Temperature-resistant high-yield strain Gannong morchella esculenta A11 and application thereof

Abstract

The invention relates to a temperature-resistant high-yield strain Gannong morchella esculenta A11 and application thereof, and belongs to the technical field of edible mushrooms. The name of the strain disclosed by the invention is Gannong morchella A11, and the strain is classified and named as a protoplast fusion strain Morchella importuna * Mes-12amp of morchella esculenta and morchella vulgaris, and the protoplast fusion strain is a protoplast fusion strain Morchella importuna * Mes-12amp of morchella esculenta. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the strain is CGMCC NO.40670. The invention further discloses a preparation method of the strain. The Jiannong morchella A11 bred by the method is high in hypha growth speed, large in ascocarp differentiation number, high in fruiting yield and particularly tolerant to early spring short-time high temperature, a new high-quality strain is provided for cultivation of morchella in middle-subtropical regions in the south of China, and the Jiannong morchella A11 has good potential of serving as a genetic diversity breeding material for breeding subtropical thermophilic morchella.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungi, and specifically relates to a temperature-resistant high-yield Morchella strain Gannong Morchella A11 and an application thereof. Background Art

[0002] Morchella Dill.ex Pers.:Fr. belongs to the phylum Ascomycota, class Discomycetes, order Pezizales, family Morchellaceae. It has the characteristics of tender meat, unique flavor, rich vitamins and amino acids, and high edible value. It is recognized as one of the rare edible fungi. At the same time, Morchella also contains active polysaccharides, phenolic compounds, ascorbic acid and vitamin D, etc., and has medicinal functions such as anti-tumor, anti-oxidation, and protection of internal organs.

[0003] According to incomplete statistics, the cultivation area of ​​morels in China has reached 250,000 mu in 2022, and the cultivation area in Jiangxi Province was about 6,000 mu at the beginning of 2023. Despite this, it is difficult to achieve stable and high yields every year in the production of morels, mainly because morels are low-temperature fungi, the most suitable temperature for mycelial growth is 15-22°C, the temperature for ascocarp differentiation is 10-12°C, and the most suitable temperature for growth is 12-16°C. Jiangxi Province is located in the subtropical climate zone. From November of morel cultivation to February and March of the following year, high temperatures of more than 20°C often appear on the surface for a short period of time, causing a large number of young mushroom caps to turn red and stop growing. Some young mushrooms only have the base of the stalk swollen but not the cap, with small flowers and low yields, more deformed mushrooms, and even dead mushrooms. The phenomenon of reduced production or even total crop failure occurs from time to time, which seriously affects the enthusiasm of mushroom farmers. There is an urgent need for medium-temperature morel production seeds suitable for the subtropical climate zone.

[0004] There are many species of Morchella in nature. According to the latest data, there are 313 classification names under the genus Morchella (including species, subspecies and variants), but only 8 species can be cultivated artificially, and only 3 strains can be cultivated commercially on a large scale, namely Morchella tiliacea, Morchella liumei and Morchella qimei, all of which are black Morchella. It is still difficult to conquer the domestication and cultivation of wild yellow Morchella subfamily species. According to the wild yellow Morchella we picked from Jiangxi and the mushroom production time in mid-to-early April, this wild species is a good material for breeding genetic diversity of subtropical thermophilic Morchella. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a temperature-resistant high-yield Morchella (Morchella importuna × Mes-12 & Mes-22) strain, Gannong Morchella A11. The mycelium growth rate of the strain is fast, the number of ascocarp differentiation is large, the mushroom yield is high, and in particular, it can tolerate short-term high temperatures in early spring, providing a new high-quality strain for the cultivation of Morchella in the southern subtropical region.

[0006] The present invention relates to the following technical solutions:

[0007] The present invention provides a temperature-resistant and high-yield strain, Gannong Morchella A11, which is classified and named as the protoplast fusion strain of Morchella importuna × Mes-12 & Mes-22, and the strain has been deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​June 25, 2023, and its preservation number is: CGMCC NO.40670.

[0008] Furthermore, the Gannong Morchella A11 is obtained by using the cultivated black morel JFRL01-107 and the wild yellow morel JFRL01-161 as parents and adopting the protoplast interspecific fusion technology.

[0009] Furthermore, the black morel JFRL01-107 is a cultivated species Morchella importuna for production in Jiangxi. Pure cultured mycelium is obtained through tissue separation, purification and identification. The ITS sequence of the strain is shown in SEQ ID NO: 1.

[0010] Furthermore, the yellow morel JFRL01-161 is a temperature-resistant wild yellow morel Mes-12 & Mes-22 collected from Yingtan, Jiangxi Province and obtained by tissue separation. Pure cultured mycelium is obtained through tissue separation, purification and identification. The ITS sequence of the strain is shown in SEQ ID NO: 2.

[0011] Furthermore, the ITS sequence of the protoplast fusion strain Morchella importuna×Mes-12&Mes-22 of the Gannong Morchella A11 Morchella importuna and Morchella flavescentis is shown in SEQ ID NO:3.

[0012] The present invention also provides an ISSR primer set for identifying the protoplast fusion strain of Morchella importuna×Mes-12&Mes-22 of Morchella importuna A11 and Morchella edulis. The primer set consists of 6 primers, and the nucleotide sequences are shown in SEQ ID NOs: 4-9 respectively.

[0013] The present invention also provides an application of the Gannong Morchella A11 as a parent in breeding.

[0014] The present invention also provides a fruiting body obtained by cultivating the Gannong Morchella A11.

[0015] The present invention also provides mycelium and / or spores obtained by culturing the Gannong Morchella A11.

[0016] The present invention also provides application of the fruiting body and / or mycelium and / or spore in food or health product processing.

[0017] The present invention also provides a food or health product, wherein the food contains the Gannong Morchella A11 and / or mycelium and / or fruiting body and / or spores obtained by culturing the strain.

[0018] The present invention also provides a cultivation method of the Gannong Morchella A11, which is characterized by comprising the following steps:

[0019] (1) culturing the Morchella strain A11, inoculating the obtained stock into a cultivar culture medium, and culturing at 18±2° C. for 18-22 days to obtain a cultivar;

[0020] (2) making a sowing furrow on the bed, breaking the cultivated seeds obtained in step (1) into small pieces and scattering them into the covering soil, and then covering the covering soil with a black mulch film;

[0021] (3) After sowing, when the mycelium has grown all over the bed to form a "fungus frost", start placing exogenous nutrient bags, make an oblique cut on one side of the nutrient bag, place the cut side flat on the bed, and gently compact it;

[0022] (4) When the mycelium and fungus frost begin to turn yellow, the film can be removed and water can be poured. After pouring the water, a small arch shed covered with film can be built in time to allow primordia to form and differentiate on the soil surface. When the young mushrooms grow to slightly unfold, the film can be removed.

[0023] Furthermore, the temperature during the growth period of the young mushrooms is controlled at 8-20°C, the soil moisture content is controlled at 20-28%, and the air humidity is controlled at 85-90%.

[0024] Furthermore, in step (1), the cultivation medium is composed of the following raw materials in percentage by mass: 75% wheat, 23% sawdust, 1% lime and 1% gypsum.

[0025] Furthermore, in step (2), the thickness of the covering soil is 3 cm.

[0026] Furthermore, in step (3), the exogenous nutrient bag is composed of the following raw materials in percentage by mass: 60% wheat grains, 38% corn cobs, 1% lime, and 1% gypsum.

[0027] Compared with the prior art, this application has the following beneficial effects:

[0028] The temperature-resistant high-yield Morchella strain Gannong Morchella A11 (CGMCCNO.40670) bred by the present invention has a fast mycelial growth rate, a large number of differentiated ascocarps, and a high mushroom yield. Compared with the parent, the yield of the strain of the present invention can be increased by more than 33%. In addition, the Gannong Morchella strain A11 (Morchella importuna×Mes-12&Mes-22) of the present invention tolerates short-term high temperatures in early spring, provides a new high-quality strain for Morchella cultivation in the mid-subtropical region of southern my country, and has good potential as a genetic diversity breeding material for breeding subtropical thermophilic Morchella. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 These are pictures of Morchella protoplasts; the left picture is Morchella esculenta JFRL01-107, and the right picture is Morchella flavescentis JFRL01-161.

[0031] Figure 2 These are the morphological pictures of the fruiting bodies in the mushroom fruiting test; the left picture is Gannong Morchella A11 (CGMCCNO.40670), and the right picture is Morchella esculenta JFRL01-107.

[0032] Figure 3 This is the antagonism diagram between the fusion strain Morchella edulis A11 (CGMCC NO.40670) and its parent; the left side is Morchella edulis JFRL01-107, the right side is Morchella flavescentis JFRL01-161, and the bottom is Morchella edulis A11 (CGMCC NO.40670).

[0033] Figure 4 It is a UPGMA cluster diagram constructed based on the ISSR electrophoresis pattern. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples. The experimental methods used in the following examples are all conventional methods in the art unless otherwise specified; the materials and reagents used are all reagents and materials obtained from commercial sources unless otherwise specified.

[0035] It is particularly noted that JFRL01-107 (Morchella importuna) is a cultivated Morchella importuna collected from the cultivation base of Lingxi Village, Guixi City, Jiangxi Province, and JFRL01-161 (Mes-12 & Mes-22) is a wild Morchella yellow, collected from Lingxi Village, Guixi City, Jiangxi Province.

[0036] Lytic enzyme was purchased from Guangdong Bide Biotechnology Co., Ltd.; snail enzyme was purchased from Beijing Solebow Technology Co., Ltd.

[0037] Example 1 Breeding of temperature-resistant high-yield Morchella strains

[0038] The breeding of a temperature-resistant high-yield Morchella strain comprises the following steps:

[0039] (1) Taking the front end of the vigorously growing mycelium on the PDA medium, inoculating it into a 250 mL triangular flask filled with complete yeast liquid medium (CYM), the bottling volume is 100 mL, and 5-6 7 mm fungus blocks are added to each bottle, and cultured at 25° C. in the dark for 3 days; picking the mycelium under sterile conditions, and drying the water with sterile filter paper, so as to obtain fresh mycelium of Morchella;

[0040] (2) Take fresh mycelium of Morchella esculenta JFRL01-107 and Morchella fusca JFRL01-161, and perform enzymolysis at a ratio of 200 mg wet weight to 1 mL enzyme solution. The enzymolysis temperature of Morchella esculenta and Morchella fusca is 30° C. and the enzymolysis time is 3 h. The enzymolysis of Morchella esculenta mycelium described in the present invention is as follows: 0.6 mol / L mannitol is used as an osmotic stabilizer, and enzymolysis is performed under the action of 2% lytic enzyme and 1% snail enzyme;

[0041] (3) After the enzymatic hydrolysis is completed, the protoplasts are filtered through a double-layer lens paper to remove the mycelial fragments, and the protoplasts are collected by centrifugation at 3000 rpm for 10 min. The protoplasts are washed with 0.6 mol / L mannitol osmotic agent, and the protoplasts are collected by centrifugation. The washing is repeated twice. Finally, the protoplasts are suspended in the osmotic agent to obtain a purified protoplast suspension (see protoplasts). Figure 1 );

[0042] (4) The purified protoplast suspension was adjusted to 10 5 / mL, take 2mL of the protoplast suspension of Morchella serrata, of which 1mL is heat-inactivated at 50℃ for 150s, and 1mL is inactivated by vertical irradiation at 30cm under a 30w ultraviolet lamp for 120s; take 2mL of the protoplast suspension of Morchella flavescentis, of which 1mL is heat-inactivated at 50℃ for 120s, and 1mL is inactivated by vertical irradiation at 30cm under a 30w ultraviolet lamp for 90s;

[0043] (5) The protoplast suspensions of different species and different inactivation methods were mixed in a volume ratio of 1:1, centrifuged at 3000 rpm for 5 min, and the supernatant was removed. An equal volume of 30% PEG solution preheated at 30°C was slowly added along the wall of the centrifuge tube, gently shaken to mix, kept at 30°C for 20 min, centrifuged at 2000 rpm for 10 min, and the supernatant was removed. The mixture was washed with sterile 0.6 mol / L mannitol osmotic agent and centrifuged twice;

[0044] (6) 200 μL of the fused protoplasm suspension was spread on a solid soft agar medium containing 0.6 mol / L mannitol and cultured at 24°C in the dark;

[0045] (7) After microcolonies grow on the regeneration medium, pick the microcolonies and transfer them to PDA medium to obtain a target fusion strain, namely, Gannong Morchella A11 (CGMCC NO.40670).

[0046] Wherein, the complete yeast liquid medium (CYM) in step (1) is: 20 g glucose, 2 g peptone, 1 g yeast extract, 0.5 g magnesium sulfate, 0.46 g potassium dihydrogen phosphate, 1 g dipotassium hydrogen phosphate, 0.6 mol / L mannitol, 1000 mL water, pH natural;

[0047] In step (5), the protoplast suspensions of different species inactivated in different ways are mixed in a volume ratio of 1:1, and the combinations are: UV-inactivated Morchella esculenta × heat-inactivated Morchella edulis; heat-inactivated Morchella esculenta × UV-inactivated Morchella edulis;

[0048] The regenerated solid soft agar medium in step (6) is composed of: 20 g glucose, 2 g peptone, 1 g yeast extract, 0.5 g magnesium sulfate, 0.46 g potassium dihydrogen phosphate, 1 g dipotassium hydrogen phosphate, 12 g / L agar, 0.6 mol / L mannitol, 1000 mL water, and natural pH.

[0049] Example 2: Identification of a thermotolerant and high-yielding Morchella strain

[0050] 1. Morphological identification

[0051] Gannong Morchella A11 (CGMCC NO.40670, Morchella importuna×Mes-12&Mes-22) ascocarp (see Figure 2 (Left picture) The cap is dark brown or yellowish brown with a yellow tip, 5-8 cm high, 3-6 cm wide, conical, and the ascocarp has 12 to 17 disorderly longitudinal ridges and shallow transverse ridges; the stipe is 2-6 cm long, 1-3 cm thick, white to light brown, cylindrical, slightly swollen at the base, and hollow; the ascus is columnar, each containing eight spores, arranged in a single longitudinal arrangement.

[0052] Morchella tiliacea JFRL01-107 fruiting body (see Figure 2 The fruiting body (pictured on the right) is smaller, with a dark brown or light brown cap, 4-6 cm high, 3-5 cm wide, conical, and the ascocarp has 11 to 17 disorderly longitudinal ridges and shallow transverse ridges; the stipe is 2-5 cm long, 1-2 cm thick, white, cylindrical, and hollow; the ascus is columnar, each containing eight spores, arranged in a single longitudinal arrangement.

[0053] 2. Antagonism test

[0054] The parent strains JFRL01-107 and JFRL01-161 and the fusion strain Gannong Morchella A11 (CGMCC NO.40670) were inoculated onto PDA plates and cultured in the dark at 24°C for 3-5 days. It can be seen at the junction of hyphae that Gannong Morchella A11 (CGMCC NO.40670) and the two parent strains have antagonistic lines, and the hyphae at the antagonistic lines are slightly raised (see Figure 3 ), indicating that the fusion strain A11 has certain differences from both parents and belongs to different strains.

[0055] 3. Identification of the relationship between the fusion strain and the parent strain (ISSR)

[0056] The genomic DNA of three strains of the parent strains JFRL01-107, JFRL01-161 and Gannong Morchella A11 (CGMCCNO.40670) of the present invention were extracted using a novel plant genomic DNA extraction kit CW0531 (Beijing Kangwei Century Company), as shown in the sequences SEQ ID NO: 1-3, and 6 ISSR primer sets (see the table below) were selected for PCR amplification. The ISSR primer sequences are as follows:

[0057]

[0058] The reaction system for ISSR PCR amplification was: 2×Taq Master Mix 12.5μL, ddH2O 5μL, primer 1.5μL, DNA template 1μL; ISSR PCR reaction conditions were: 94℃ pre-denaturation for 5min, 94℃ denaturation for 45s, 53℃ annealing for 45s, 72℃ extension for 60s, 35 cycles; 72℃ extension for 7min, and storage at 4℃.

[0059] The bands were read according to their mobility on the electrophoresis spectrum, with the presence of bands recorded as "1" and the absence of bands recorded as "0". Ambiguous bands were discarded, and a data matrix was established in an Excel table. UPGMA (Unweighted Pair-Group Mean Average) cluster analysis was performed using the software Ntsys v2.1e.

[0060] From the UPGMA cluster diagram (see Figure 4 ) can be seen from the data that at the similarity level coefficient of 0.29, they are divided into two groups, one is the yellow morel JFRL01-161, and the other is composed of JFRL01-107 and the fusion strain Gannong Morel A11. At the similarity level coefficient of 0.70, the parent Morchella esculenta JFRL01-107 and the fusion strain Gannong Morel A11 are divided into different branches, indicating that there is a large gap between the two strains, that is, A11 is a new strain different from the parent.

[0061] Example 3: Mycelial growth rate of a temperature-resistant high-yield Morchella strain

[0062] The activated Gannong Morchella A11 and the two parent strains were punched with a 7mm borer for later use, inoculated onto PDA culture medium, cultured at 24°C in the dark, and the growth of mycelium was recorded every day.

[0063] The results showed that the mycelial growth rate of Gannong Morchella A11 strain was 15.41±0.37 mm / d, while the mycelial growth rates of JFRL01-107 and JFRL01-161 were 12.16±0.41 mm / d and 12.84±0.16 mm / d, respectively. The mycelial growth rate of Gannong Morchella A11 was significantly increased.

[0064] Example 4: Verification of the number of young mushroom buds and yield of a temperature-resistant high-yield Morchella strain

[0065] (1) inoculating the original seed obtained by culturing Morchella strain (CGMCC NO.40670) into a cultivar culture medium, and culturing at 18±2°C for 18-22 days to obtain a cultivar;

[0066] (2) making a sowing furrow on the bed, breaking the cultivated seeds obtained in step (1) into small pieces and scattering them into the covering soil, and then covering the covering soil with a black mulch film;

[0067] (3) After sowing, when the mycelium has grown all over the box and formed a "fungus frost", start placing exogenous nutrient bags, make three oblique cuts on one side of the nutrient bag, place the cut side of the nutrient bag flat on the box, and gently compact it so that the morel mycelium can directly contact the culture medium in the nutrient bag;

[0068] (4) When the mycelium and fungus frost begin to turn yellow, the film can be removed and water can be poured. After pouring the water, a small arch shed covered with film can be built in time. The primordium will form on the soil surface and differentiate. When the young mushrooms grow slightly, the film can be removed. The temperature during the growth period of young mushrooms should be controlled at 8-20℃, the soil moisture content should be controlled at 20-28% during the fruiting period, and the air humidity should be controlled at 85-90%.

[0069] The cultivation conditions were recorded in detail during the cultivation process. The results showed that the number of young mushrooms of Gannong Morchella A11 was 128.00±7.21 / m 2 , the total yield is 351.90±10.83g / m 2 No primordia or young mushrooms were observed in the parent Morchella edulis JFRL01-161, and the number of young mushrooms produced by the parent Morchella edulis JFRL01-107 was 240.00±72.11 / m 2 The total yield is 264.51±70.18g / m 2 Compared with its parent, the juvenile mushroom production density of Gannong Morchella A11 is moderate, the fruiting body is larger, the mushroom shape is better, and the yield can be increased by more than 33%.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present application may have various modifications and variations. Any modification, replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A thermotolerant high-yield strain, Gannong Morchella A11, characterized in that: The strain is Gannong Morchella A11, which is classified and named as the protoplast fusion strain of Morchella importuna×Mes-12&Mes-22 of Morchella importuna and Morchella lutea, and is preserved in the General Microbiology Center of China Microbiological Culture Collection Administration, with the preservation number: CGMCCNO.40670.

2. The Gannong Morchella A11 according to claim 1, characterized in that The Gannong Morchella A11 is obtained by using cultivable black Morchella JFRL01-107 and wild yellow Morchella JFRL01-161 as parents through the use of protoplast interspecific fusion technology.

3. The Gannong Morchella A11 according to claim 2, characterized in that: The black morel JFRL01-107 is a cultivated species Morchella importuna for production in Jiangxi, and the yellow morel JFRL01-161 is a temperature-resistant wild yellow morel Mes-12 & Mes-22 collected from Yingtan, Jiangxi and obtained by tissue separation.

4. Use of the Gannong Morchella A11 described in claim 1 as a parent in breeding.

5. The fruiting body obtained by cultivating the Gannong Morchella A11 described in claim 1.

6. Mycelium and / or spores obtained by culturing the Morchella A11 described in claim 1.

7. Use of the fruiting body according to claim 5 and / or the mycelium and / or spores according to claim 6 in food or health product processing.

8. A food or health product, characterized in that: The food contains the Gannong Morchella A11 described in claim 1 and / or mycelium and / or fruiting bodies and / or spores obtained by culturing the Gannong Morchella A11.

9. The cultivation method of Gannong Morchella A11 according to claim 1, characterized in that: The following steps are involved: (1) Cultivating the Morchella strain according to claim 1, inoculating the obtained stock into a cultivar culture medium, and culturing at 18±2° C. for 18-22 days to obtain a cultivar; (2) making a sowing furrow on the bed, breaking the cultivated seeds obtained in step (1) into small pieces and scattering them into the covering soil, and then covering the covering soil with a black mulch film; (3) After sowing, when the mycelium has grown all over the bed and formed a fungus frost, start placing exogenous nutrient bags, make an oblique cut on one side of the nutrient bag, place the cut side flat on the bed, and gently compact it; (4) When the mycelium and fungus frost begin to turn yellow, the film can be removed and water can be poured. After pouring the water, a small arch shed covered with film can be built in time to allow primordia to form and differentiate on the soil surface. When the young mushrooms grow to slightly unfold, the film can be removed.

10. The cultivation method according to claim 9, wherein the temperature during the growth period of young mushrooms is controlled at 8-20°C, the soil moisture content is controlled at 20-28%, and the air humidity is controlled at 85-90%.

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