A method for rapidly obtaining a large amount of DSE mycelium inoculants comprises the following specific steps:
(1) preparation of DSE pinophyta piscivora H93 inoculant: after the strain DSE Exophiala piscicola H93 is activated, hyphae are inoculated into a culture dish containing a PDA culture medium, and the preparation method of the PDA culture medium is as follows: preparing 200g of potato, 20g of glucose, 20g of agar and tap water to a constant volume for 1L, performing dark inverted culture at 29 ℃ for 14 days, and punching a plurality of hypha blocks along the edge of a mycelium by using a puncher to serve as an inoculant for later use, wherein the hypha blocks are round blocks with the diameter of 7 mm;
(2) preparation of solid medium: mixing coarse bran for feed with garden vermiculite in equal volume to obtain a mixture, adding tap water, uniformly mixing, wherein the mass ratio of the mixture to the tap water is 1:1.6, transferring the uniformly mixed wheat bran-vermiculite mixed matrix into 250mL triangular bottles, filling 150mL of the uniformly mixed wheat bran-vermiculite mixed matrix into each triangular bottle, sealing the triangular bottles by using an aseptic PVA film, sterilizing at 121 ℃ for 25min, and naturally cooling at room temperature;
(3) mass fermentation of mycelia: inoculating the prepared DSE Exophiala pisciophila H93 hypha blocks in the step (1) into the culture medium cooled in the step (2), inoculating 3 hypha blocks with the diameter of 7mm into each triangular bottle, smashing and uniformly mixing the inoculated hypha blocks by using an inoculating needle, sealing by using an aseptic PVA film, placing the triangular bottles in the dark at 29 ℃ for 20 days, culturing for the first 10 days without shaking to avoid damaging the integral structure of the hypha, after a small amount of hypha grows out, shaking the triangular bottles every 2 days from the 11 th day to uniformly mix the culture medium in the bottles so that the hypha is uniformly distributed and absorb sufficient nutrition, mixing the hypha and wheat bran into a whole after 20 days, basically fully growing the triangular bottles, and ensuring that the fresh weight of the hypha in each triangular bottle is 46.248g, thereby obtaining a large amount of DSE Exophiala pisciophila H93 hypha.
The DNA of the mycelium cultured by the wheat bran is extracted by adopting a urea extraction method, the DNA is sent to a sequencing company for sequence determination after detection, the obtained sequence is subjected to Blast comparison with the sequence on NCBI, the similarity is 100 percent, and the strain is proved to have no mutation in the rapid expanding culture process, short time and large quantity.
Comparative example 1
Preparing 1L PDA culture medium by using 200g of potatoes, 20g of glucose, 20g of agar and tap water to fix the volume, uniformly pouring the PDA culture medium into a test tube and a culture dish, sterilizing at high temperature for 20min, naturally cooling at room temperature, then inoculating the activated DSE Exophiala pisciophila H93 strain into the test tube and the culture dish containing the PDA culture medium by using an inoculating needle respectively, placing in a dark inversion culture at 27-29 ℃, culturing for 30 days, wherein the cultured mycelia in each test tube or culture dish are shown in figure 2, the fresh weight of the mycelia cultured in each test tube is 0.688g, the fresh weight of the mycelia cultured in each culture dish is 1.290g and is far less than the number of the mycelia cultured in examples 1-3, corn 500 plants are planted in a field DSE inoculation experiment, the same inoculation amount only needs 216 triangular flasks of wheat bran-vermiculite solid culture mycelia, if the test tubes or the culture dishes are used for culturing, 7752 culture dishes and 14535 test tubes are needed, the workload is increased, and the cost is increased.
Comparative example 2
Preparing 1L PDA culture medium by using 200g of potatoes, 20g of glucose, 20g of agar and tap water at constant volume, uniformly pouring the PDA culture medium into a triangular flask with the same volume as that in the example 1-3, filling the triangular flask into 150mL of matrix, sterilizing at high temperature for 20min, naturally cooling at room temperature, inoculating a DSE Exophiala piscicola H93 hypha block with the same volume as that in the example 1 into the triangular flask containing the PDA culture medium by using an inoculating needle, and placing the triangular flask at 27-29 ℃ for dark inverted culture; after 30 days of culture, the number of mycelia of DSE Exophiala pisciophila H93 cultured in PDA medium in the triangular flask was much smaller than that cultured with wheat bran-vermiculite mixed matrix solid in the same volume of triangular flask.
Establishment of symbiotic system of DSE Exophiala piscivora H93 and corn:
selecting corn seeds (Huiyan No. four) with consistent size, placing the corn seeds in an ethanol solution with the mass fraction of 75%, soaking for 5min, taking out the seeds, and cleaning for 3 times by using sterile water; placing the seeds in a 10% sodium hypochlorite solution, soaking for 10min, taking out the seeds, and washing for 3 times by using sterile water; placing the seeds with the sterilized surfaces on sterile moist filter paper, placing 2-3 seeds in each bottle, culturing in the dark at 22-23 ℃ for 3-4 days, germinating, transferring into an illumination incubator, placing the seeds into the illumination incubator, wherein the illumination intensity is 18000Lux, the illumination time and the culture temperature are 16/8H, 25/22 ℃ and the relative air humidity is 70%, culturing for 5-7 days, growing into 6-8 cm sterile seedlings, selecting sterile seedlings with consistent growth, placing 30g of vermiculite into each nutrition bag, planting one sterile seedling, placing 20g of the DSE Exophiala piscicola H93 block inoculant expanded in the embodiment 1-3 into each corn sterile seedling, adding 20g of vermiculite, adding sterile purified water to just soak the seedlings, placing the seedlings into the illumination incubator, wherein the illumination intensity is 18000Lux, the illumination time and the culture temperature are 16/8H, 25/22 ℃ and the relative air humidity is 70%, in the culture process, 20mL of sterile purified water is added every 3 days, and the corn root is just soaked and wetted each time.
After culturing for 20 days (as shown in figure 3), taking out the maize seedlings, washing the roots of the maize seedlings with deionized water, putting lateral roots of the maize seedlings into a test tube, adding a KOH solution with the mass concentration of 10% to completely immerse the roots, putting the test tube into a water bath kettle to dissociate at 90 ℃ until the root samples are transparent, performing lactic acid neutralization, acid fuchsin dyeing and lactic acid glycerol decoloring, then preparing slices, and observing the colonization condition of DSE under an OLYPUS-BX51 microscope. FIG. 4 is a micrograph of DSE Exophiala piscivora H93 after colonization of maize roots; FIG. 5 is a photomicrograph of DSE Exophiala piscicola H93 after colonization of the corn roots; it can be seen that there are a number of spores and hyphae colonizing the corn roots and forming microsclerotia, a typical structural feature of DSEs, with a distinct septum in the silk.
Meanwhile, taking a part of lateral roots of the maize seedlings, cutting a root sample into segments of about 0.5cm by using sterile scissors, randomly selecting 20 segments, pasting the segments on a PDA culture medium, carrying out 5 segments on each culture dish, carrying out dark inversion culture on all the culture dishes at 28 ℃ for 25 days, carrying out a tieback experiment, picking mycelia growing near the root segments in the culture dishes by using an inoculating needle after 25 days, placing the mycelia on a glass slide with a drop of water to prepare a glass slide, and observing the colonization condition of the tieback experiment mycelia under an OLYPUS-BX51 microscope. The observation of a slide made of mycelium cultured from a corn root sample in a back grafting experiment under a microscope also only separates a DSE strain with the same morphological characteristics as an inoculant, which indicates that the DSE Exophiala piscicola H93 expanded by the method is successfully colonized at the corn root, and the bacteria inoculated from the colonized root sample are only the mycelium of the DSE Exophiala piscicola H93.
The mycelia expanded in comparative examples 1 and 2 were colonized on the corn roots according to the above-mentioned method, and the phenomena shown in FIGS. 4 and 5 were observed under a microscope after the mycelia were colonized on the corn, indicating that the cultured mycelia were identical in performance.
The morphological characteristics and the molecular analysis result are integrated, so that the method for quickly obtaining the massive dark septate endophytic fungi (DSE) mycelium inoculant is successfully constructed, is quicker, simpler and more efficient than the method for culturing the DSE mycelium inoculant by using the conventional test tube culture, culture dish culture and conventional culture medium, is simple and convenient to operate, has less workload, and provides a simple method for batch production of the DSE mycelium inoculant or field test inoculation of DSE mycelium.
Claims (5)
1. A method for rapidly obtaining a large amount of DSE mycelium inoculant is characterized by comprising the following specific steps:
(1) activating the DSE strain, inoculating hyphae into a culture dish containing a PDA culture medium, carrying out dark inverted culture at the temperature of 27-29 ℃ for 14-16 days, and taking hypha blocks along the edges of the mycelium for later use;
(2) mixing wheat bran and garden vermiculite in equal volume to obtain a mixture, adding tap water, transferring the uniformly mixed wheat bran-vermiculite mixed matrix into a triangular flask, and sealing the opening with an aseptic PVA (polyvinyl alcohol) membrane for sterilization;
(3) inoculating the hypha blocks in the step (1) into triangular flasks filled with the culture medium in the step (2) in an aseptic workbench, inoculating 3-5 hypha blocks in each triangular flask, crushing and uniformly mixing the inoculated hypha blocks by using an inoculating needle, sealing the triangular flasks by using a sterile PVA film, placing the triangular flasks in a dark environment at 27-29 ℃ for culturing without shaking for the first 10 days, shaking the culture medium in the triangular flasks and the uniform flasks every 2 days from the 11 th day, and growing hyphae in the triangular flasks after 20 days to obtain a large amount of DSE mycelia.
2. The method for rapidly obtaining a mass of DSE mycelium inoculant according to claim 1, wherein the mycelium blocks of step (1) are round blocks with a diameter of 5-7 mm.
3. The method for rapidly obtaining a mass of DSE mycelium inoculant according to claim 1, wherein the bran of step (2) is wheat bran or semolina bran.
4. The method for rapidly obtaining a large amount of DSE mycelium inoculant according to claim 1, wherein the mass ratio of the mixture in step (2) to the tap water is 1: 1.4-1.6.
5. The method for rapidly obtaining a mass of DSE mycelium inoculant according to claim 1, wherein the step (2) of sterilizing is performed at 121 ℃ for 20-25 min, and the mixture is naturally cooled to room temperature after sterilizing.
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Application publication date: 20191220 |