Space-carried dictyophora rubrovolvata strain kungs-5 and application thereof

Abstract

The invention relates to a spaceflight-carried dictyophora rubrovolvata strain kungs-5 and application thereof, and belongs to the technical field of strain breeding. The spaceflight-carried dictyophora rubrovolvata strain No.5 in the application is preserved in the China General Microbiological Culture Collection Center on August 9, 2024, the preservation number is CGMCC No.41429, the classification name is dictyophora rubrovolvata, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Mycelia of the dictyophora rubrovolvata strain Kungplant No. 5 are attached, are white and lint-shaped, are light red and amaranth after 10-15 days, and are rapid in germination, rapid in growth, high in vitality, early in fungus egg formation, high in density, rapid in differentiation and suitable for industrial cultivation on common mother strain, stock strain and cultivar culture media of dictyophora rubrovolvata.

Description

Technical Field

[0001] The present application relates to the technical field of strain breeding, and in particular to a space-borne red-stalked bamboo fungus strain Kunzhi No. 5 and its application. Background Art

[0002] Red stem bamboo fungus belongs to the Phallales, Phallaceae, and Dictyophora Phallus ) fungi. Wild red bamboo fungus is mainly distributed in Yunnan, Guizhou and Sichuan. The polysaccharides in the fruiting bodies of red bamboo fungus are mainly composed of glucose, galactose, mannose and xylose, and have obvious in vitro antioxidant activity. It is known as the "Queen of Mushrooms" and "Flower of Fungi". The mycelium of red bamboo fungus grows slowly, has poor genetic stability, is easy to degenerate, has a long cultivation cycle (150-180 days), high cost, low yield (yield 500-600kg / mu), and few commercial mushrooms.

[0003] The technical personnel in this field have always been pursuing to improve the growth rate of the mycelium of Dictyophora rubra (including mother strains, original strains and cultivated strains), the commercial rate of fruiting bodies and the yield, which has great application value. Summary of the invention

[0004] The present application provides a red-stemmed bamboo fungus strain Kunzhi No. 5, which has attached mycelium, white and fluffy, and turns light red or purple after 10 to 15 days. It has the characteristics of fast germination, fast growth, strong vitality, early egg formation, high density, and fast differentiation on the culture medium of the universal mother strain, original strain and cultivated strain of red-stemmed bamboo fungus.

[0005] The red-tuber bamboo fungus strain Kunzhi No. 5 in this application was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on August 9, 2024, with the deposit number CGMCC No.41429, and the classification name is red-tuber bamboo fungus ( Phallus rubrovolvatus ), the storage address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Specifically, the new strain of Dictyophora rubra Kunzhi No. 5 in the present application is obtained by the following breeding method: On July 4, 2019, the inventors collected a number of red-stemmed bamboo fungus fruiting bodies in the wild in Daxi Village, Eshan County, Yuxi City, Yunnan Province, and obtained pure strains through conventional fruiting body tissue separation of edible fungi. After primary screening, re-screening, regional tests and production tests, the strain intended for space travel (No. S14-20) was selected.

[0007] On June 5, 2022, the red-tubed bamboo fungus strain numbered S14-20 was carried into space by the Shenzhou 14 manned spacecraft, and returned to the ground on December 4, 2022 with the Shenzhou 14 manned spacecraft; the S14-20 strain was activated, the activated mycelium was ground, and the single root fragments were separated and cultured to obtain strains numbered S142001-S1420500, and further screened according to conventional breeding methods for edible fungi, and obtained excellent strains with fast mycelium growth rate, fast bamboo egg differentiation, high yield, strong resistance and stable mushroom production, numbered Kunzhi No. 5.

[0008] The present application also provides an application of the above-mentioned red-suspension bamboo fungus strain Kunzhi No. 5 in the cultivation and production of red-suspension bamboo fungus. Applying the strain Kunzhi No. 5 provided in the present application to the cultivation and production of red-suspension bamboo fungus can significantly shorten the cultivation cycle, improve the quality and per-acre yield of red-suspension bamboo fungus commercial mushrooms, and has broad application prospects.

[0009] The beneficial effects of this application are: Compared with the strain S14-20 that was not cultivated on space flight, the Kunzhi No. 5 strain has an attached mycelium, which is white and fluffy, and turns light red or purple after 10 to 15 days. It germinates and grows quickly, has strong vitality, forms eggs early, has a large density, differentiates quickly, and has high collection and single-plant yield. It has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The above and other objects, features and advantages of the present application will become more apparent through a more detailed description of exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0011] Figure 1 It is a schematic diagram of the morphology of the strain Kunzhi No. 5 obtained in the present invention; Figure 2 This is a bacterial egg diagram of the strain Kunzhi No. 5 obtained in the present invention; Figure 3 This is a picture of the fruiting body of the strain Kunzhi No. 5 obtained in the present invention. DETAILED DESCRIPTION

[0012] The embodiments of the present application will be described in more detail below with reference to examples. Although the embodiments of the present application are shown in the examples, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and the scope of the present application can be fully communicated to those skilled in the art.

[0013] Example 1 This example illustrates the breeding process of the red-tip bamboo fungus strain Kunzhi No. 5, and the specific steps are as follows: (1) On July 4, 2019, the inventors collected multiple fruiting bodies of Dictyophora rubra in the wild in Daxi Village, Eshan County, Yuxi City, Yunnan Province, and obtained pure strains through conventional fruiting body tissue separation of edible fungi. After primary screening, secondary screening, regional trials and production trials, strains (numbered S14-20) were selected for space travel. The strains were identified as Dictyophora rubra by conventional ITS molecular biology of edible fungi. The sequence is as follows: GGGCGCGGATCCCTTTTTCCCCGGACGACGCGAGACTTATCACAACGTCTTCGAGGGGACTGGAAACCTCGCCGATGAATTTGAAGACGAGCCTTCACCGACGGGCAAAGGCAAGACCGTCCAAGTCCGAAAA AAGAGGGCCCCCCTTTTCACAAGGGACCCTCCTTCCGATGAGACTTCACGACACTCAAACAGGCATGCTCCTCGGAATGCCGAGGAGCGCAAGGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATT (2) The strain was carried into space on the Shenzhou XIV manned spacecraft on June 5, 2022, and returned to the ground in the Shenzhou XIV manned spacecraft on December 4, 2022. After being fair-labeled, it was brought back to the Culture Collection Center of the Kunming Institute of Botany, Chinese Academy of Sciences for relevant research.

[0014] (3) The S14-20 strain was activated, the activated mycelium was ground, and the single root fragments were separated and cultured to obtain the numbers S142001-S1420500. The mycelium growth rate, growth potential, consistency, stability, antagonism and salt tolerance of these 500 strains were further studied with reference to "GB / T 21125-2007 Technical Specifications for the Selection of Edible Fungi Varieties". Multi-point and multiple strain fruiting screening tests were carried out according to the conventional cultivation method of red-suspension bamboo fungus. A red-suspension bamboo fungus strain was obtained, which has the characteristics of fast mycelium growth, resistance to foreign bacteria, fast liquid fermentation culture, early egg formation, and obvious fruiting tide of fruiting bodies, achieving the purpose of domestication and breeding of excellent strains. The strain was named "Kunzhi No. 5". The strain was preserved in the Kunming Institute of Botany, Chinese Academy of Sciences (KUNCC) using the 10% glycerol + distilled water preservation method.

[0015] (4) The strain Kunzhi 5 of the red-tipped bamboo fungus was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on August 9, 2024, with the deposit number: CGMCC No.41429, and the classification name is red-tipped bamboo fungus ( Phallus rubrovolvatus ), the storage address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0016] like Figure 1 and Figure 2 As shown, the characteristics of the red bamboo fungus strain Kunzhi No. 5 in this embodiment are as follows: 1. Morphological characteristics The eggs grow in scattered or clusters, with a diameter of 3 to 6 cm and a weight of 70 to 180 g. The mature fruiting body consists of the cap (including the skirt), stipe and peduncle. The cap is bell-shaped with a perforation at the top. The mature spore fluid is foul-smelling and adheres to the surface of the cap in dark green or bluish-brown. The veil (skirt) is white, reticular, circular, oval or polygonal, 7 to 10 cm long and 8 to 15 cm in diameter. The stipe is white, columnar and spongy, tender and crisp, hollow in the middle, slightly smaller at the top, 10 to 25 cm long and 3 to 6 cm in diameter. The peduncle is purple-red, 3 to 5 cm high and 5 to 10 cm in diameter. Figure 1 shown.

[0017] 2. Biological characteristics 1) Preparation of mother species, original species and cultivated species: Prepare mother species, original species and cultivated species according to the general preparation method of mother species, original species and cultivated species of wood-decaying fungi and set them aside.

[0018] 2) Select the cultivation environment: soil temperature 20-25℃, relative air humidity 80-85%, soil moisture content 45-60%, light intensity 100-2000lux, carbon dioxide concentration 400-2000ppm.

[0019] 3) Cultivate using conventional red-stemmed bamboo fungus factory cultivation methods: ① Use a knife to cut the surface of the bag obtained by the conventional red-stemmed bamboo fungus cultivation bag making method and remove the bag; ② Place the cultivated seeds along the bed surface with a spacing of 8 to 10 cm between sticks; ③ Immediately after sowing, evenly cover with 5 cm of fine soil with a pH of 5 to 6; ④ Maintain air temperature at 20 to 25°C, relative air humidity at 80 to 85%, soil moisture at 45%, and culture for 30 to 45 days.

[0020] 4) Management of mushroom production: When the primordium further grows to form bamboo fungus eggs, control the air temperature at 20-25°C, relative humidity at 75-85%, soil moisture at 58%, light intensity at 500 lux, carbon dioxide concentration at 400-600 ppm, and cultivate for 35 days.

[0021] 5) Harvesting: When the mature bamboo fungus eggs grow bamboo fungus fruiting bodies from the top, they can be harvested. Figure 3 This is a picture of the fruiting body of strain Kunzhi No. 5.

[0022] 3. Genetic characteristics The genomic DNA of the red bamboo fungus strain Kunzhi 5 was first amplified and sequenced using the universal primers ITS4 / ITS5 for fungi to obtain the following molecular fragments, the sequence of which is shown in SEQ ID NO.3, specifically: GGGCGCGGATCCCTTTTTCCCCGGACGACGCGAGACTTATCACAACGTCTTCGAGGGGACTGGAAACCTCGCCGATGAATTTGAAGACGAGCCTTCACCGACGGGCAAAGGCAAGACCGTCCAAGTCCGAAAA AAGAGGGCCCCCCTTTTCACAAGGGACCCTCCTTCCGATGAGACTTCACGACACTCAAACAGGCATGCTCCTCGGAATGCCGAGGAGCGCAAGGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATT The fragment was sequenced by GenBank, and the result showed 100% homology with the red-tuber Dictyophora in the database, gene accession number (KF939500). The red-tuber Dictyophora strain Kunzhi 5 (preservation number CGMCC No. 41429) was identified as red-tuber Dictyophora.

[0023] Subsequently, the genome of the red bamboo fungus strain Kunzhi 5 was sequenced by conventional fungal genome sequencing methods, and shallow genome sequencing (Shallow Whole Genome Sequencing) and primer design were performed with reference to the existing technology to obtain the following specific primers: Primer_KZ5_1L:5'-GCGCTGATTTGCACTTGAGT-3' Primer_KZ5_1R:5'-GCTCTACGTCCTCTGATCGC-3' as well as Primer_KZ5_2L:5'-TTTCGGACGGTGTCAAAGTTG-3' Primer_KZ5_2R:5'-CGAGATTTGTGACTGCGTATGT-3'.

[0024] After further PCR amplification, specific DNA fragments of approximately 301 bp and 505 bp were obtained. The sequences are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively: Sequence SEQ ID NO.4, specifically: AGAGTGGAATCAAGATCATACCACGTCAAGTCCGGCGCTGATTTGCACTTGAGTTGGGCACGAGTCTATATTCAAGGGGTCGACAATTCCTAATATAGGCCGTAAAGCACGGCTCGCAATTCCAGTTTTGAAGTTCTTCGATGTTACATCGACGATGGCTGAGGGGGTAGTTTCGCTCCCTGTCCCTGCGGTCCTGGAAAGAAGATGTTAACTAGTGATTGTTTGATTCATAATCATAGAAATTACGTGGAAACTGAAAAATAGTTTTGCGTCAATTTTAGAACTTCAACTAAGCTTATAAGAGGTTACACACTAGCGATCAGAGGACGTAGAGCCTTCTCAACAGGCAGACCCTTTCCAATGGGAGGGCTTGTAAA Sequence SEQ ID NO.5, specifically: TCGTTTCCATAAACACTGTATGTACGCTCTGCAGGGTCTATACGAACTGATTTAACTCCAGCAGAGGCATGTCTTGGACGCGAGACGGGTTTCGGACGGTGTCAAAGTTGTGCTCAAGCTGGTTAATACTGATCAAGAAGAAATGCCCATAGGTCTCTATTTATCGTCTGCC TTGCTTCGCTCTGATCCTAGAAACATTGTGCCTCCCATTCTCGATGTTATTCCAATGCCTAATAACGACGTTTTTCGCCTTTATTGTGATGCCCTTGTTGAGGCATTTTGATCACCCGTATTTTCAGCGAAAGATTGAATGCTTTGAGGCATTTTATCTAATGCTAGAGGTAAT TCTGATTTGTCCCATCTTTTTCTCGATGCTTATAATATTCCTGTAGGGAATAGACTTCATGCATGAGCATAATGTTGCTCACAGGTAAATTGTAAAAAGCTCCGCACAAATGTTGCTAATCTCATGTTTAGAGATGCCTGTTTGTTCAACTTTTGTATGGATGCGACAAGAT TGATTCCTAGAGGATTTCATTTCGCTAGAGATTGCACATATGATGGCGTACATTACATACGCAGTCACAAATCTCGAAAGGCCGTTGGACCAATGAAATATTACTGGATTGATTTCGACCTTTCTATCCGATTTCCCAGCTTCGAGAAACGATATCCGGTAGTTGGAAAGTTT Specific gene detection was performed on the red-sprung bamboo fungus strain Kunzhi No. 5 bred after being carried on the Shenzhou 14 manned spacecraft, the strain not carried on board, and the common red-sprung bamboo fungus on the market. During amplification, primers Primer_KZ5_1L / Primer_KZ5_1R or Primer_KZ5_2L / Primer_KZ5_2R were added to the corresponding amplification system respectively, and the reaction was detected by 2% agarose gel electrophoresis after the reaction was completed.

[0025] 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, template DNA 50 ng; the reaction conditions were as follows: pre-denaturation at 95°C for 3 min, followed by 95°C for 60 s, 60°C for 40 s, and 72°C for 30 s, 35 cycles; and finally extension at 72°C for 10 min.

[0026] The sequencing of the PCR amplification reaction products was sent to Beijing Biotech Biotechnology Co., Ltd., Shanghai Shenggong Bioengineering Technology Service Co., Ltd. and Yingjun Biotechnology Co., Ltd. for sequencing. The sequencer used for sequencing was ABI3730 DNA sequencer, and the sequencing primers were the same as the PCR amplification primers. Only the Kunzhi No. 5 strain can obtain the following DNA sequence bases. The unloaded strains or the common red bamboo fungus in the market cannot amplify the following DNA sequence bases.

[0027] The DNA sequence bases obtained by sequencing are shown in SEQ ID NO.1, specifically: AGAGTGGAATCAAGATCATACCACGTCAAGTCCGGCGCTGATTTGCACTTGAGTTGGGCACGAGTCTATATTCAAGGGGTCGACAATTCCTAATATAGGCCGTAAAGCACGGCTCGCAATTCCAGTTTTGAAGTTCTTCGATGTTACATCGACGATGGCTGAGGGGGTAGTTTCGCTCCCTGTCCCTG CGGTCCTGGAAAGAAGATGTTAACTAGTGATTGTTTGATTCATAATCATAGAAATTACGTGGAAACTGAAAAATAGTTTTGCGTCAATTTTAGAACTTCAACTAAGCTTATAAGAGGTTACACACTAGCGATCAGAGGACGTAGAGCCTTCTCAACAGGCAGACCCTTTCCAATGGGAGGGCTTGTAAA The DNA sequence bases obtained by sequencing are shown in SEQ ID NO.2, specifically: TCGTTTCCATAAACACTGTATGTACGCTCTGCAGGGTCTATACGAACTGATTTAACTCCAGCAGAGGCATGTCTTGGACGCGAGACGGGTTTCGGACGGTGTCAAAGTTGTGCTCAAGCTGGTTAATACTGATCAAGAAGAAATGCCCATAGGTCTCTATTTATCGTCTGCC TTGCTTCGCTCTGATCCTAGAAACATTGTGCCTCCCATTCTCGATGTTATTCCAATGCCTAATAACGACGTTTTTCGCCTTTATTGTGATGCCCTTGTTGAGGCATTTTGATCACCCGTATTTTCAGCGAAAGATTGAATGCTTTGAGGCATTTTATCTAATGCTAGAGGTAAT TCTGATTTGTCCCATCTTTTTCTCGATGCTTATAATATTCCTGTAGGGAATAGACTTCATGCATGAGCATAATGTTGCTCACAGGTAAATTGTAAAAAGCTCCGCACAAATGTTGCTAATCTCATGTTTAGAGATGCCTGTTTGTTCAACTTTTGTATGGATGCGACAAGAT TGATTCCTAGAGGATTTCATTTCGCTAGAGATTGCACATATGATGGCGTACATTACATACGCAGTCACAAATCTCGAAAGGCCGTTGGACCAATGAAATATTACTGGATTGATTTCGACCTTTCTATCCGATTTCCCAGCTTCGAGAAACGATATCCGGTAGTTGGAAAGTTT Only the Kunzhi No. 5 strain can amplify the unique DNA fragment using the specific primers.

[0028] Example 2 Cultivation Comparative Test In this example, the red-tubeled bamboo fungus strain Kunzhi No. 5 was cultivated and compared with the strain S14-20 that was not carried on space flight, and the mycelial growth rate, liquid fermentation culture time, egg formation time, egg maturity time, commercial mushroom quantity and yield of the mother strain and cultivated strain of the test strain were investigated.

[0029] The seed formula used in this example is: 200 g potato (sliced, boiled and filtered), 20 g glucose, 4 g peptone, 2.5 g ammonium sulfate, 70 g broadleaf sawdust, 25 g agar, 1 L distilled water, sterilized at 121° C. for 30 min, pH 5 after sterilization.

[0030] The cultivar formula used in this embodiment is: 77% broadleaf wood chips (used after fermentation), 18% wheat bran, 2% soybean powder, 1% sucrose, 1% gypsum, 0.5% magnesium sulfate, 0.5% potassium dihydrogen phosphate, and pH 5.5 after sterilization.

[0031] The liquid strain (original strain) formula used in this embodiment is: 70g of broad-leaved tree sawdust (diameter 1-3mm), 5g of wheat bran, 5g of soybean meal, 20g of brown sugar, 1g of gypsum, 0.2g of potassium dihydrogen phosphate, 0.3g of magnesium sulfate, 1L of distilled water, sterilized at 121°C for 30min, pH 5 after sterilization.

[0032] Furthermore, the cultivation method of the red bamboo fungus Kunzhi No. 5 strain in this embodiment comprises the following steps: 1) Preparation of mother species, original species and cultivated species: According to the general preparation method of wood-decaying fungi, the above formula is made into corresponding mother species, original species and cultivated species for standby use.

[0033] 2) Select the cultivation environment: soil temperature 20-25℃, relative air humidity 80-85%, soil moisture content 45-60%, light intensity 100-2000lux, carbon dioxide concentration 400-2000ppm.

[0034] 3) Conventional red-stemmed bamboo fungus factory cultivation process: ① Use a knife to cut the surface of the bag obtained by the conventional red-stemmed bamboo fungus cultivation bag making method and remove the bag; ② Place the cultivation seeds along the bed surface with a spacing of 8 to 10 cm between sticks; ③ Immediately after sowing, evenly cover with 5 cm of fine soil with a pH of 5 to 6; ④ Maintain an air temperature of 20 to 25°C, a relative air humidity of 80 to 85%, a soil moisture content of 45%, and culture for 30 to 45 days.

[0035] 4) Management of mushroom production: When the primordium further grows to form bamboo fungus eggs, control the air temperature at 20-25°C, relative humidity at 75-85%, soil moisture at 58%, light intensity at 500 lux, carbon dioxide concentration at 400-600 ppm, and cultivate for 30-35 days.

[0036] 5) Harvesting: When the mature bamboo fungus eggs grow bamboo fungus fruiting bodies from the top, they can be harvested.

[0037] It should be noted that in Example 2, when comparing the cultivation of Kunzhi No. 5 and G14-20, the only difference was the strain itself, and the other cultivation conditions were the same.

[0038] The cultivation results are shown in the following table: It can be seen from the above table that compared with the cultivation of strain S14-20 that was not carried by space, the full growth time of the original strain, the full growth time of the cultivated strain, the full growth time of the nutrient bag, the number of primordia, the number of young mushrooms formed and the yield of Kunzhi No. 5 strain were significantly improved.

[0039] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A space-borne red-tipped bamboo fungus strain Kunzhi No. 5, characterized in that: It was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on August 9, 2024, with the deposit number CGMCC No.41429 and the classification name of Dictyophora rubra ( Phallus rubrovolvatus ), the storage address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. An application of the strain Kunzhi No. 5 described in claim 1 in the cultivation and production of Dictyophora rubra.

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