A strain of Morchella sextelata JYN13 and its breeding method

Abstract

The present invention provides a strain of Morchella sextelata JYN13 and a breeding method thereof, belonging to the technical field of edible fungi. The Morchella sextelata JYN13 has been deposited in the China Center for Type Culture Collection on February 21, 2023, with a deposit address of Wuhan University, Wuhan, China, and a deposit number of CCTCC NO: M 2023169. Field tests show that the Morchella sextelata JYN13 provided by the present invention has strong adaptability, can be planted in many places, and has a yield of more than 650 kg / mu, with a maximum yield of 1220 kg / mu.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungi, and in particular relates to a Morchella sextelata JYN13 and a breeding method thereof. Background Art

[0002] Morchella spp. belongs to the phylum Ascomycota, class Pezizomycetes, family Morchellaceae, genus Morchella Dill.: Pers. Morchella occupies an important role in the world's food culture. It is not only delicious and crispy, but also has good medicinal value such as anti-tumor, anti-cancer, anti-fatigue, anti-oxidation, immunity enhancement and whitening. At present, the varieties widely promoted and applied are mainly ladder-shaped Morchella (M. importuna), six-sister Morchella (M. sextelata) and a small amount of seven-sister Morchella (M. eximia). The typical characteristics of the six-sister Morchella are that the ascocarp is conical, the overall color of the cap is darker, the pits are brown-black, and the ridges are black to dark black when mature. The pits on the surface of the cap are arranged nearly vertically, and the vertical and horizontal ridges are obvious. It also has obvious advantages in resisting high and low temperature fluctuations when cultivated artificially.

[0003] In recent years, with the continuous development of the Morchella cultivation industry, there have been problems such as single high-quality seed sources, aging of strains, weak resistance to diseases and pests, and the stability of Morchella cultivation is not high. Nearly 70% of practitioners cannot make stable profits every year. That is, the single variety and insufficient strain quality have become one of the key factors for the instability of Morchella cultivation. After searching, there are still few research reports on Morchella hybrid breeding. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a strain of Morchella sextelata JYN13 and a breeding method thereof. The Morchella sextelata JYN13 has strong adaptability, can be planted in many places, and has an acreage yield of more than 650 kg, and the highest acreage yield can reach 1220 kg.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The invention provides a Morchella sextelata JYN13, which is preserved in China Center for Type Culture Collection with a preservation number of CCTCCNO: M 2023169.

[0007] The present invention also provides a breeding method for the Morchella sextelata JYN13, which is obtained by breeding through a multi-spore hybridization method.

[0008] Preferably, the parent strain selected by the polyspore hybridization method is wild Morchella liliiflora.

[0009] The present invention also provides application of the Morchella sextelata JYN13 in cultivation and production.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention obtains a new strain of high-yield Morchella liliumei through multi-spore separation, multi-spore hybridization, hybrid cultivation and fruiting, breeding and identification; the inventors have carried out adaptive cultivation of Morchella liliumei JYN13 in various parts of the country, and the results show that compared with the parent and similar strains, the yield and resistance are significantly increased, especially in the northern region, the highest yield can reach 1220 kg / mu, in Henan, Shandong, Shanxi, Jiangsu, Anhui and Hebei and other places, the yield is more than 650 kg / mu; and the variety is also suitable for factory-based indoor cultivation, with a yield of more than 1.5 kg / m 2 .

[0012] Biological Deposit Description

[0013] Morchella sextelata JYN13 is deposited in the China Center for Type Culture Collection, with the deposit address at Wuhan University, Wuhan, China. The deposit date is February 21, 2023, and the deposit number is CCTCC NO: M2023169. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of ITS phylogenetic analysis of the parent strain 2016-19 and Morchella liumei JYN13, where the sequences with JQ numbers represent publicly published data downloaded from the NCBI public database;

[0015] Figure 2 The results of mating type gene detection of Morchella liumei JYN13, where M is the DL2000 standard molecular marker, and the sizes of the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, respectively; P is the parent strain 2016-19, H is Morchella liumei JYN13, the left picture is the Mat1-1-1 target band, and the right picture is the Mat1-2-1 target band;

[0016] Figure 3 This is the result diagram of antagonism experiment;

[0017] Figure 4 This is the SRAP polymorphism fingerprint of Morchella japonica JYN13 and its parent strain 2016-19. The left lanes are from left to right, and the two lanes form a group. The left lane is the parent strain 2016-19, and the right lane is Morchella japonica JYN13. The primer groups from left to right are: ME1×EM6, ME1×EM7, ME2×EM3, ME2×EM4, ME3×EM5, ME3×EM7, ME4×EM7, ME5×EM4, ME5×EM6, ME5×EM7, ME6×EM4, ME7×EM4, ME7×EM6; the characteristic bands are marked by white arrows.

[0018] Figure 5 These are the morphological characteristics of the Liumei Morel JYN13 at different stages. The left picture shows the morphological characteristics of the small mushrooms of the Liumei Morel JYN13 cultivated in North China, and the right picture shows the morphological characteristics of the mature ascocarps of the Liumei Morel JYN13 cultivated in the Huanghuai River Basin. DETAILED DESCRIPTION

[0019] The invention provides a Morchella sextelata JYN13, which is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M 2023169.

[0020] The breeding method of the six-sister morel JYN13 is as follows: collecting wild six-sister morel specimens and isolating the parent strain 2016-19; cultivating the parent strain 2016-19 to obtain ascocarps, and selecting robust ascocarps with a pagoda shape and obvious cracks in the cap ridges and pits; culturing the robust ascocarps to obtain a multi-sporic hybrid strain; determining the growth rate, hypha density, colony color, number of sclerotia, life span and other parameters of the multi-sporic hybrid strain and the parent strain 2016-19, and selecting a strain with the above parameters significantly better than the parent strain 2016-19 as an excellent parent strain; cultivating the excellent parent strain, selecting ascocarps with a pagoda shape, thick flesh, brown-black or dark-brown cap color, white stipe, stipe height less than 3 cm, and no diseases and insect pests as hybrid mushroom ascocarps; performing tissue separation and purification culture on the stipes of the hybrid mushroom ascocarps to obtain the six-sister morel JYN13.

[0021] The ascocarp of the Six Sister Morel JYN13 is conical, with a medium to large conical cap, a top, brown-black or black, obvious vertical and horizontal ridges, and obvious pits; the junction between the cap and the stipe is concave; the stipe is cylindrical or swollen at the base, with slight grooves at the base, and is white; a single mushroom weighs 28.0 to 52.5 g; the mycelium is cylindrical with a smooth surface, and the sclerotia are small granular.

[0022] The present invention also provides a method for breeding the Morchella sextelata JYN13, which is obtained by breeding through a multi-spore hybridization method.

[0023] In the present invention, the parent strain selected by the polyspore hybridization method is preferably wild Morchella esculenta.

[0024] The present invention also provides application of the Morchella sextelata JYN13 in cultivation and production.

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Example 1 Breeding of parent strains

[0027] 1.1 Collection and isolation of parent strains

[0028] In March 2016, wild Morchella liumei specimens were collected in Qingchuan County, Sichuan Province, and dried naturally. The surface of the ascocarp of the specimen was wiped with a 75% alcohol cotton ball to remove debris and dust. Then, a 0.5 cm thick piece of the stem was cut from the middle of the stem. 2 The tissue blocks were washed and soaked in sterile water, then sterilized in 75% alcohol for 60 seconds, quickly taken out and washed away the adsorbed alcohol in sterile water, and cut into 2 mm pieces with a sterile scalpel on sterile filter paper. 3 Small pieces were placed on CYM solid medium (formula: yeast extract 2g / L, peptone 2g / L, K2HPO4 1g / L, MgSO4 0.5g / L, KH2PO4 0.46g / L, glucose 20g / L, agar powder 20g / L) and cultured at 21°C for 2 days, and the germinated colonies were picked and placed on a new CYM slant to obtain a pure culture strain, numbered 2016-19. The pure culture parent strain 2016-19 was placed in CYM liquid medium and cultured for 7 days to obtain mycelium.

[0029] 1.2 Molecular identification of parent strains

[0030] The CTAB method was used to extract genomic DNA. The specific steps are as follows:

[0031] The mycelium ground by liquid nitrogen was lysed with CTAB lysis buffer (formula: CTAB 2%, PVP 2%, Tris-HCl 100mmol / L, EDTA 20mmol / L, NaCl 1.4mol / L) to obtain a crude DNA extract, which was then deproteinized with phenol: chloroform: isoamyl alcohol (volume ratio of 25:24:1), and then ethanol precipitation and RNAase digestion were used to remove RNA contamination to obtain pure genomic DNA. The obtained DNA was diluted to 50ng / μL and stored at 4°C for later use.

[0032] ITS-1 / ITS-4 universal primers were used to amplify the ITS region of the sample to obtain the target band. The amplification reaction system was: PCR Buffer 1×, MgCl2 1.50 mM, dNTPs Mix 0.25 mM, primers 1.00 mM each, rTaq Polymerase 1.5 U, template DNA 50 ng; amplification reaction conditions were: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 58°C annealing for 30 s; 72°C extension for 60 s, 34 cycles, and finally 72°C extension for 5 min.

[0033] The sequencing of the target band was completed by Beijing Qingke Biotechnology Co., Ltd. through Sanger sequencing; the sequencing results were quality controlled, and after removing low-quality bases, they were submitted to the NCBI public database and the sequence number was obtained as OQ691650.

[0034] The sequences were compared by NCBI blastn, and the results showed that the parent strain 2016-19 and M. sextelata had 100% similarity.

[0035] The reference sequence was downloaded from the NCBI public database, and MAFFT was used for sequence alignment. IQtree2 was then used to automatically detect the best model and construct a phylogenetic tree. The boostrap was set to 1000, and the tree file was visualized using FigTree_v1.4.3. The results are shown in Figure 1 shown.

[0036] The results showed that the test strain was clustered with Morchella importuna, while the nearby Morchella importuna and Morchella eximia were clustered in independent developmental branches. Combined with the morphological characteristics of the ascocarps and phylogenetic analysis, the experimental strain can be confirmed to be Morchella eximia.

[0037] Example 2 Cultivation of parent strain 2016-19

[0038] A cultivation method of parent strain 2016-19, the steps are as follows:

[0039] (1) Use 150kg / mu of quicklime to disinfect the soil and plow the soil to a depth of 25cm. Then, plow the land into beds with a width of 1m and a spacing of 0.2cm. Before sowing, adjust the soil moisture to 28% and the temperature to 16°C.

[0040] (2) The cultivar of the parent strain 2016-19 was sown in rows, covered with soil at a height of 3 cm and then covered with a mulch film with a thickness of 0.6 silk, and the shading rate of the shading net was adjusted to 85-90%, and the bacteria were cultured at 4°C and 22% humidity for 45 days;

[0041] (3) 5 days after sowing, place exogenous nutrient bags on the bed at a quantity of 1800 bags / mu. 25 days after sowing, uncover the ground film, and when the highest temperature of the soil during the day rises to 8°C, water the mushrooms (the amount of watering is based on the saturation of the soil within 15 cm), and keep the air humidity at 95%, the soil temperature at 8°C, and the soil moisture at 33%. 7 days after mushroom induction, adjust the air humidity to 85-95%, the soil temperature to 4-10°C, the soil moisture to 25-28%, and the surface temperature to 4-12°C for primordium maintenance; 10 days later, adjust the air humidity to 75-90%, the soil temperature to 6-10°C, the soil moisture to 22-25%, and the surface temperature to 6-12°C for young mushroom maintenance and mature cultivation, and harvest fresh mushrooms.

[0042] The preparation method of the cultivar is as follows:

[0043] (a) The parent strain 2016-19 was inoculated into a modified PDA medium and cultured at 21°C for 6 days to obtain the mother strain;

[0044] (b) inoculating the mother seed onto the stock seed matrix and culturing at 21° C. for 13 days to obtain the stock seed;

[0045] (c) The original seed was inoculated onto the cultivar substrate and cultured at 18°C ​​for 15 days to obtain the cultivar.

[0046] The formula of the improved PDA medium is: 200 g / L potato boiled juice, 20 g / L glucose, 15 g / L wheat bran juice, 20 g / L agar powder, 1 L distilled water, natural pH; obtained by high pressure steam sterilization at 121° C. for 30 min;

[0047] The formula of the original seed matrix is: 15% wheat, 82.5% sawdust, 1% lime, 1.5% gypsum; obtained by high pressure steam sterilization at 121°C for 150 minutes;

[0048] The formula of the cultivation substrate is: 33% wheat, 64.5% sawdust, 1% lime, 1.5% gypsum; obtained by high pressure steam sterilization at 121°C for 210 minutes;

[0049] The formula of the exogenous nutrient bag is: 64.5% wheat, 32.5% corn cob, 1% lime, 1.5% gypsum, and 0.5% potassium dihydrogen phosphate; the bag is obtained by high-pressure steam sterilization at 121° C. for 210 minutes.

[0050] Example 3 Construction of polysporic hybrid strains

[0051] Among the ascocarps obtained by cultivation in Example 2, the robust ascocarps with pagoda-shaped ascocarps and obvious cracks in the cap ridges and pits were selected, and the ascocarps were hung about 3 cm above the sterile culture dish in the clean bench, and the whole ascocarps and the culture dish were covered with a large clean beaker of 5L specification, and kept overnight at room temperature of 20-25°C. The next morning, a large number of light white ascospore prints could be observed on the culture dish below.

[0052] Under sterile conditions, use an inoculation needle to dip spores and streak on CYM solid culture medium. Culture at a constant temperature of 21°C. After 2 days, a large number of dense germination colonies can be observed in the streaked area.

[0053] Dense and robust colonies were picked up and placed on new CYM slant medium, and purified and cultured at 21°C for 7 days to obtain candidate multi-sporic hybrid strains. A total of 20 multi-sporic hybrid strains were selected for subsequent screening and evaluation.

[0054] The growth rate, mycelial density, colony color and number of sclerotia of the polysporic hybrid strain and the parent strain 2016-19 were tested respectively by the cross-streak method on a 9 cm diameter plate containing PDA culture medium; and the lifespan of the polysporic hybrid strain and the parent strain 2016-19 was determined by referring to the subculture method described in the invention titled “A method for identifying aging of morel strains” with patent application number “CN201710407468.6”.

[0055] The polysporic strains with significantly better traits than the parent strain 2016-19 were selected as candidate excellent parent strains. A total of 5 excellent parent strains were selected from 20 polysporic hybrid strains, with less colony pigments, uniform colony growth rate, large sclerotium volume, and mycelial lifespan of the excellent parent strains greater than 2400h and total growth distance greater than 150cm.

[0056] Example 4 Selection of Morchella sextelata JYN13

[0057] In 2018-2020, the candidate excellent parent strains in Example 3 were cultivated in the planting base in Jianyang City, Sichuan Province, the Huangpi Morchella planting base in Wuhan City, and Guangyuan City, Sichuan Province according to the method of Example 2. The mycelial growth state, sclerotial state and aging rate of the candidate excellent parent strains were recorded and measured in detail, the fungus state after sowing, the occurrence time of fungus frost, the mycelial feeding state, the full bag time, the nutrient loss rate of the exogenous nutrient bag, the time of fungus frost disappearance, the ground temperature when the primordium occurs, the primordium density, the primordium survival rate, the mature picking time, the yield, and the occurrence of diseases and insect pests were recorded after the exogenous nutrient bags were placed.

[0058] Appearance characteristics: mushroom-shaped pagoda, height 12.0-14.5cm, widest part 5.5-6.5cm, thick flesh, stipe thickness 2.5-4.0mm, cap color brown-black or dark brown, stipe white, stipe height less than 3-3.5cm, free from diseases and insect pests.

[0059] The ascocarps obtained by cultivating the superior parent strains meeting the above-mentioned appearance characteristics were selected as hybrid mushroom ascocarps. The stipes of the hybrid mushroom ascocarps were tissue separated by the method of Section 1.1 of Example 1, wherein the culture medium was CYM solid culture medium. Dense and front-end neat germination colonies were selected and inoculated into CYM slant culture medium for purification culture to obtain a hybrid strain, which was labeled as JYN13.

[0060] Example 5 Cultivation of Morchella sextelata JYN13

[0061] A cultivation method of Morchella liumei JYN13, comprising the following steps:

[0062] (1) Use 150kg / mu of quicklime to spread evenly on the land surface, then plow to 30cm deep, and then arrange the land into 1.2m wide and 0.2cm spacing. Before sowing, adjust the soil moisture to 22-25% and the maximum temperature to 15-16℃;

[0063] (2) Sowing the cultivar of Morchella japonica JYN13 by row sowing or broadcasting, covering with soil with a thickness of 3 cm, and then covering with a mulch film with a thickness of 0.6 silk; the shading rate of the shading net is 85%; then entering the fungus cultivation stage, the fungus cultivation cycle is preferably greater than 45 days;

[0064] (3) 10 days after sowing, place exogenous nutrient bags on the bed at a quantity of 1800-2000 bags / mu. 35 days after sowing, uncover the ground film. When the highest temperature of the soil during the day rises to 6-8°C, water the mushrooms (the amount of watering is based on the water saturation of the soil within 15 cm), and keep the air humidity at 85-95%, the soil temperature at 4-10°C, and the soil moisture at 23-25%. 5 days after mushroom induction, adjust the air humidity to 80-90%, the soil temperature at 4-10°C, the soil moisture at 2-25%, and the surface temperature at 4-15°C for primordium maintenance. 15 days later, maintain the air humidity at 75-95%, the soil temperature at 4-12°C, the soil moisture at 22-24%, and the surface temperature at 4-16°C for small mushroom maintenance and mature culture, and harvest fresh mushrooms.

[0065] The preparation method of the cultivar is as follows:

[0066] (a) inoculating the Morchella lilii JYN13 onto a modified PDA medium and culturing at 21° C. for 10 days to obtain a mother strain;

[0067] (b) inoculating the mother seed onto the stock seed matrix and culturing at 21° C. for 13 days to obtain the stock seed;

[0068] (c) The original seed is inoculated onto the cultivar substrate and cultured at 18°C ​​for 20 days to obtain the cultivar.

[0069] The formula of the improved PDA medium is: 200 g / L potato juice, 18 g / L glucose, 15 g / L bran juice, 20 g / L agar powder, 1 L distilled water, natural pH; obtained by high pressure steam sterilization at 121° C. for 30 min;

[0070] The formula of the original seed matrix is: 15% wheat, 82.5% sawdust, 1% lime, 1.5% gypsum; obtained by high pressure steam sterilization at 121°C for 150 minutes;

[0071] The formula of the cultivation substrate is: 33% wheat, 64.5% sawdust, 1% lime, 1.5% gypsum; obtained by high pressure steam sterilization at 121°C for 210 minutes;

[0072] The formula of the exogenous nutrient bag is: 64.5% wheat, 32.5% corn cob, 1% lime, 1.5% gypsum, and 0.5% potassium dihydrogen phosphate; the bag is obtained by high-pressure steam sterilization at 121° C. for 210 minutes.

[0073] Test Example 1: Detection of strain mating type gene

[0074] The parent strain 2016-19 and the hybrid strain JYN13 were cultured according to the methods of 1.1 and 1.2 in Example 1 to obtain mycelium and genomic DNA. Referring to the amplification method of the mating type gene described in "Experimental cultivation and polarity analysis of single spore and hybrid population of Morchella tiliacea" (Liu Wei, Cai Yingli, He Peixin, et al. Experimental cultivation and polarity analysis of single spore and hybrid population of Morchella tiliacea [J]. Fungal Research, 2019, 17(01): 43-49. DOI: 10.13341 / j.jfr.2018.1238.), the mating type gene of the parent strain 2016-19 and the hybrid strain JYN13 was detected using Mat1-1-1 and Mat1-2-1 primers, and the reaction was detected by 1.5% agarose gel electrophoresis after completion.

[0075] The amplification system was as follows: PCR Buffer 1×, MgCl2 1.50 mM, dNTPs Mix 0.25 mM, primers 1.00 mM each, rTaq Polymerase 1.5 U, and template DNA 50 ng; the amplification conditions were as follows: pre-denaturation at 95°C for 3 min, followed by 95°C for 30 s, 60°C for 20 s, and 72°C for 30 s, for 34 cycles; and finally extension at 72°C for 5 min.

[0076] The results are as follows Figure 2 As shown, the results showed that both the parent strain 2016-19 and the hybrid strain JYN13 contained two mating type genes.

[0077] Test Example 2 Antagonism Experiment

[0078] The activated strains were inoculated in pairs 3 cm apart on the central axis of a 9 cm diameter plate containing PDA solid medium. The pairing combinations included Morchella japonica JYN13×Morchella japonica JYN13, Morchella japonica JYN13×parent strain 2016-19, and parent strain 2016-19×parent strain 2016-19. After constant temperature culture at 21°C for 14 days, the antagonism of the paired strains was observed and recorded. The results are shown in Figure 3 shown.

[0079] The results showed that the strains could fuse and there was no antagonistic line, while the six-sister morel JYN13 and the parent strain 2016-19 had obvious antagonistic lines, indicating that the six-sister morel JYN13 obtained in the experiment was a new strain different from the parent strain 2016-19.

[0080] Experimental Example 3 SRAP molecular identification of Morchella jyn13 and parent strain 2016-19

[0081] Referring to the contents of Sections 1.1 and 1.2 of Example 1, Morchella japonica JYN13 and the parent strain 2016-19 were cultured to obtain the corresponding mycelium and DNA.

[0082] The SRAP primer set described in "Construction of Molecular Genetic Linkage Map of Lentinus edodes" (Liu Wei. Construction of Molecular Genetic Linkage Map of Lentinus edodes [D]. Huazhong Agricultural University, 2011.) was used to perform SRAP (sequence-related amplification polymorphism) amplification on Morchella japonica JYN13 and the parent strain 2016-19. The amplified products were electrophoresed by 6% polyacrylamide denaturing gel and silver stained to statistically analyze the polymorphism. The results are as follows Figure 4 shown.

[0083] Wherein, the SRAP primer sets are ME1×EM6, ME1×EM7, ME2×EM3, ME2×EM4, ME3×EM5, ME3×EM7, ME4×EM7, ME5×EM4, ME5×EM6, ME5×EM7, ME6×EM4, ME7×EM4, ME7×EM6;

[0084] The PCR amplification system was: PCR Buffer 1×, MgCl2 2.0 mM, dNTPs Mix 0.6 mM, upstream and downstream primers 0.4 μM each, rTaq Polymerase 1.75 U, template DNA 50 ng; the amplification conditions were: pre-denaturation at 94°C for 5 min, followed by 5 low-temperature annealing cycles: denaturation at 94°C for 1 min, renaturation at 35°C for 1 min, and extension at 72°C for 1 min; followed by 35 cycles: denaturation at 94°C for 1 min, renaturation at 50°C for 1 min, and extension at 72°C for 1 min; and finally fill-in at 72°C for 7 min.

[0085] The amplification results showed that the 13 pairs of SRAP primer sets can well amplify the parent strain 2016-19 and the six-sister morel JYN13, among which the parent strain 2016-19 produced a total of 107 characteristic bands. Compared with the parent strain 2016-19, the six-sister morel JYN13 had one more characteristic band in ME2×EM3 and ME7×EM6, respectively, compared with the parent strain 2016-19, and the band sizes were about 325bp and 315bp, respectively. This shows that the strain obtained by multi-spore hybridization in the present invention is a new strain that is significantly different from the parent strain 2016-19, and the primer combination and fingerprint map tested can be used for the identification of the hybrid strain.

[0086] Test Example 4 Field Test

[0087] The hybrid strain JYN13 and the parent strain 2016-19 were cultivated in Xinjin base in Chengdu, Sichuan Province, Wuhan base in Hubei Province, Zhengzhou, Henan Province, Kaifeng, Henan Province and Xingtai, Hebei Province respectively by the method of Example 2, and the cultivation schemes were all carried out in vegetable greenhouses. The results are shown in Table 1.

[0088] Table 1 Yield of hybrid strains

[0089]

[0090] As shown in Table 1, compared with the parent strain 2016-19, the yield of the hybrid strain Morchella japonica JYN13 was significantly improved, with the increases of 34.4% and 37.6% in the two bases respectively, and it performed well in large-scale (5 to 30 acres) production, with the highest yield reaching 1220 kg.

[0091] Experimental Example 5 Physiological and cultivation characteristics of the hybrid strain Morchella liumei JYN13

[0092] According to the method of Example 5, the hybrid strain Morchella liumei JYN13 was cultivated in the North China region and the Huanghuai River Basin, and the physiological and cultivation characteristics of the hybrid strain Morchella liumei JYN13 were observed. Figure 5 shown.

[0093] The results showed that the ascocarps of Liumei Morchella JYN13 were conical, medium to large, 10.1-16.8 cm high, and 2.5-4.5 cm at the widest part; the cap was conical, with a pointed tip, brown-black or black, with obvious vertical and horizontal ridges and pits; the junction between the cap and the stipe was concave, 3-5 mm; the stipe was cylindrical or swollen at the base, with slight grooves at the base, and was white; the weight of a single mushroom was 28.0-52.5 g, the diameter of the cap at the widest part was 2.5-4.5 cm, and the length of the cap was 6.7-12.8 cm; the stipe was 2.2-3.4 cm wide and 2.2-3.4 cm long, and the dry-to-dry ratio of fresh mushrooms was 7.0-8.4:1.

[0094] The hyphae are cylindrical and smooth, with particles of varying sizes on the surface of some hyphae. The hyphae diameter is 6.0-21.8μm, the cell length is 65-205μm, the nucleus is multi-nucleated, the septum is obvious, the initial hyphae is colorless, and the old hyphae are brown or brownish yellow. The hyphae are small granular, and they occur around the inoculation block on the PDA medium, with a diameter of 1-3mm. A large number of hyphae can be condensed into sheet-like hyphae, which are white in the early stage and light yellow or light brown in the later stage. Asexual spores are produced 5-7 days after sowing. They are fine white powder in the early stage, and then they occur in large quantities. The bottleneck produces spores, with 4-6 asexual spore stalks, and the asexual spores are spherical, 3.5-4.5μm.

[0095] The octoprosporangium has a spore size of 18-25×10-17μm, is oval, and has a smooth, homogeneous cell surface; the spore print is bright yellow; the ascus is 200-300×5-26μm; cylindrical with a rounded apex, a pointed tip, and colorless; the lateral filaments are 175-320×5-15μm, cylindrical with a rounded to nearly club-shaped, nearly conical, or nearly spindle-shaped apex, with septa, and appear colorless in a 2% KOH test; the setae on the sterile ridge are 50-180×5-25μm, with septa, and appear brown to tan in a 2% KOH test, and the apical cells are cylindrical with a rounded apex, nearly spindle-shaped, capitate, nearly conical, or nearly spindle-shaped.

[0096] The optimum culture temperature of Morchella japonica JYN13 in PDA medium is 21℃, the average growth rate is 1.65±0.2cm / d, the colonies are round, the front ends are neat, the aerial hyphae are obvious, the amount of hyphae is moderate, and the color is colorless, white, and light white at the beginning. As the culture time increases, it gradually deepens to light yellow-white and light yellow, and no serious pigmentation is produced.

[0097] The optimum culture temperature in the wheat grain sawdust culture medium is 21°C, and the average growth rate is about 1.54±0.15cm / d. The mycelium is white at the beginning and slightly sparse. As the culture time increases, the mycelium density increases. Sclerotia begin to form when the mycelium grows to nearly full bag. They are small granular, white at the beginning, and light yellow to light brown in the later stage. There are more sclerotia in the middle and upper part of the bag. Using plastic or glass original seed bottles, the amount of sclerotia is significantly greater.

[0098] The germination conditions and characteristics of Liumei Morchella JYN13: The best sowing temperature is when the soil temperature is less than 16℃. 24 hours after sowing, fine hyphae can be seen sprouting from the surface of the seed block. After 2 to 3 days, an initial fine hyphae network can be formed. After 4 to 5 days, an obvious hyphae layer can be formed, which is dense and colorless or light white in the early stage.

[0099] Conditions and characteristics of the formation of fungal frost of Liumei Morel JYN13: Fungal frost is formed 5 to 7 days after sowing. In the early stage, the fungal frost is sparse white powder. Later, as time goes by, the mycelium exposed to the surface will all form large pieces of white powdery fungal frost. The fungal frost begins to change color to slightly yellow or light yellow-white 35 to 40 days after its formation, accompanied by signs of fading.

[0100] Conditions and characteristics of primordium formation of Morchella JYN13: Primordia begin to form when the highest ground temperature reaches 6-8℃. The primordia are initially white and spherical, densely packed. The initial primordia are fleshy white and spherical, then grow upward to a cone or rod shape, with the base swollen. The surface temperature is 8-14℃, which is the best development temperature. It can grow to 1cm in about 8-12 days and enter the young mushroom differentiation stage.

[0101] The conditions and characteristics of ascocarp development of Liumei Morchella JYN13: The temperature for ascocarp development is 6-18℃, the most suitable surface temperature is 10-15℃, the air humidity is 80-95%, the soil humidity is 20-25%, and it takes 16-22 days from a young mushroom of about 1cm to mature. The higher the ambient temperature, the faster the ascocarp development. The young ascocarp has a broad conical cap, which is gray-black to gray-brown, and the stipe is white and cylindrical with a slightly swollen base. As time goes by, the pits on the cap surface become more prominent, and the longitudinal ridges on the cap surface are arranged up and down as a whole, slightly staggered and curved, with thick ridges and a lighter color than that of young mushrooms. The mature ascocarp is conical, brown-black, with obvious and deep pits, thin and forked longitudinal ridges, inclined transverse ridges and staggered longitudinal ridges, cap 6.7-12.8cm, 2.5-4.5cm at the widest point, stipe cylindrical, short, slightly swollen base, occasionally with small grooves, 2.2-3.4cm wide, 2.2-3.4cm long. In conventional field cultivation, the average yield of fresh mushrooms can reach 1220kg / mu at the highest, and an average of 650kg / mu over a large area of ​​the North China Plain.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Six-sister Morel ( Morchella sextelata ) The application of JYN13 in cultivation production is characterized by: The cultivation and production location is Xingtai City, Hebei Province; the cultivation and production is field cultivation; the six-sister morel Morchella sextelata JYN13 is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 2023169.

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