CN113875502A - Morchella strain preservation and activation method - Google Patents
Morchella strain preservation and activation method Download PDFInfo
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- CN113875502A CN113875502A CN202110146187.6A CN202110146187A CN113875502A CN 113875502 A CN113875502 A CN 113875502A CN 202110146187 A CN202110146187 A CN 202110146187A CN 113875502 A CN113875502 A CN 113875502A
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- 238000004321 preservation Methods 0.000 title claims abstract description 48
- 241000221638 Morchella Species 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 42
- 230000004913 activation Effects 0.000 title claims abstract description 18
- 238000007710 freezing Methods 0.000 claims abstract description 19
- 230000008014 freezing Effects 0.000 claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims abstract description 19
- 239000002689 soil Substances 0.000 claims abstract description 18
- 230000003213 activating effect Effects 0.000 claims abstract description 9
- 239000004576 sand Substances 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 238000010257 thawing Methods 0.000 claims abstract description 7
- 238000012360 testing method Methods 0.000 claims description 46
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 11
- 238000007789 sealing Methods 0.000 claims description 6
- 238000003860 storage Methods 0.000 claims description 6
- 238000005056 compaction Methods 0.000 claims description 3
- 239000002655 kraft paper Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 2
- 241000894007 species Species 0.000 claims 3
- 238000011161 development Methods 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 239000012153 distilled water Substances 0.000 description 14
- 238000000926 separation method Methods 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 7
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 241000576755 Sclerotia Species 0.000 description 4
- 241001558929 Sclerotium <basidiomycota> Species 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 230000005059 dormancy Effects 0.000 description 3
- 230000035784 germination Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000012364 cultivation method Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000235349 Ascomycota Species 0.000 description 1
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 1
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 description 1
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000221662 Sclerotinia Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229910052602 gypsum Inorganic materials 0.000 description 1
- 239000010440 gypsum Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000003864 humus Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000000123 paper Substances 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
Classifications
-
- 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
- A01G18/40—Cultivation of spawn
-
- 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
- A01G18/20—Culture media, e.g. compost
-
- 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
- A01G18/30—Accessories for use before inoculation of spawn, e.g. sterilisers
-
- 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
- A01G18/50—Inoculation of spawn
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of strain preservation, in particular to a method for preserving and activating a morchella strain. The method comprises the following steps: (1) preparing a strain preservation culture medium; (2) inoculating and culturing; (3) pretreating strains; (4) freezing and preserving; (5) and (5) thawing and activating. The morchella strain preservation and activation method provided by the invention adopts suitable soil as a main raw material of a morchella strain preservation culture medium, is supplemented with plant ash, sand grains and the like for hypha culture, is preserved by a freezing pretreatment and freezing method, and can achieve normal hypha growth, normal fruiting and yield per mu of more than 150kg after strain activation is carried out when needed, thereby effectively promoting the stable and healthy development of the morchella industry.
Description
Technical Field
The invention relates to the technical field of strain preservation, in particular to a method for preserving and activating a morchella strain.
Background
At present, the cultivation mode of the morchella is mainly that soil is directly sown for spawn running, exogenous nutrition is supplemented at a proper time node, the cultivation method is simple and easy to implement, and some cultivators are tentatively named as being as simple as planting Chinese cabbages. The key point of spawn running is that the spawn, morchella is different from other edible fungi, belongs to the phylum of ascomycete and the class of sclerotinia, and is characterized by rapid propagation, and the spawn can be completely cultivated in 4-6 days under the condition of normal temperature (the spawn can be completely cultivated in 10-15 days for mushrooms and the like). But the aging and the degeneration of the morchella are also quick, the morchella can be stored for 30 days at the temperature of 0-4 ℃ at most, and can be stored for only 8-10 days at room temperature; and the aged and degenerated strains have little or no fruiting during cultivation.
According to statistics of relevant persons in the industry, in 2015/2016 years, the total amount of nationwide cultivation is 25000 mu, only about 40% of the total profit or the total cost is kept, and 60% of the total profit or the total cost is the loss, which is the main reason for the problem of strains. Moreover, other edible fungi such as mushrooms and the like are bred into a good variety which can be continuously used for 5-8 years, if the preservation method is proper, the service life is longer, but the morchella is not suitable, the breeding is generally separated in the current year, the breeding time in the next year is missed if a mushroom test is carried out in the current year, the strains preserved in the previous year are not dared to be used at all, and many people have been deeply taught.
At present, the preservation form of the morchella strains is still a preservation method of other edible fungus strains, and the preservation method mainly comprises the following steps: the method comprises the steps of slant preservation, distilled water preservation, mineral oil preservation, filter paper preservation, freeze drying, liquid nitrogen ultra-low temperature preservation and the like, but the application effect is not ideal; the research on domestic morchella is mainly carried out in China, but the strains separated in the same year are not subjected to fruiting tests, so that the success rate of cultivation is difficult to ensure, and the breeding principle of normal edible fungi is violated; therefore, the preservation of the toadstool strains is the bottleneck of the development of the toadstool industry.
Disclosure of Invention
The invention provides a method for preserving and activating a morchella strain, which adopts natural soil added with partial auxiliary materials as a morchella mycelium carrier, makes the morchella mycelium dormant in a freezing mode, ensures the normal activity and biological characteristics of the morchella mycelium after the dormancy is released, and achieves the purpose of continuous use of the morchella strain.
The technical scheme of the invention is realized as follows:
a morchella strain preservation and activation method comprises the following steps:
(1) preparing a strain preservation culture medium;
taking suitable soil, adding plant ash and sand with particle size of 1mm, adding water to adjust water content to 28%, filling into a test tube, wherein the filling amount is 1/3 of the length of the test tube, and leveling the surface but forbidding compaction; plugging the air port with silica gel, bundling, sealing with kraft paper, and autoclaving;
(2) inoculating and culturing;
under aseptic conditions, inoculating strains into the test tube, sealing the test tube, and culturing in a biochemical incubator at 18 + -1 deg.C until the test tube is full of Morchella mycelia; wherein, the strain is inoculated on the surface of the soil culture medium when being inoculated;
(3) pretreating strains;
when the test tube is almost full of morchella mycelium, placing the morchella mycelium into a refrigerator at 0-4 ℃ for precooling treatment, wherein the treatment time is 48 hours;
(4) freezing and preserving;
after the pretreatment time reaches 48 hours, transferring the test tube to a freezing refrigerator at the temperature of-18 to-20 ℃ for freezing storage;
(5) thawing and activating
When needed, the test tube stored in the freezing refrigerator is moved into a refrigerator with the temperature of 0-4 ℃ for unfreezing, and the unfreezing time is 48 hours; after the thawing process is completed, the test tube is transferred into a biochemical incubator at 18 +/-1 ℃ for activation for 48 hours, and then the preserved seeds are transferred to an activation culture medium for culture, and the overgrown strains can be used.
Preferably, the culture medium in the step 1 is prepared from the following components in percentage by mass: 80% of soil, 10% of plant ash and 10% of large sand grains.
Preferably, the inoculated strain in the step 2 is a robust strain of the mother strain to be preserved within 48 hours of growing the test tube.
The invention has the beneficial effects that:
the morchella strain preservation and activation method provided by the invention adopts suitable soil as a main raw material of a morchella strain preservation culture medium, is supplemented with plant ash, sand grains and the like for hypha culture, is preserved by a freezing pretreatment and freezing method, and can achieve normal hypha growth, normal fruiting and yield per mu of more than 150kg after strain activation is carried out when needed, thereby effectively promoting the stable and healthy development of the morchella industry.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to a morchella strain preservation and activation method, which comprises the following steps:
(1) preparing a strain preservation culture medium;
taking suitable soil, adding plant ash and large sand, adding water to adjust the water content to 28%, filling into a test tube, wherein the filling amount is 1/3 of the length of the test tube, and leveling the surface but forbidding compaction; plugging the air port with silica gel, bundling, sealing with kraft paper, and autoclaving;
(2) inoculating and culturing;
under aseptic condition, inoculating strain into the test tube, sealing the test tube, and culturing in a biochemical incubator at 18 + -1 deg.C until the test tube is full of Morchella mycelia; wherein, the strain is inoculated on the surface of the soil culture medium when being inoculated;
(3) pretreating strains;
when the test tube is almost full of morchella mycelium, placing the morchella mycelium into a refrigerator at 0-4 ℃ for precooling treatment, wherein the treatment time is 48 hours;
(4) freezing and preserving;
after the pretreatment time reaches 48 hours, transferring the test tube to a freezing refrigerator at the temperature of-18 to-20 ℃ for freezing storage;
(5) thawing and activating
When needed, the test tube stored in the freezing refrigerator is moved into a refrigerator with the temperature of 0-4 ℃ for unfreezing, and the unfreezing time is 48 hours; after the thawing process is finished, the test tube is moved into a biochemical incubator at the temperature of 18 +/-1 ℃ for activation treatment, and the test tube can be used after the activation time is 48 hours.
According to experimental research, the hypha of the morchella can resist the low temperature of-20 ℃ in soil without death, and the metabolic level of the hypha of the morchella is reduced to the minimum at the temperature. Therefore, the temperature of the frozen preservation is preferably 18 +/-1 ℃, and the temperature is designed according to the lowest growth temperature of the wild morchella mycelium.
Preferably, the culture medium in the step 1 is prepared from the following components in percentage by mass: 80% of soil, 10% of plant ash and 10% of large sand grains.
Preferably, the inoculated strain in the step 2 is a robust strain of the mother strain to be preserved within 48 hours of growing the test tube.
Examples comparative experiments
1. Materials and methods
1.1 Material test strains selection Hionly corporation breeds domesticated six sister series breed H2.
1.2 methods
1.2.1 slant storage of PDA enriched Medium
1.2.2 PDA enriched Medium distilled Water preservation method
1.2.3 isolation of strains that have not undergone cryopreservation
1.2.4 frozen dormancy preservation: according to the mass percentage: 80% of suitable soil, 10% of plant ash and 10% of sand with the diameter of 1mm are added, water is added to adjust the water content to 28%,
1.3 formula of culture medium for activating mother seeds: according to the mass percentage: 16% of potatoes, 1.5% of glucose, 1% of plant ash, 1% of humus soil, 1.5% of agar and 79% of water.
The formula of the stock culture medium is as follows: according to the mass percentage: 35% of wheat grains, 40% of rice hulls, 12% of wheat bran, 10% of garden soil, 2% of gypsum, 1% of lime and 60% of water content.
2. Test results
2.1 propagation of mother seed
Tests show that the germination time of the strains on the activated mother culture medium is the fastest in the tests, and is only 48 hours; the separation time is 72 hours; the slant preservation method is 96 hours, the preservation with distilled water is the worst, and the germination is carried out within 120 hours; the fastest and slowest phases differ by 72 hours. The slant storage, the distilled water storage and the separation subculture of the growth rate of hyphae were all substantially the same and were 0.625mm, but the test method was 0.78mm, with a difference of 0.155 mm. The growth vigor of the morchella mycelia of various preservation methods is strong and dense, and only the strains preserved in distilled water are sparse. The tube filling time of hyphae on a culture medium with the length of 150mm is the fastest by a test method, and is only 144 hours; 192 hours after separation and subculture; the slant is stored for another time, which is 216 hours, the distilled water is slowest to store the strains, and the tube is filled after 240 hours; the fastest and slowest phases differ by 96 hours. The four preservation methods have the appearance of the sclerotia, but the number of the sclerotia is more, the strains for the test and separation subculture appear more, and the slant and the distilled water are less for preservation. The time for sclerotium formation test was the earliest and was 192 hours; the separation time is 210 hours after subculture; the slant is stored for 164 hours again, and the distilled water is stored for 288 hours at the latest; the difference was significant 96 hours between the earliest and latest.
2.2 propagation of stock and cultivar
As can be seen from the table above, the germination time of the strains preserved by different preservation methods on the culture medium is the fastest in the test, and is only 2 days; separating subculture and preserving the inclined plane for 3 days; the strain preserved by the distilled water preservation method is slowest and germinates in 4 days; the fastest and slowest phases differ by 2 days. The morchella mycelia of the strains preserved by various preservation methods are strong in growth vigor, and only the strains preserved by distilled water are sparse. The hypha full-bag time of the strains preserved by various preservation methods is the fastest in the test, and is only 19 days; the separation and subculture time is 23 days; the slope is preserved for 25 days; the strain is preserved in distilled water at the slowest and is filled in the bag after 30 days; the fastest and slowest comparisons differ by 11 days. The strains preserved by various preservation methods have sclerotium, but the number of the strains is divided into more strains for test and separation subculture, and the number of the strains for slant preservation and distilled water preservation is less. The time of sclerotia formation was 19 days at the earliest of the test; the separation and subculture time is 23 days; the slope is preserved for 25 days; the distilled water is stored for 30 days at the latest; the difference between the earliest and latest was 11 days, and the difference was significant. The strains preserved by various preservation methods have no pollution phenomenon in the propagation process.
2.3 cultivation fruiting test
The method is characterized in that the method is carried out on the strains expanded and propagated by the four preservation methods in the same morchella production shed room under the same soil condition, climate condition, cultivation method and the like, and the main evaluation indexes comprise indexes of bed surface conidium appearance time, conidium bed full time, conidium density, hypha entering time after an exogenous nutrition bag is placed, hypha bag full time of the exogenous nutrition bag, whether yellow brown sclerotia is formed, time for forming needle-shaped primordium under the condition of proper temperature, time from forming mushroom buds to harvesting, morchella fresh product yield and the like.
The results of the tests are given in the following table:
according to the comprehensive analysis of the data, the early sclerotium formation time, the large sclerotium quantity and the vigorous hypha activity are important factors for the excellent morchella strains, and are the guarantee of the production yield and the product quality. Production fruiting tests prove that the success and failure of cultivation can be directly influenced by the strain preservation method, and the strains preserved in distilled water and preserved on an inclined plane are almost not obtained or fail, so that the two conventional preservation methods are not feasible. Although the yield of the new isolated subculture strain can be said to be past, the method is not practical because the method is complicated to operate and has high error rate when the strain is isolated in the same year and the method is used in northern areas of China for insufficient time. Therefore, the tested morchella preservation method effectively solves the difficulty of morchella preservation, and can ensure that the yield of the fresh morchella reaches more than 150kg per mu.
In conclusion, the method for preserving and activating the toadstool strains provided by the invention makes the toadstool strains dormant in a freezing mode, and ensures the normal activity and biological characteristics of the toadstool strains after the dormancy is released, thereby achieving the purpose of continuously using the toadstool strains.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (3)
1. A morchella strain preservation and activation method is characterized by comprising the following steps:
(1) preparing a strain preservation culture medium;
taking suitable soil, adding plant ash and sand with particle size of 1mm, adding water to adjust water content to 28%, filling into a test tube, wherein the filling amount is 1/3 of the length of the test tube, and leveling the surface but forbidding compaction; plugging the air port with silica gel, bundling, sealing with kraft paper, and autoclaving;
(2) inoculating and culturing;
under aseptic conditions, inoculating strains into the test tube, sealing the test tube, and culturing in a biochemical incubator at 18 + -1 deg.C until the test tube is full of Morchella mycelia; wherein, the strain is inoculated on the surface of the soil culture medium when being inoculated;
(3) pretreating strains;
when the test tube is almost full of morchella mycelium, placing the morchella mycelium into a refrigerator at 0-4 ℃ for precooling treatment, wherein the treatment time is 48 hours;
(4) freezing and preserving;
after the pretreatment time reaches 48 hours, transferring the test tube to a freezing refrigerator at the temperature of-18 to-20 ℃ for freezing storage;
(5) thawing and activating
When needed, the test tube stored in the freezing refrigerator is moved into a refrigerator with the temperature of 0-4 ℃ for unfreezing, and the unfreezing time is 48 hours; after the thawing process is completed, the test tube is moved into a biochemical incubator at 18 +/-1 ℃ for activation treatment, the activation time is 48 hours, then the preserved seeds are transferred to an activation culture medium for culture, and the overgrown strains can be used.
2. The morchella strain preservation and activation method according to claim 1, wherein the culture medium in the step 1 is prepared from the following components in percentage by mass: 80% of soil, 10% of plant ash and 10% of large sand grains.
3. The morchella species preservation and activation method according to claim 1, wherein the inoculated species in the step 2 is a robust species of the mother species to be preserved within 48 hours of the growth in a test tube.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2885395A1 (en) * | 2012-08-20 | 2015-06-24 | Chr. Hansen A/S | Method for freeze drying a bacteria-containing concentrate |
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| CN110663452A (en) * | 2019-11-07 | 2020-01-10 | 郝哲 | Morchella strain preservation method |
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2021
- 2021-02-02 CN CN202110146187.6A patent/CN113875502A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2885395A1 (en) * | 2012-08-20 | 2015-06-24 | Chr. Hansen A/S | Method for freeze drying a bacteria-containing concentrate |
| CN104762208A (en) * | 2015-03-18 | 2015-07-08 | 景洪宏臻农业科技有限公司 | Boletus aereus stock culture low-temperature preservation method |
| CN108812079A (en) * | 2018-05-30 | 2018-11-16 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | A kind of hickory chick strain separating method |
| CN110663452A (en) * | 2019-11-07 | 2020-01-10 | 郝哲 | Morchella strain preservation method |
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| Title |
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| 党建章: "《发酵工艺教程 第2版》", 31 July 2016, 中国轻工业出版社 * |
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