CN110140591B - Morchella high-yield strain and application thereof - Google Patents
Morchella high-yield strain and application thereof Download PDFInfo
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Abstract
The invention discloses a high-yield morchella strain, which is named as Morchella shannon No.1 and is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation numbers as follows: CGMCC NO. 17677. The Morchella esculenta No.1 bred by the method has the advantages of high hypha growth speed and high fruiting yield, and provides a new high-quality strain for Morchella esculenta cultivation in northern areas.
Description
Technical Field
The invention relates to the technical field of edible fungi, and particularly relates to a novel morchella high-yield strain and application thereof.
Background
Morchella is named because its pileus is shaped like morchella, is a general name for all species of Morchella, and does not refer to a specific species. Morchella (Morchella Dill. ex Pers.: Fr.) belongs to Ascomycota (Ascomycota), Discomycetes (Discomycetes), Pezizales (Pezizales), Morchella (Morchellacaceae).
All the species of morchella are rare food and medicinal fungi, and have high nutritional and medicinal values. In europe, morchella is considered to be a delicious edible fungus next to truffles; in north america, it is considered the best food. In our country, the book Ben Cao gang mu of Ming Dynasty Li Shizhen records "sweet, cold, non-toxic, benefiting intestines and stomach, resolving phlegm and benefiting qi". The morchella sporocarp is crisp and tender in meat quality and delicious in taste, contains 7 essential amino acids for human bodies, a plurality of vitamins, high-content iron, zinc and a plurality of mineral elements, and is a delicious dish on a dining table. The morchella contains a large amount of polysaccharide, and has strong anti-tumor and anti-cancer effects; contains melanin formation inhibitor, and has skin caring, antiaging, and antioxidant effects.
Morchella is naturally distributed in a wide range and various types, and the morchella is reported to comprise 68 types. The difference of the growth environment conditions of the morchella is large, and the biological characteristics are easily influenced by environmental factors, so that the difficulty of artificial cultivation of the morchella is greatly increased. Research reports that, among many Morchella species, 4 species of Morchella esculenta are successfully cultivated at the present stage, including Morchella esculenta (Morchella importuna), Morchella hexakista (Morchella sexteta), and Morchella esculenta (Morchella septielata), and Morchella rubra (Morchella rufbrunna) is cultivated more in the united states. The morchella terrapin and the morchella hexameina are two most widely cultivated species at present in China.
At present, the cultivation technology of morchella is still in an initial stage, and still has a plurality of problems. The source of the strain is undefined, the seed source is mixed, most of the cultivated strains mainly come from wild domesticated strains, the overall yield is low, the strains are unstable, and the repeatability is poor; the cultivation management technology is relatively extensive, relatively accurate technical parameters are lacked, especially in northern areas of China, high-yield strains and cultivation technologies for cultivating morchella are lacked, and the method is a main problem for limiting the development of the morchella industry.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a morchella japonica high-yield strain morchella japonica No. 1. The strain has high hypha growth speed and high fruiting yield, and provides a new high-quality strain for Morchella cultivation in northern areas.
Specifically, the invention relates to the following technical scheme:
the high-yield morchella strain provided by the invention is named as morchella sannong No.1, and the strain is preserved in the China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Xilu No.1 Beichen of Chaoyang district in Beijing) in 28 days 4 months in 2019, and the preservation number is as follows: CGMCC NO. 17677.
The strain is prepared by taking black morchella strains M10 and M20 as parents and adopting a protoplast fusion breeding technology, wherein the strain M10 is morchella ladder bred by Sichuan academy of agricultural sciences, and the strain M20 is morchella hexameiensis.
The Morchella esculenta No.1 provided by the invention has the advantages that the fruiting body is single or group, a few of morchella esculenta grows in clusters, the pileus is conical, occasionally, the pileus is oval, the length is 5-12 cm, and the width is 2-7 cm. The mushroom cap surface has obvious pits and criss-cross ridges, the ridge surface is smooth and fragile, the color is dark brown when young and tender, and the dark brown is nearly black after old. The mushroom meat is white and fleshy, 1-3 mm thick, coarse in inner wall and provided with white powdery particles. The stipe is cylindrical, the height is 3-8 cm, the diameter is 2-4 cm, the color is white to light brown, the surface is smooth when the stipe is young, the surface has powdery particles after the stipe is old, and the basal part of the stipe is expanded and hollow.
The hyphae are white in the initial stage, are brown after maturation, grow vigorously on aerial hyphae, are brown and cotton-like in colonies, and are easy to generate brown sclerotium when cultured on a PDA culture medium for 5-7 days.
The morchella shannon No.1 of the invention has excellent biological properties of high hypha growth speed and high fruiting yield; compared with the parent strain, the yield of the strain can be improved by more than 65%.
The morchella strain (CGMCC NO.17677) claimed by the invention comprises a strain corresponding to CGMCC NO.17677 and also comprises a progeny which is produced by the propagation of the strain and has the same genetic and/or morphological characters with the strain.
The application of the morchella strain (CGMCC NO.17677) as a parent for breeding also belongs to the protection scope of the invention.
The fruit body obtained by cultivating the morchella strain (CGMCC NO.17677) also belongs to the protection scope of the invention.
The mycelium and/or spore obtained by culturing the morchella (CGMCC NO.17677) also belong to the protection scope of the invention.
According to one aspect of the invention, the invention also relates to a cultivation method of the morchella strain (CGMCC NO.17677), which comprises the following steps:
(1) inoculating stock seed obtained by culturing Morchella strain (CGMCC NO.17677) into culture medium, and culturing at 22-24 deg.C for 10-15 days to obtain culture seed;
(2) sowing seed ditches on the ridge surface, sowing the cultivated seeds obtained in the step (1), covering soil, covering with a film after covering the soil, and moisturizing;
(3) after seeding, when hypha grows to fill the surface of the furrow to form fungus frost, a nutrition bag containing a culture material is placed, an opening is cut on one surface of the nutrition bag, the cut surface of the nutrition bag is horizontally placed on the surface of the furrow, and the morchella hypha can directly contact the culture material in the nutrition bag;
(4) placing the nutrition bag for 35-45d, and removing the nutrition bag; uncovering the film after the soil surface begins to form an original base; the temperature of the fruiting body is controlled to be 8-20 ℃ in the growing period, the water content of the soil in the fruiting period is controlled to be 20-28%, and the air humidity is controlled to be 85-90%.
Preferably, in the step (1), the culture medium of the cultivar consists of the following raw materials in percentage by mass:
40% of wheat grains, 45% of sawdust, 10% of humus soil, 3% of wheat bran, 1% of lime and 1% of gypsum.
Preferably, in the step (2), 20% of turf is added into the soil covering material; the thickness of the covering soil is 3 cm.
Preferably, in the step (3), the culture material is composed of the following raw materials by mass percent:
30% of wheat grains, 68% of wood chips, 1% of lime and 1% of gypsum.
At present, many techniques in the cultivation of morchella are not clear and concrete, and the cultivation conditions are extensive, so that the yield and the quality of the finally obtained morchella are unstable. Aiming at the high-yield morchella strains, the cultivation technology of the morchella strains is researched and optimized, and technical support is provided for cultivation of morchella in northern areas.
According to the method for cultivating the morchella esculenta No.1, a certain amount of turf is added into the soil covering material, the turf is loose in texture, the effect of improving the air permeability and the water retention of soil is remarkable, and sufficient oxygen and proper humidity can be provided for the growth of morchella esculenta hyphae in the soil. The number and the yield of the effective fruit bodies are increased along with the increase of the thickness of the covering soil, and the number and the yield of the effective fruit bodies of the morchella are highest when the thickness of the covering soil is 3 cm. When the thickness of the covering soil layer continues to increase, the number of effective fruit bodies and the yield begin to decrease. The use of the exogenous nutrition bag is a milestone in the artificial cultivation exploration process of the morchella esculenta, so that the high yield of the morchella esculenta cultivation can be realized. As an important technology in the cultivation technology, the placing of the exogenous nutrition bag needs to be deeply researched, and researches show that the placing time of the nutrition bag is in positive correlation with the forming time of the morchella anlage, and the forming time of the anlage is delayed along with the delay of the placing time of the nutrition bag. The nutrition bag can provide nutrition for the growth of hyphae, the hyphae on the soil surface grow vigorously, and the hyphae can spread around from the opening of the nutrition bag. The earlier the nutrition bag is placed, the more vigorous the hypha grows, and after the hypha grows for a certain time, the nutrition bag needs to be removed, and primordium can be formed under the appropriate temperature and humidity conditions. The time for withdrawing the nutrition bag is too early, hypha does not fully utilize the nutrition bag, and the nutrition bag has small promotion effect on fruiting and later growth of sporocarp; the disease and insect pests are easily infected after the nutrition bag is removed too late, and certain loss is caused to the yield of the morchella; multiple experimental researches show that the optimal placing time is that the nutrition bag is moved after being placed for 35-45 days, and the yield of the morchella esculenta can be remarkably improved.
According to one aspect of the invention, the invention also relates to the application of the fruit body obtained by the morchella strain (CGMCC NO.17677) in food processing.
According to one aspect of the invention, the invention also relates to a food product comprising a strain as described above;
and/or:
the mycelium and/or fruiting body obtained by the strain cultivation.
The invention has the beneficial effects that:
the morchella strain (CGMCC NO.17677) bred by the invention has high hypha growth speed and high fruiting yield, and provides a new high-quality strain for morchella cultivation in northern areas.
Drawings
FIG. 1: the fusion strain R5 antagonizes the phenomenon of the parent.
FIG. 2: and (3) constructing a phylogenetic tree based on ITS.
FIG. 3: primer pair Me9-Em8 maps the fusion strain R5 and the SRAP of the parent strain.
FIG. 4: and (3) fruiting of the fusion strain R5.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
Description of terms:
fruiting body: is the spore-forming structure of higher fungi, i.e. fruit body, and is composed of organized mycelium.
Primordia: before the edible mushrooms grow out, the small particles formed by winding and twisting the hyphae grow up to form the mushrooms.
Hypha: a single tubular filament, the structural unit of most fungi.
Mycelium: many hyphae aggregate together to form the trophozoite of the fungus, i.e., the mycelium.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If the experimental conditions not specified in the examples are specified, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, and the like used in the following examples are commercially available unless otherwise specified.
The formula of the culture medium used in the examples of the present invention is as follows:
PDA culture medium: 200g of potato (peeled), 20g of glucose, 20g of agar and 1L of water.
Regeneration culture medium: 200g of potato (peeled), 20g of glucose, 109.3g of mannitol, 20g of agar and 1L of water.
Stock culture medium: 90% of wheat grains, 8% of wheat bran, 1% of lime and 1% of gypsum; (weight percent).
Culture medium of cultivar: 40% of wheat grains, 45% of sawdust, 10% of humus soil, 3% of wheat bran, 1% of lime and 1% of gypsum; (weight percent).
The culture material formula in the nutrition bag is as follows: 30% of wheat grains, 68% of sawdust, 1% of lime and 1% of gypsum; (weight percent).
Example 1: breeding of morchella high-yield strain
Black morchella strains M10 and M20 were selected as parents for protoplast fusion. The strain M10 is a strain of morchella terrapin bred by agricultural academy in Sichuan province, the strain M20 is morchella hexameica, and the public can obtain the strain from Shandong agricultural university. And (4) breeding the morchella high-yield strains by a protoplast interspecific fusion technology.
1. Preparing protoplasts:
(1) cultivation of mycelia
And respectively taking 3-4 fungus cakes on a PDA culture medium by using a puncher, inoculating the fungus cakes in the culture medium stuck with glass paper, and culturing for 1-2 days at 25 ℃.
(2) Preparation of enzyme solution
Weighing a certain amount of lywallzyme (Guangdong institute of microorganisms) and dissolving in 0.6mol/L MgSO4In the osmotic pressure stabilizer, a 0.22 mu m microporous membrane is used for filtration sterilization (the enzyme solution is prepared at present), and the concentration of the muramidase is 3 percent.
(3) Protoplast preparation
Picking out Morchella mycelium in culture medium, washing with sterile water twice, and adding 0.6mol/L MgSO4And (4) washing twice, and sucking dry the filter paper and placing the filter paper in prepared enzyme liquid, wherein the volume of the enzyme liquid is 3-5 times of that of the wet mycelia. And (3) violently shaking the centrifugal tube to disperse hyphae, and then placing the centrifugal tube in a shaking table for enzymolysis at the temperature of 32 ℃ at 80r/min for about 3 hours. And detecting enzymolysis every half hour, sucking 10 mu l of enzymolysis liquid on a blood counting plate, observing a protoplast release mode under a microscope, and counting.
(4) Protoplast purification
After the enzymolysis is finished, the enzyme solution is absorbed and filtered by a cell sieve with the aperture of 40 mu m to remove residual hyphae. Centrifuging the filtrate at 4500r/min for 10min, discarding supernatant, precipitating with 0.6mol/L MgSO4And washing twice. Adding proper amount of 0.6mol/L MgSO4Preparing into suspension, and measuring the final yield of the protoplast by using a blood counting plate.
2. Protoplast fusion:
(1) the purified M10 strain protoplast was suspended in 0.6mol/L MgSO4In the solution, the concentration is adjusted to 105one/mL. Sucking 200 μ L protoplast, uniformly coating on a plate regeneration medium under aseptic condition, uncovering at a distance of 10cm below 30W (253.7nm) ultraviolet lamp, and vertically irradiating for 24 min.
(2) The purified M20 strain protoplast was suspended in 0.6mol/LMgSO4In the solution, the concentration is adjusted to 105one/mL. Sucking 1mL of the solution, putting the solution into a 2mL centrifuge tube, and simultaneously putting the centrifuge tube into a constant-temperature water bath box at 55 ℃ for heat inactivation treatment for 20 min; gently shake the centrifuge tube 1 time every 2min to heat the protoplasts evenly.
(3) Suspending the two parent protoplasts treated in the steps (1) and (2) in 0.5-1 mL (according to the amount of enzyme filtered out each time) of each protoplast in 0.6mol/L MgSO4In the solution, the concentration is adjusted to 105Mix 1:1 after order of magnitude of one/mL. With 0.6mol/LMgSO4Eluting the mixed solution for 2 times, centrifuging at 2000r/min for 10min to obtain protoplast precipitate, and metering to 1 mL.
Preheating the protoplast precipitate in a water bath at 30 ℃, and dropwise and slowly adding 1mL of PEG-CaCl with a certain concentration (30-45%) along the tube wall by using a pipette2Cosolvent, mixing, and performing protoplast fusion reaction in 30 deg.C water bath for 20 min. After the fusion reaction is finished, centrifuging at low speed of 2000r/min for 10min, removing supernatant, and then using 0.6mol/L MgSO4The penetration stabilizer of (2) was washed to remove PEG toxicity as much as possible.
Coating 100 mu L of diluent on a regeneration culture medium, placing the regeneration culture medium in a constant temperature incubator at 25 ℃ for light-proof culture, regularly observing the growth condition of bacterial colonies, and selecting a fusion strain with thick hypha and high growth speed as a target strain, wherein the number of the fusion strain is R5.
Example 2: identification of high-yield strains of morchella
1. Morphological identification:
the fused strain R5 has the advantages of single-born or group-born fruiting bodies, few fasciculations, conical pileus, occasional oval shape, 5-12 cm long and 2-7 cm wide. The mushroom cap surface has obvious pits and criss-cross ridges, the ridge surface is smooth and fragile, the color is dark brown when young and tender, and the dark brown is nearly black after old. The mushroom meat is white and fleshy, 1-3 mm thick, coarse in inner wall and provided with white powdery particles. The stipe is cylindrical, the height is 3-8 cm, the diameter is 2-4 cm, the color is white to light brown, the surface is smooth when the stipe is young, the surface has powdery particles after the stipe is old, and the basal part of the stipe is expanded and hollow.
The hyphae are white in the initial stage, are brown after maturation, grow vigorously on aerial hyphae, are brown and cotton-like in colonies, and are easy to generate brown sclerotium when cultured on a PDA culture medium for 5-7 days.
2. Antagonistic test:
and respectively inoculating the parent strain and the strain R5 screened after protoplast fusion on the plate, culturing the parent strain and the strain R5 at a constant temperature of 25 ℃ at a distance of 2cm, and observing whether an obvious antagonistic line is generated or not within 3-5 days. If hyphae can be fused and grown and are connected into one piece, the hyphae are of the same species, and if antagonistic lines are obvious, the strain properties are different, and new characters can be generated for effective fusion strains, and the results are shown in figure 1.
As can be seen from FIG. 1, the obtained fusion strain R5 and the amphiphile have antagonistic lines, and a large number of sclerotia are generated near the antagonistic lines, which indicates that the fusion strain R5 and the amphiphile have certain differences and belong to different strains.
3. And (3) carrying out affinity identification on the fusion strain and the parent strain:
ITS identification is carried out on the fusion strain and the parent thereof to construct a phylogenetic tree (see figure 2), and the parent M10 and the fusion strain R5 are gathered on one branch as can be seen from figure 2, which shows that the fusion strains R5 and M10 are closer in relationship.
The fusion strain R5 and the parent strain thereof are molecularly marked by using an SRAP technology, and the primer pair used for SRAP molecular identification is as follows: me5-Em17, Me9-Em6, Me9-Em8, Me9-Em9, Me10-Em3, Me10-Em12, Me10-Em17, Me11-Em3, Me11-Em17 and Me13-Em10, the sequences are as follows:
Me5:5'-TGAGTCCAAACCGGAAG-3'；(SEQ ID NO.1)
Me9：5'-TGAGTCCAAACCGGAGG-3'；(SEQ ID NO.2)
Me10:5'-TGAGTCCAAACCGGAAA-3'；(SEQ ID NO.3)
Me11:5'-TGAGTCCAAACCGGAAC-3'；(SEQ ID NO.4)
Me13:5'-TGAGTCCAAACCGGAAG-3'；(SEQ ID NO.5)
Em3:5'-GACTGCGTACGAATTGAC-3'；(SEQ ID NO.6)
Em6:5'-GACTGCGTACGAATTGCA-3'；(SEQ ID NO.7)
Em8：5'-GACTGCGTACGAATTCAC-3'；(SEQ ID NO.8)
Em9:5'-GACTGCGTACGAATTCAG-3'；(SEQ ID NO.9)
Em10:5'-GACTGCGTACGAATTCAT-3'；(SEQ ID NO.10)
Em12:5'-GACTGCGTACGAATTCTC-3'；(SEQ ID NO.11)
Em17:5'-TGTGGTCCGCAAATTTAG-3'。(SEQ ID NO.12)
the band amplified by the primer pair Me9-Em8 for fusion strain R5 and the parent is shown in FIG. 3. The similarity matrix was obtained by analysis using NTSYS-PC software, as shown in table 1.
Table 1: similarity coefficient matrix of fusion strain based on SRAP molecular marker
Based on the results of morphological identification, antagonistic test and molecular identification of the strains, the fusion strain R5 is identified as a new strain of morchella esculenta, named as Morchella shannon No. 1. And the strain is subjected to biological preservation, and the preservation information is as follows:
reference biological material (strain): morchella shannong No.1
And (3) classification and naming: morchella terraced (Morchella importuna)
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 2019.4.28.
registration number of the preservation center: CGMCC NO. 17677.
Example 3: comparison of hyphal growth rates
The fusion strain R5 of the present invention and parent strains M10 and M20 were cultured on a plate to measure the growth rate thereof, and the hyphal morphology was observed. The results of the hyphal growth rate are shown in Table 2.
Hypha growth rate (cm/d) [ (colony diameter-0.4 cm)/2 ]/days of culture
(0.4cm is the diameter of the mushroom cake)
Table 2: hypha growth speed of fused strain and parent strain
As can be seen from Table 2, the hyphal growth rate of the fusion strain R5 was superior to that of the parent strain. Moreover, the hyphae of the fusion strain R5 grow vigorously and are thick.
Example 4: cultivation yield verification of morchella strain (CGMCC NO.17677)
(1) Site selection and processing
Cleaning stones, sundries, weeds and other plant roots in the field, solarizing for 10 days, spreading lime, preparing soil and building bed with the specification of 0.9 multiplied by 1m2The distance between the ridges is 0.3m, and the ridge surface is 0.1m higher than the ground. Irrigating the soil with large water infiltration.
(2) Strain production
Inoculating the activated strain to PDA culture medium, and culturing at 25 deg.C for 5 days for later use.
Weighing a proper amount of wheat grains, cleaning, boiling with strong fire until most of the wheat grains are transparent and have no white core, and immediately pouring into cold water for cooling. Filtering out excessive water, adding 2% wheat bran and 2% gypsum, and stirring. Packaging with original seed bottle, wherein the packaging amount of each bottle is 2/3 bottles, and sealing with air-permeable sealing film. Sterilizing at 126 deg.C for 120min, cooling, inoculating the cultured mother strain, and culturing at 23 deg.C to obtain stock.
Weighing the culture seeds, prewetting, uniformly mixing, adjusting the water content to about 65%, putting into a polypropylene fungus bag, sterilizing for 120min at 126 ℃, cooling, inoculating with the stock, and culturing for 15-20 d at 23 ℃ for later use.
(3) Seeding and hypha growth period management
At 0.9X 1m2Three seeding ditches with depth of 5cm are uniformly arranged on the ridge surface, the strain is sowed and covered with soil with thickness of 3cm, and a film is covered after the soil is covered with soil for moisture preservation.
During the growth period of hyphae, the soil humidity is mainly managed and is controlled to be 20-25%.
(4) Nutrition bag
About 1 week after sowing, hypha will grow over the surface of the ridge to form fungus frost, and nutrient bags are placed on the ridge surface, 6 nutrient bags are evenly placed on each ridge surface, and the specification of the nutrient bags is 12cm multiplied by 24 cm. Two openings of about 10cm are scribed on one surface of the nutrition bag, one surface of the scribed opening of the nutrition bag is flatly placed on the surface of the ridge, and the nutrition bag is lightly compacted, so that the morchella mycelium can directly contact the culture material in the 'exogenous nutrition bag'. Hyphae slowly grow into the 'exogenous nutrition bag' and absorb nutrition, and are transmitted to hyphae in the soil layer.
(5) Fruiting management
The hypha slowly grows over the nutrition bag, the hypha turns from white to yellow, and the nutrition bag is moved away after being placed for 35 d. After the soil surface starts to form primordia, the film is uncovered. The temperature range of the fruiting body in the growing period is controlled to be 8-20 ℃ as much as possible. And enhancing the temperature increasing measure when the temperature is low. When the temperature exceeds 22 ℃, ventilation and temperature reduction are carried out in time. During ventilation, strong wind is not suitable for blowing the surface of the soil layer, so that water on the surface of the soil is easily lost, primordium is easily dead, and pileus of the fruiting body becomes sharp or small. The water content of the soil in the fruiting period is controlled to be 20-28%, and the air humidity is preferably 85-90%.
(6) Pest control
In the cultivation period of the morchella, the amount of infectious microbes such as agaricus, discodermus, slime mold and the like is small, and the infectious diseases can be removed in time. When the soil humidity is too high, white mould is easily formed on the fruit body, so that primordium and young mushrooms start to rot, and at the moment, good ventilation is kept, the soil humidity and the air humidity are reduced, and the propagation and the diffusion of pathogenic bacteria are inhibited. During fruiting, yellow plates are hung to attract winged insects and prevent mushroom mosquitoes, mushroom flies and the like, and the sugar and vinegar liquid is used to attract fruit flies, black cutworms and the like.
(7) Harvesting and data statistical analysis
When the ridge and the pit ridge of the ascomycete cap of the morchella esculenta are clear, the cap meat is thick and elastic and spores are not ejected, and the morchella esculenta can be harvested. When the fruiting bodies are collected, the stipes are cut and picked off at the place close to the ground along the horizontal direction by a knife, and the neatness of the fruiting bodies is kept to ensure the commodity characters. After picking, the residual mushroom feet are slowly cleaned, and other primordia and young mushrooms are prevented from being damaged. Mature fruiting bodies must be harvested in time to avoid the effect of reducing the quality of the product. And observing and recording the formation time of the bacterial frost and the primordium during the management period, finally counting and analyzing the cell yield, and calculating the yield per unit area.
The parent strain is used as a control, the fused strain R5 (namely morchella sannong No. 1) is subjected to fruiting verification, and the cultivation method is the same as the above. The test results are shown in Table 3.
Table 3: fruiting verification test result of strain (CGMCC NO.17677)
As can be seen from table 3, the yield of fusion strain R5 was significantly higher than the parent M10 and M20, indicating that fusion strain R5 is a high yielding morchella.
Example 5: second generation fruiting verification
And (4) obtaining strains after fruiting verification, selecting fruiting body tissues with good shapes for isolated culture, and carrying out seed production and sowing in a winter greenhouse for cultivation. And (4) counting the fruiting yield performance of the mushroom, and further verifying the stability of high yield, wherein the cultivation method is the same as that in example 4. The test results are shown in Table 4, and the fruiting site of the strain R5 is shown in FIG. 4.
Table 4: second generation fruiting verification test result of strain (CGMCC NO.17677)
As can be seen from Table 4, the selectionSeparating and culturing fruiting body of good shape during R5 (Morchella esculenta No. 1), culturing again, forming primordia at 40d after seeding strain R5, and obtaining good fruiting effect with yield of 218.60kg/667m2The fruiting performance is stable, which shows that the strains R5 have better high-yield genetic stability.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
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Claims (7)
1. The high-yield morchella esculenta strain is named as morchella esculenta No.1, is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has the preservation number as follows: CGMCC number 17677; the black morchella strains M10 and M20 are used as parents for protoplast fusion, and a protoplast interspecific fusion technology is adopted; the strain M10 is morchella terrapin bred by agricultural academy in Sichuan province, and the strain M20 is morchella hexameiica.
2. Use of the morchella strain of claim 1 as a parent for breeding.
3. Fruiting body obtained by cultivating Morchella strain according to claim 1.
4. Mycelium and/or spores obtained by culturing the morchella strain according to claim 1.
5. The method for cultivating the morchella strain according to claim 1, comprising the following steps:
(1) inoculating stock seed obtained by culturing Morchella esculenta No.1 with preservation number of CGMCC NO.17677 into culture medium of cultivar, and culturing at 22-24 deg.C for 10-15d to obtain cultivar;
(2) sowing seed ditches on the ridge surface, sowing the cultivated seeds obtained in the step (1), covering soil, covering with a film after covering the soil, and moisturizing;
(3) after seeding, when the surface of the furrow is full of the toadstool hyphae to form fungus frost, starting to place a nutrition bag containing a culture material, cutting an opening on one surface of the nutrition bag, and horizontally placing the cut surface of the nutrition bag on the surface of the furrow, so that the toadstool hyphae can directly contact the culture material in the nutrition bag;
(4) placing the nutrition bag for 35-45d, and removing the nutrition bag; uncovering the film after the soil surface begins to form an original base; the temperature of the fruiting body in the growing period is controlled to be 8-20 ℃, the water content of the soil in the fruiting period is controlled to be 20-28%, and the air humidity is controlled to be 85-90%;
in the step (1), the culture medium of the cultivar comprises the following raw materials in percentage by mass:
40% of wheat grains, 45% of sawdust, 10% of humus soil, 3% of wheat bran, 1% of lime and 1% of gypsum;
in the step (2), 20% of turf is added into the soil covering material, and the soil covering thickness is 3 cm;
in the step (3), the culture material is composed of the following raw materials by mass percent:
30% of wheat grains, 68% of wood chips, 1% of lime and 1% of gypsum.
6. Use of the fruit body of claim 3 in food processing.
7. A food product comprising the morchella strain of claim 1;
and/or:
the mycelium and/or fruiting body obtained by the strain cultivation.
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| CN1793315A (en) * | 2005-12-08 | 2006-06-28 | 云南菌苑科技有限公司 | Screening for strain of steepletop hickory chick and process for preparing strain thereof |
| KR20150140145A (en) * | 2014-06-05 | 2015-12-15 | 전라북도 | A artificial culture method for mass producing mycelium of morchella esculenta |
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