CN110604004A - Method for separating strains by dried morchella sporocarp - Google Patents
Method for separating strains by dried morchella sporocarp Download PDFInfo
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- CN110604004A CN110604004A CN201910894907.XA CN201910894907A CN110604004A CN 110604004 A CN110604004 A CN 110604004A CN 201910894907 A CN201910894907 A CN 201910894907A CN 110604004 A CN110604004 A CN 110604004A
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- morchella
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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Abstract
The invention discloses a method for separating strains through dried morchella sporocarp, which comprises the steps of firstly soaking the dried morchella sporocarp in 5% glucose solution, then soaking the morchella sporocarp in 75% alcohol for 2-8s, then soaking the morchella sporocarp in 5% glucose solution for 3-12s, then cutting the morchella sporocarp into small tissue blocks and inoculating the tissue blocks on a PDA culture medium containing streptomycin, wherein the final concentration of the streptomycin is 300 mu g/L, culturing the tissue blocks at a constant temperature of 18-22 ℃ for 2-3d, and finally picking tip hyphae and inoculating the tip hyphae on a new PDA culture medium containing the streptomycin for purification for 1-2 times. The invention has the advantages that: (1) hyphae can germinate rapidly, and hyphae can grow after 3d of culture; (2) the purification times are less, and only 1-2 times of purification is needed; (3) only one culture medium needs to be prepared, and the operation is simple.
Description
Technical Field
The invention relates to a method for separating toadstool strains, in particular to a method for separating the strains through dried sporocarp of toadstool, and belongs to the technical field of microorganisms.
Background
Morchella esculenta is a rare edible and medicinal fungus, and although artificial cultivation is realized at present, the yield is extremely unstable, so that the price of the morchella esculenta is always high.
The acquisition of the strain is a key step in the production of the morchella esculenta. Currently, morchella separation mainly uses fresh sporocarp as a separation material, one is a spore separation method, and the other is a tissue separation method.
The fresh morchella sporocarp is easy to rot, so that the strain is obtained in a very strong seasonal manner, and the strain is not easy to obtain if the fruiting season is missed.
In addition, fresh fruit bodies are used as separation materials, and morchella esculenta is taken from a field and carries a large amount of mixed bacteria including bacteria and fungi, so that the strains are easily polluted by the mixed bacteria when being separated.
Chinese patent CN104686196A discloses a method for preserving and separating morchella strains by drying fruit bodies in the shade, which records the method comprising the following steps:
a. and (3) strain preservation: selecting mature, healthy and strong morchella esculenta which is free from diseases and insect pests, full in pileus, fully expanded in pileus upper edges and pits, milky in stipe, normal in shape and moderate in volume in 3-4 months, spreading and placing in a place with good ventilation, 10-15 ℃ and dark and dry, and turning over at intervals until the sporocarp is completely dried; packaging with paper bags, and storing in dry dark and cool place;
b. strain separation: when strains need to be used, taking 0.5-1cm under aseptic condition2Drying fruiting body pileus tissue in shade, soaking in sterile water, softening completely, placing into sterilized mortar, adding sterile water 1ml, grinding to obtain tissue suspension, and inoculating the tissue suspension in the center of wheat grain culture medium; culturing at 15-25 deg.C in dark for 10-20 days until the morchella aerial mycelium or sclerotia grows out, selecting aerial mycelium or sclerotia with inoculating needle, inoculating to PDA culture medium, culturing again, and repeatedly purifying on PDA culture mediumCulturing until pollution-free strains are obtained.
However, in the process of storing and separating the morchella strains by drying the fruiting bodies in the shade, the morchella needs to be cultured for 10-20 days at 15-25 ℃ in the dark to grow aerial hypha or sclerotium, the culture time is long, and the production is influenced.
In addition, this method requires preparation of a wheat grain medium in addition to a PDA medium in the process of storing and isolating morchella species by drying fruit bodies in the shade, and requires a large number of purification times, which makes the operation complicated.
Drawings
FIG. 1 is a photograph of tissue block culture 3d in example 1;
FIG. 2 is a photograph of mycelia obtained after 2-time purification of the mycelia in FIG. 1;
FIG. 3 is a photograph of tissue block culture 3d in example 2;
FIG. 4 is a photograph of the mycelium obtained after 1 purification of the mycelium in FIG. 3;
FIG. 5 is a photograph of tissue block culture 3d in example 3;
FIG. 6 is a photograph of mycelia obtained after 2-time purification of the mycelia in FIG. 5;
FIG. 7 is a photograph of tissue block culture 3d in example 4;
FIG. 8 is a photograph of mycelia obtained after 2-time purification of the mycelia in FIG. 7;
FIG. 9 is a photograph of tissue block culture 3d in example 5;
FIG. 10 is a photograph of tissue block culture 3d in example 6.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the method which has the advantages that hyphae can be rapidly germinated when strains are separated from dried morchella esculenta sporocarp, the purification times are few, and the operation is simple.
In order to achieve the above object, the present invention adopts the following technical solutions:
a method for separating strains through dried morchella sporocarp is characterized by comprising the following steps:
(I) collecting and drying Morchella sporocarp
Selecting high-quality morchella in the morchella harvesting season, airing the morchella until the fruiting bodies are completely dried, and then putting the morchella into a sample bag to be stored in a refrigerator at 4 ℃ for later use;
(II) pretreatment of fruit body
Soaking dried morchella esculenta fruiting body in 5% glucose solution, soaking in 75% ethanol solution for 2-8s after the fruiting body is completely softened, taking out, soaking in 5% glucose solution for 3-12s, sucking out the glucose solution on the morchella esculenta with sterile gauze, and rapidly baking the morchella esculenta above the flame of an alcohol lamp for 3 times;
(III) isolation of the bacterial species
Cutting the processed morchella sporophore into small tissue blocks, inoculating on PDA culture medium containing streptomycin, culturing at constant temperature for 2-3 days, picking tip mycelium, inoculating on new PDA culture medium containing streptomycin, and purifying for 1-2 times.
The method for separating the strains by drying the morchella esculenta sporocarp is characterized in that in the step (I), the morchella esculenta is placed in a dry and well-ventilated environment for airing.
The method for separating strains by using dried morchella sporocarp is characterized in that in the step (three), the processed morchella sporocarp is cut into small tissue blocks with the size of 0.5 cm.
The method for separating the strains by drying the morchella esculenta sporocarp is characterized in that in the third step, the culture temperature is 18-22 ℃.
The invention has the advantages that:
(1) firstly, a 5% glucose solution is used for soaking and drying sporocarp, so that the normal form of the sporocarp is maintained, then 75% alcohol is used for treating morchella sporocarp soaked in the glucose solution, so that not only can the mixed bacteria on the surface of the sporocarp be effectively killed, but also the germination activity of sporocarp cells is not lost, and finally 5% glucose solution is used for soaking, so that the alcohol residue is eluted, and the nutrient substances required by hypha germination are ensured, therefore, the hypha can germinate rapidly, and the hypha can grow after being cultured for 3 days;
(2) the morchella sporocarp soaked in the glucose solution is treated by using 75% alcohol, so that the mixed bacteria on the surface of the sporocarp are effectively killed, the purification times are few, and only 1-2 times of purification is needed;
(3) only one culture medium, namely a PDA culture medium with streptomycin, needs to be prepared, so the operation is simple.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
Example 1
First, morchella sporocarp collection and drying
Selecting normal-shape, moderate-size, robust and disease and pest-free morchella in the morchella harvesting season, placing the morchella in a dry and well-ventilated environment, airing until the fruiting bodies are completely dried, then placing the morchella in a sample bag, and storing the morchella in a refrigerator at 4 ℃ for later use.
Second, pretreatment of fruit body
Firstly, soaking dried morchella esculenta sporocarp in 5% (w/v) glucose solution, and after the morchella esculenta is completely softened (taking for 8min), soaking the dried morchella esculenta sporocarp in 75% (v/v) ethanol solution for 2 s. Then, the morchella was taken out and put into a new glucose solution with a concentration of 5% (w/v) again to be soaked for 3 seconds. Finally, the glucose solution on the morchella was blotted dry with sterile gauze and placed over an alcohol burner flame to rapidly bake 3 times back and forth.
Thirdly, separating strains
Cutting the processed morchella sporophore into tissue blocks with the size of 0.5cm, inoculating the tissue blocks on a PDA culture medium containing streptomycin, wherein the final concentration of the streptomycin is 300 mug/L, and placing the culture dish in a constant temperature incubator at 18 ℃ for culture. By 3d, hyphae started to germinate, see FIG. 1.
At this time, the tip mycelium was picked and inoculated on a new PDA medium (final concentration of streptomycin is 300. mu.g/L) containing streptomycin for purification, and purified 2 times to obtain a relatively pure morchella mycelium, as shown in FIG. 2.
Example 2
First, morchella sporocarp collection and drying
Selecting normal-shape, moderate-size, robust and disease and pest-free morchella in the morchella harvesting season, placing the morchella in a dry and well-ventilated environment, airing until the fruiting bodies are completely dried, then placing the morchella in a sample bag, and storing the morchella in a refrigerator at 4 ℃ for later use.
Second, pretreatment of fruit body
Firstly, soaking dried morchella esculenta sporocarp in 5% (w/v) glucose solution, and after the morchella esculenta is completely softened (taking for 8min), soaking the dried morchella esculenta sporocarp in 75% (v/v) ethanol solution for 4 s. Then, the morchella was taken out and put in a new 5% (w/v) glucose solution again to be soaked for 6 s. Finally, the glucose solution on the morchella was blotted dry with sterile gauze and placed over an alcohol lamp flame to rapidly bake 3 times back and forth.
Thirdly, separating strains
Firstly, cutting the processed morchella sporocarp into tissue blocks with the size of 0.5cm, inoculating the tissue blocks on a PDA culture medium containing streptomycin, wherein the final concentration of the streptomycin is 300 mug/L, and placing a culture dish in a constant temperature incubator at 18 ℃ for culture. By 3d, hyphae started to germinate, see FIG. 3.
At this time, the tip hyphae were picked and inoculated on a new PDA medium containing streptomycin (final concentration of streptomycin is 300. mu.g/L) for purification, and purified 1 time to obtain relatively pure Morchella mycelium, see FIG. 4.
Example 3
First, morchella sporocarp collection and drying
Selecting normal-shape, moderate-size, robust and disease and pest-free morchella in the morchella harvesting season, placing the morchella in a dry and well-ventilated environment, airing until the fruiting bodies are completely dried, then placing the morchella in a sample bag, and storing the morchella in a refrigerator at 4 ℃ for later use.
Second, pretreatment of fruit body
Firstly, soaking dried morchella esculenta sporocarp in 5% (w/v) glucose solution, and after the morchella esculenta is completely softened (taking for 8min), soaking the dried morchella esculenta sporocarp in 75% (v/v) ethanol solution for 6 s. Then, the morchella was taken out and put in a new 5% (w/v) glucose solution again to be soaked for 9 s. Finally, the glucose solution on the morchella was blotted dry with sterile gauze and placed over an alcohol lamp flame to rapidly bake 3 times back and forth.
Thirdly, separating strains
Firstly, cutting the processed morchella sporocarp into tissue blocks with the size of 0.5cm, inoculating the tissue blocks on a PDA culture medium containing streptomycin, wherein the final concentration of the streptomycin is 300 mug/L, and placing a culture dish in a constant temperature incubator at 18 ℃ for culture. By 3d, hyphae started to germinate, see FIG. 5.
At this time, the tip hyphae were picked and inoculated on a new PDA medium (final streptomycin concentration 300. mu.g/L) containing streptomycin for purification, and purified 2 times to obtain relatively pure Morchella mycelium, see FIG. 6.
Example 4
First, morchella sporocarp collection and drying
Selecting normal-shape, moderate-size, robust and disease and pest-free morchella in the morchella harvesting season, placing the morchella in a dry and well-ventilated environment, airing until the fruiting bodies are completely dried, then placing the morchella in a sample bag, and storing the morchella in a refrigerator at 4 ℃ for later use.
Second, pretreatment of fruit body
Firstly, soaking dried morchella esculenta sporocarp in 5% (w/v) glucose solution, and after the morchella esculenta is completely softened (taking for 8min), soaking the dried morchella esculenta sporocarp in 75% (v/v) ethanol solution for 8 s. Then, the morchella was taken out and put into a new 5% (w/v) glucose solution again to be soaked for 12 s. Finally, the glucose solution on the morchella was blotted dry with sterile gauze and placed over an alcohol lamp flame to rapidly bake 3 times back and forth.
Thirdly, separating strains
Firstly, cutting the processed morchella sporocarp into tissue blocks with the size of 0.5cm, then inoculating the tissue blocks on a PDA culture medium containing streptomycin, wherein the final concentration of the streptomycin is 300 mug/L, and finally placing a culture dish in a constant temperature incubator at 18 ℃ for culture. By 3d, hyphae started to germinate, see FIG. 7.
At this time, the tip hyphae were picked and inoculated on a new PDA medium (final streptomycin concentration 300. mu.g/L) containing streptomycin for purification, and purified 2 times to obtain relatively pure Morchella mycelium, see FIG. 8.
Example 5
First, morchella sporocarp collection and drying
Selecting normal-shape, moderate-size, robust and disease and pest-free morchella in the morchella harvesting season, placing the morchella in a dry and well-ventilated environment, airing until the fruiting bodies are completely dried, then placing the morchella in a sample bag, and storing the morchella in a refrigerator at 4 ℃ for later use.
Second, pretreatment of fruit body
Firstly, soaking dried morchella esculenta fruiting body in sterile water until the fruiting body is completely softened (8 min), and then soaking in 75% (v/v) ethanol solution for 4 s. Taking out Morchella esculenta, and soaking in sterile water for 6 s. Finally, the sterilized water on the morchella was blotted with a sterile gauze, and the resulting product was quickly baked 3 times back and forth above the flame of an alcohol burner.
Thirdly, separating strains
Cutting the processed morchella sporophore into tissue blocks with the size of 0.5cm, inoculating the tissue blocks on a PDA culture medium containing streptomycin, wherein the final concentration of the streptomycin is 300 mug/L, and placing the culture dish in a constant temperature incubator at 18 ℃ for culture. When the culture reached 3d, no hyphae were germinated, as shown in FIG. 9.
As is clear from comparative examples 1 to 5, when dried morchella esculenta fruiting bodies were treated with a 5% glucose solution, not only was the maintenance of the normal morphology of the fruiting bodies facilitated, but also nutrients required for hypha germination were ensured, so hyphae could germinate rapidly, and hyphae could grow after 3 days of culture.
Example 6
First, morchella sporocarp collection and drying
Selecting normal-shape, moderate-size, robust and disease and pest-free morchella in the morchella harvesting season, placing the morchella in a dry and well-ventilated environment, airing until the fruiting bodies are completely dried, then placing the morchella in a sample bag, and storing the morchella in a refrigerator at 4 ℃ for later use.
Second, pretreatment of fruit body
Firstly, dried morchella esculenta fruiting body is soaked in 5% (w/v) glucose solution until it is completely softened (8 min for use), and then it is soaked in sterile water for 4 s. Taking out Morchella esculenta, and soaking in new 5% (w/v) glucose solution for 6 s. Finally, the glucose solution on the morchella was blotted dry with sterile gauze and placed over an alcohol burner flame to rapidly bake 3 times back and forth.
Thirdly, separating strains
Firstly, cutting the processed morchella sporocarp into tissue blocks with the size of 0.5cm, then inoculating the tissue blocks on a PDA culture medium containing streptomycin, wherein the final concentration of the streptomycin is 300 mug/L, and finally placing a culture dish in a constant temperature incubator at 18 ℃ for culture. By 3d, the growth of the undesired bacteria around the tissue mass began, see FIG. 10.
It can be seen from comparison of examples 1 to 5 that when dried morchella esculenta fruiting bodies are treated with 75% alcohol, the mixed bacteria on the surfaces of the fruiting bodies can be effectively killed, and a foundation is laid for reducing the subsequent purification times.
As regards the incubation temperature, it has been tested that this range is possible between 18 ℃ and 22 ℃.
It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.
Claims (4)
1. A method for separating strains through dried morchella sporocarp is characterized by comprising the following steps:
(I) collecting and drying Morchella sporocarp
Selecting high-quality morchella in the morchella harvesting season, airing the morchella until the fruiting bodies are completely dried, and then putting the morchella into a sample bag to be stored in a refrigerator at 4 ℃ for later use;
(II) pretreatment of fruit body
Soaking dried morchella esculenta fruiting body in 5% glucose solution, soaking in 75% ethanol solution for 2-8s after the fruiting body is completely softened, taking out, soaking in 5% glucose solution for 3-12s, sucking out the glucose solution on the morchella esculenta with sterile gauze, and rapidly baking the morchella esculenta above the flame of an alcohol lamp for 3 times;
(III) isolation of the bacterial species
Cutting the processed morchella sporophore into small tissue blocks, inoculating on PDA culture medium containing streptomycin, culturing at constant temperature for 2-3 days, picking tip hyphae after hyphae germinate, inoculating on new PDA culture medium containing streptomycin, and purifying for 1-2 times.
2. The method for separating strains from dried fruiting bodies of Morchella esculenta according to claim 1, wherein in the step (one), the Morchella esculenta is dried in a dry and well ventilated environment.
3. The method for separating strains from dried fruiting bodies of Morchella esculenta according to claim 1, wherein in the third step, the processed Morchella esculenta fruiting bodies are cut into small tissue pieces with a size of 0.5 cm.
4. The method for isolating strains from dried fruiting bodies of Morchella esculenta according to claim 1, wherein in the third step, the cultivation temperature is 18-22 ℃.
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Cited By (4)
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| CN112425447A (en) * | 2020-10-20 | 2021-03-02 | 安徽乐永园农业科技有限公司 | Cultivation method for artificially cultivating morchella |
| CN112449988A (en) * | 2020-10-20 | 2021-03-09 | 安徽乐永园农业科技有限公司 | Breeding and transferring method suitable for morchella |
| CN112655468A (en) * | 2020-10-20 | 2021-04-16 | 安徽乐永园农业科技有限公司 | Process suitable for fungus picking spores to prevent mixed bacteria |
| CN115530007A (en) * | 2022-11-03 | 2022-12-30 | 井冈山市君先竹荪种植专业合作社 | Method and equipment for separating dried morchella esculenta |
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