Pholiota adipose strain HK6

Abstract

The invention discloses a pholiota adipose strain HK6, and belongs to the technical field of edible fungus research. The preservation number of the pholiota adipose strain HK6 is CGMCC (China General Microbiological Culture Collection Center) NO.41675, the pholiota adipose strain HK6 is named as pholiota adipose by taxonomy, the preservation date is November 28, 2024, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The space mutation pholiota adipose strain HK6 is obtained through screening, the biological efficiency of the space mutation pholiota adipose strain HK6 reaches 82%, the length of the stipe is long, the space mutation pholiota adipose strain HK6 is suitable for industrialized production, the space mutation technology enriches germplasm resources of pholiota adipose, the pholiota adipose strain suitable for industrialized production is obtained, and a new thought is provided for breeding of new edible fungus varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungi research, and in particular relates to a Pholiota adzuki agaric strain HK6. Background Art

[0002] Pholiota adipose (Fr) Quel belongs to Fungi, Basidiomycetes, Agaricales, Strophariaceae, and Pholiota genus. It is also known as fat scale umbrella and willow mushroom. The flesh of Pholiota adipose is tender and delicious, and is rich in protein, vitamins, minerals and other nutrients. Various extracts of Pholiota adipose also have important medicinal value. Pholiota adipose total flavonoids are natural antioxidants, and Pholiota adipose crude polysaccharides have the function of enhancing immunity. Therefore, Pholiota adipose is regarded as a fungus with medicinal value. Pholiota adipose has bright color and unique appearance, which is suitable for facility construction, soil covering under forests or factory planting. As a high-value-added edible fungus, it has been greatly welcomed in leisure agriculture and rural tourism. However, compared with its close edible fungus, Pholiota nameko, the content of fruiting body and ash, amino acids and certain trace elements all show greater superiority, but there is a significant gap in market share. Therefore, we still have great development potential in the fields of breeding, cultivation and promotion of Pholiota adipose.

[0003] Overall, my country's edible fungus seed industry lags far behind production demand, and there is still a long way to go in breeding. At present, common edible fungus breeding methods include domestication breeding, hybrid breeding, mutation breeding, and molecular breeding. Space breeding is a form of mutation breeding, which is a breeding method that carries the starting strain into space on a returning spacecraft and uses beneficial genetic variations to select new varieties. Summary of the invention

[0004] In the early stage, our team bred the excellent Pholiota adzuki variety HS5 through the separation, identification and variety experiment of wild strain tissue. This invention uses HS5 as the test material, carries out mutagenesis on it through space travel, and evaluates the difference in somatic cell incompatibility, growth rate, agronomic traits, etc. of the returned space-borne mutagenesis strain. The influence of space mutagenesis on the biological characteristics of Pholiota adzuki binuclear strains is explored to enrich the mutant library of Pholiota adzuki and provide new germplasm for Pholiota adzuki breeding.

[0005] The invention provides a Pholiota adiposa strain HK6, the preservation number of the Pholiota adiposa strain HK6 is CGMCCNO.41675, and the taxonomic name is Pholiota adiposa.

[0006] Preferably, the Pholiota adiposa strain HK6 has at least one of the following characteristics:

[0007] (1) P. adiposa strain HK6 and P. adiposa strain HS5 have stable antagonism;

[0008] (2) The growth rate of P. aurantifolia strain HK6 was faster than that of P. aurantifolia strain HS5;

[0009] (3) The yield of P. aurantifolia strain HK6 was higher than that of P. aurantifolia strain HS5;

[0010] (4) The biological efficiency of P. aurantifolia strain HK6 is higher than that of P. aurantifolia strain HS5;

[0011] (5) The biological efficiency of P. amanita floribunda strain HK6 was higher than that of P. amanita floribunda strain HS5;

[0012] (6) The cap diameter of P. amanita strain HK6 is smaller than that of P. amanita strain HS5;

[0013] (7) The stipe length of P. amanita strain HK6 is longer than that of P. amanita strain HS5;

[0014] (8) The stipe diameter of P. amanita strain HK6 is smaller than that of P. amanita strain HS5;

[0015] (9) The cap hardness of P. amanita floribunda strain HK6 is greater than that of P. amanita floribunda strain HS5.

[0016] The present invention also provides a cultivation method of the Pholiota adzuki strain HK6, wherein the Pholiota adzuki strain HK6 produces mushrooms at 22-28°C.

[0017] Preferably, the culture temperature of the Pholiota adzuki strain HK6 during the fruiting body stage is room temperature.

[0018] The present invention also provides application of the Pholiota adzuki strain HK6 in Pholiota adzuki breeding.

[0019] The present invention also provides the fruiting body, mycelium and / or spore of the Pholiota adzuki strain HK6.

[0020] The invention also provides the protoplasts of the Pholiota adzuki strain HK6.

[0021] The invention also provides a mushroom stick containing the Pholiota adiposa strain HK6.

[0022] The present invention also provides the use of the Pholiota adzuki mushroom strain HK6 in preparing Pholiota adzuki mushroom fruiting bodies and / or Pholiota adzuki mushroom mycelium and / or Pholiota adzuki mushroom spores.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention screened and obtained a Phellodendron floribunda strain with a biological efficiency of 82% and a long stipe, which is a space-induced mutagenesis strain "HK6" suitable for factory production. The space mutagenesis technology enriched the germplasm resources of Phellodendron floribunda and obtained Phellodendron floribunda strains suitable for factory production, providing new ideas for the breeding of new edible fungi varieties.

[0025] Description of biological preservation of space variant strain Pholiota adiposa HK6:

[0026] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0027] Deposit number: CGMCC NO.41675;

[0028] Deposit date: November 28, 2024;

[0029] Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0030] Taxonomic name: Pholiota adiposa. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram of the antagonistic reaction between the 10th generation of the HS5 mutagenesis strain and each generation in Example 1.

[0032] Figure 2 The results of the significance analysis of the growth rate differences of the HS5 space-induced mutant strain in Example 1 are shown.

[0033] Figure 3 The results of the fruiting test of the control strain and the induced strain in Example 1 are shown. DETAILED DESCRIPTION

[0034] Example 1

[0035] 1 Materials and methods

[0036] 1.1 Test strains

[0037] Test strain: Pholiota adiposa HS5 (CK strain) is a wild strain from the willow tree in Zizhuyuan Park, Haidian District, Beijing. It was selected through tissue separation, identification and variety comparison tests to obtain Beijing's crop variety identification (identification number: Jingpinjianjun 2013007). It is now in the Edible Fungi Research Laboratory of the Institute of Plant Protection, Beijing Academy of Agricultural and Forestry Sciences (preservation number: BIPP21160005).

[0038] 1.2 Test culture

[0039] 1.2.1 Comprehensive PDA medium: 200 g of potatoes were cooked and filtered through gauze to obtain the filtrate, 20 g of glucose, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate, 5 g of peptone, 10 mg of vitamin B1, 20 g of agar, and the volume was adjusted to 1 L with deionized water. Sterilize at 121°C for 30 min.

[0040] 1.2.2 Cultivation medium formula: 50% cottonseed hull, 30% sawdust, 18% wheat bran, 2% lime, and 67% water content.

[0041] 1.3 Main instruments

[0042] GR110DR high-pressure vertical sterilizer (ZEALWAY, USA), laboratory pure water system (Shanghai Hetai Instrument Co., Ltd.), HS-1300-U clean bench (Suzhou Antai Air Technology Co., Ltd.), ABS digital display vernier caliper (Yantai Lvlin Tools Co., Ltd.), DSC-H50 digital camera (Sony Corporation, Japan), precision electronic balance (Shanghai Hengji Scientific Instrument Co., Ltd.), BSP-250 biochemical incubator (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), hardness tester (FR-5102).

[0043] 1.4 Spaceflight mutagenesis of Pholiota adzuki strain HS5

[0044] The CK strain was inoculated into a sterilized cryopreservation tube containing 1 mL of comprehensive PDA medium, cultured in a dark incubator at 25°C for 3 days, and carried on the spacecraft Shenzhou XII for space mutagenesis. The launch orbit of the Shenzhou XII spacecraft is 200 km (perigee altitude) × 348 km (apogee altitude), the orbital inclination is 41.3°, and the operation time is from June 17 to September 17, 2021, with a 92-day on-board. For the tip isolation of the space return strain, only 1-2 cells of the front segment of the hyphae were isolated, and 50 Pholiota adiposa HS5 space strains (HK strains) were obtained and numbered HK 1-HK 50 respectively.

[0045] 1.5 Test methods

[0046] 1.5.1 Subculture

[0047] In order to explore the genetic stability of mutation breeding, the 50 isolated HK and CK strains were recorded as the first generation and cultured at 25°C. When the hyphae tips of the first generation grew to 2 / 3 of the plate, the hyphae tips of the 50 HK and CK strains were punched with a sterilized 6mm puncher, and the bacterial cake was inoculated into the center of a quantitative 20mL comprehensive PDA plate, recorded as the second generation, and then transferred to the 10th generation.

[0048] 1.5.2 Antagonism analysis

[0049] Punch holes in the test strains with a sterilized 6mm puncher, and use the four-point inoculation method to inoculate the 1st, 5th, and 10th generation HK strains and the 10th generation CK strain on the same integrated PDA plate. Culture at a constant temperature of 25°C. About 16 days after inoculation, observe whether there is an antagonistic line between the strains and the degree of antagonism, and take photos and record them.

[0050] 1.5.3 Determination of growth rate

[0051] The growth rates of the strains in the 1st, 5th, and 10th subcultures were measured using the "cross" streaking method. After the mycelia germinated, a cross was drawn on the back of the Petri dish. The growth tips were marked with scales every 2 days, and the lines were streaked a total of 4 times. An ABS digital vernier caliper was used for measurement, and 3 replicate Petri dishes were set for each strain.

[0052] 1.5.4 Fruiting experiment

[0053] Using CK as the control, mutagenic strains that had stable antagonism with the CK strain and whose growth rate was always faster than that of CK were selected. The mycelia on the Petri dish were inoculated into sterilized fruiting bags (polyethylene fungus bags of 170 mm × 330 mm). The cultivation substrate formula for Pholiota adiposa in 1.2.2 was used, with the pH natural. The fruiting temperature was 25.5 °C (the temperature would not be changed during subsequent cultivation), the humidity was 154.2%, the carbon dioxide concentration was 532 PPM, and the light intensity was 0.3 KL. 10 bags were set as replicates for each strain. During the fruiting experiment, the temperature, humidity, and ventilation should be appropriately adjusted according to the different environments. During the fruiting process, when the mycelia filled the fungus bag, the surface of the substrate was scratched, and then the mycelia were allowed to recover for about a week. The mouth of the fungus bag was opened and covered with newspaper, and water was poured on the newspaper. When measuring the agronomic traits, 5 Pholiota adiposa fruiting bodies were randomly measured from each bag. A vernier caliper was used to measure the cap diameter (mm), cap thickness (mm), stipe length (mm), and stipe diameter (mm). A hardness tester (FR-5102) was used to measure the hardness of the cap and stipe. The yield (g) of each bag of mushrooms in each treatment was recorded, and the biological efficiency was calculated.

[0054] Biological efficiency = fresh mushroom weight / dry cultivation material weight * 100%

[0055] 1.6 Data measurement and processing

[0056] The experimental data were statistically analyzed and sorted using Excel 2019, and significant analysis and variance analysis were performed using IBM SPSS Statistics 25 software. A single-factor test was used, and the significance categories were: * indicating a significant difference, 0.01 < P < 0.05; ** indicating a significant difference, 0.001 < P < 0.01; *** indicating a highly significant difference, P < 0.001. Origin2022 software was used for plotting.

[0057] 2 Results and analysis

[0058] 2.1 Antagonism experiment

[0059] The antagonism test is simple to operate, and the antagonism results are easy to observe and analyze. It is often used to identify the differences in genetic characteristics between basidiomycete species. Through the four-point antagonism test between the CK strain and the HK strain, it was found that 10 of the first-generation HS5 mutant strains had an antagonistic effect with the CK strain, and the mutation rate was 20%; after 10 subcultures, 5 HS5 mutant strains remained stable during the subculture process, that is, they were still antagonistic to CK, and the mutation rate was 10%. The mutation rate showed a significant downward trend after the subculture of the strain, which indicates that some of the mutant strains have undergone back mutations. Table 1 shows the space-borne mutant strains that still have an antagonistic effect with CK after 10 generations of subculture. Figure 1 The more intuitive display shows that the mutagenic strains in Table 1 have obvious antagonistic lines with CK after multiple subcultures. These strains are antagonistic to CK and there is no antagonism between generations, which indicates that they have genomic mutations after being carried by space, and the mutations can be stably inherited.

[0060] Table 1 Analysis of intergenerational antagonism of space-induced mutant strains of HS5 after subculture

[0061]

[0062] Note: “+” indicates antagonism; “-” indicates no antagonism

[0063] 2.2 Analysis of mycelium growth rate

[0064] Determination of growth rate is one of the most intuitive methods for biological identification of edible fungi. By measuring the growth rate of the strain, it is possible to preliminarily determine whether the growth characteristics of the strain have mutated. The mycelium growth rate of the HS5 space-induced mutant strain was measured, and it was found that 34 of the 50 HK strains after returning from space had a significantly faster growth rate than CK, accounting for 68%. The average growth rate of these 50 HK strains was 4.82 mm / d, and the average growth rate of CK was 4.33 mm / d. Overall, the growth rate of the induced strains was 11.40% higher than that of CK. Among them, HK 50 had the fastest growth rate, which was 12.69% higher than that of CK. During the 10 subcultures, 25 HK strains always grew faster than the CK strains ( Figure 2 ), accounting for half of the total number of induced strains. This indicates that after spaceflight mutagenesis, HS5 is more likely to mutate in the direction of increasing growth rate.

[0065] 2.3 Mushroom production experiment

[0066] Five mutant strains (HK1, HK4, HK6, HK15, HK37) that have a stable antagonistic reaction with CK and whose growth rate is always faster than CK were selected for fruiting test. On this basis, with CK as the control, strains with excellent traits were screened out by comparing yield, agronomic characteristics of fruiting bodies and biological efficiency. According to the fruiting data (Table 2), the number of days for CK to fill a bag is 61 days, and the number of days for the mutant strain HK6 to fill a bag is 54 days.

[0067] This shows that HK6 grows fastest on substitute materials. The six strains grew primordia in 63 to 73 days, of which CK was 73 days. In terms of the number of entities per bag, CK had an average of 24 entities per bag, and HK1 had the highest number of entities per bag, which was 29, 20.83% higher than CK, and the number of entities per bag of other induced strains was also higher than CK. The average yield per bag of the induced strains was higher than that of CK, among which HK6 had the highest average yield per bag, 12.18% higher than CK; the average yield per bag of HK1, HK15, and HK37 was also 9.31%, 9.79%, and 10.76% higher than that of CK, respectively. In terms of biological efficiency, the biological efficiency of CK was 73%, and the strain with the highest biological efficiency was HK6, which could reach 82%. In addition, the biological efficiencies of HK1, HK15, and HK37 also reached more than 80%. Among the fruiting strains, the mutant strain HK6 has the shortest time to produce primordia, the highest average yield per bag, and the highest biological efficiency, so the agronomic traits of the mutant strain HK6 should be focused on.

[0068] According to the results of agronomic traits (Table 3), the cap diameter of CK was the largest, which was 28.04 mm. The cap diameter of the mutant strain HK6 was 19.91 mm, which was smaller than that of CK. The stipe length of HK6 was 62.89 mm, which was the longest among the 6 fruiting strains. The stipe diameter of HK6 was smaller than that of CK and HK4, but larger than that of HK37, HK1, and HK15. The cap hardness was greater than that of CK and HK4, but smaller than that of HK15, HK37, and HK1. The stipe hardness of HK6 was smaller than that of CK, but there was no significant difference. In the selection of high-yield Pholiota cap mushrooms, the most important indicator is the stipe length. Combined with Tables 2 and 3, a mutant strain HK6 with a long stipe, short fruiting time, high yield and biological efficiency was selected.

[0069] Table 2 Mushroom production statistics

[0070]

[0071] Data are mean ± SD (n = 35); different lowercase letters indicate significant differences (P < 0.05). The data on the number of entities per bag, average yield per bag, and biological efficiency were calculated for the space species at 64 days and for the CK species at 73 days.

[0072] Table 3 Agronomic traits measurement table

[0073]

[0074] The data in Table 3 are based on 64 days for aerospace species and 73 days for CK.

[0075] 3 Conclusion

[0076] The space mutagenesis method was used to carry the Pholiota admiralis strain HS5 on the "Shenzhou XII" carrier rocket for space mutagenesis. The mutated strains were separated and purified by tip to obtain 50 mutated strains. The 50 mutated strains and CK were subjected to 10 subculture tests, and were evaluated from the aspects of antagonism test, growth rate significance, fruiting test and agronomic traits. Finally, the Pholiota admiralis HK6 mutated strain was selected, and the biological efficiency of the head tide mushroom can reach more than 80%, and it has the characteristics of short fruiting time, long stipe, small cap and moderate stipe hardness, which is suitable for factory production. In summary, a mutagenesis strain HK6 with long stipe, high yield and biological efficiency was screened out.

[0077] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Pholiota adzuki strain HK6, characterized in that The deposit number of the Pholiota adiposa strain HK6 is CGMCC NO.41675, and its taxonomic name is Pholiota adiposa.

2. The Pholiota adzuki bean strain HK6 according to claim 1, characterized in that The Pholiota adzuki strain HK6 has at least one of the following characteristics: (1) P. adiposa strain HK6 and P. adiposa strain HS5 have stable antagonism; (2) The growth rate of P. aurantifolia strain HK6 was faster than that of P. aurantifolia strain HS5; (3) The yield of P. aurantifolia strain HK6 was higher than that of P. aurantifolia strain HS5; (4) The biological efficiency of P. aurantifolia strain HK6 is higher than that of P. aurantifolia strain HS5; (5) The biological efficiency of P. amanita floribunda strain HK6 was higher than that of P. amanita floribunda strain HS5; (6) The cap diameter of P. amanita strain HK6 is smaller than that of P. amanita strain HS5; (7) The stipe length of P. amanita strain HK6 is longer than that of P. amanita strain HS5; (8) The stipe diameter of P. amanita strain HK6 is smaller than that of P. amanita strain HS5; (9) The cap hardness of P. amanita floribunda strain HK6 is greater than that of P. amanita floribunda strain HS5.

3. The cultivation method of Pholiota adzuki strain HK6 according to claim 1 or 2, characterized in that: The Pholiota adzuki strain HK6 produced mushrooms at 22-28°C.

4. The cultivation method according to claim 3, characterized in that: The culture temperature of the Pholiota adzuki strain HK6 during the fruiting body stage is room temperature.

5. Use of the Pholiota adzuki strain HK6 according to claim 1 or 2 in Pholiota adzuki breeding.

6. The fruiting body, mycelium and / or spore of the Pholiota adzuki strain HK6 according to claim 1 or 2.

7. The protoplasts of the Pholiota adzuki strain HK6 according to claim 1 or 2.

8. A mushroom log containing the Pholiota adzuki strain HK6 according to claim 1 or 2.

9. Use of the Pholiota adzuki mushroom strain HK6 according to claim 1 or 2 in preparing Pholiota adzuki mushroom fruiting bodies and / or Pholiota adzuki mushroom mycelium and / or Pholiota adzuki mushroom spores.

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