A pholiota nameko strain hk6

Abstract

The application discloses a pholiota adiposa strain HK6 and belongs to the technical field of edible fungi research.The pholiota adiposa strain HK6 has a preservation number of CGMCC NO.41675, a taxonomic name of pholiota adiposa, a preservation date of November 28, 2024, and a preservation address of No.3, Institute of Microbiology, Chinese Academy of Sciences, No.1, Beichen West Road, Chaoyang District, Beijing.The application screens a spaceflight mutagenesis pholiota adiposa strain HK6, the biological efficiency of which reaches 82 %, the length of the stem is long, and the strain is suitable for factory production, the spaceflight mutagenesis technology enriches the germplasm resources of the pholiota adiposa, and the pholiota adiposa strain suitable for factory production is obtained, thereby providing a new idea for breeding of new varieties of edible fungi.

Description

Technical Field

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

[0002] Pholiota adipose (Fr) Quel belongs to the Fungi, Basidiomycetes, Agaricales, Strophariaceae, and genus Pholiota. It is also known as the fat scale umbrella and willow mushroom. Its flesh is tender and delicious, rich in protein, vitamins, minerals, and other nutrients. Its various extracts also have important medicinal properties. Pholiota adipose total flavonoids are natural antioxidants, and its crude polysaccharides have immune-boosting properties. Therefore, Pholiota adipose is considered a fungus with medicinal value. Its bright color and unique appearance make it suitable for use in infrastructure construction, forest soil mulching, and industrial cultivation. As a high-value edible fungus, it has gained great popularity in leisure agriculture and rural tourism. However, compared to its closely related edible fungus, the nameko, Pholiota nameko, it currently exhibits superior fruiting body and ash content, as well as amino acid and certain trace element content, yet its market share remains significantly lower. Therefore, there is significant potential for development in the breeding, cultivation, and promotion of Pholiota adipose.

[0003] Overall, my country's edible mushroom seed industry lags significantly behind production demand, and there's still a long way to go in mushroom breeding. Currently, common methods of edible mushroom breeding include domestication, hybridization, mutation, and molecular breeding. Space breeding, a form of mutation breeding, involves launching a starting strain into space on a reusable spacecraft and leveraging beneficial genetic variations to select new varieties. Summary of the Invention

[0004] In the past, our team bred the excellent Pholiota adidas cultivar HS5 through isolation, identification, and cultivar experimentation of wild strain tissue. This study used HS5 as the experimental material, subjected it to spaceflight mutagenesis, and evaluated the differences in somatic cell incompatibility, growth rate, and agronomic traits of the returned spaceflight-induced strains. This study explored the effects of spaceflight mutagenesis on the biological characteristics of the binuclear Pholiota adidas strain, enriched the mutant library of Pholiota adidas, and provided new germplasm for Pholiota adidas breeding.

[0005] The present invention provides a Pholiota adiposa strain HK6. The Pholiota adiposa strain HK6 has a preservation number of CGMCC NO.41675 and a taxonomic name of Pholiota adiposa.

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

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

[0008] (2) The growth speed of the Pholiota nameko strain HK6 is faster than that of the Pholiota nameko strain HS5;

[0009] (3) The yield of the Pholiota nameko strain HK6 is higher than that of the Pholiota nameko strain HS5;

[0010] (4) The biological efficiency of the Pholiota nameko strain HK6 is higher than that of the Pholiota nameko strain HS5;

[0011] (5) The biological efficiency of the Pholiota nameko strain HK6 on the Calocybe gambusina is higher than that of the Pholiota nameko strain HS5;

[0012] (6) The cap diameter of the Pholiota nameko strain HK6 is smaller than that of the Pholiota nameko strain HS5;

[0013] (7) The stem length of the Pholiota nameko strain HK6 is longer than that of the Pholiota nameko strain HS5;

[0014] (8) The stem diameter of the Pholiota nameko strain HK6 is smaller than that of the Pholiota nameko strain HS5;

[0015] (9) The cap hardness of the Pholiota nameko strain HK6 is greater than that of the Pholiota nameko strain HS5.

[0016] The application further provides a cultivation method of the Pholiota nameko strain HK6, and the Pholiota nameko strain HK6 is induced to fruit at 22-28 DEG C.

[0017] Preferably, the cultivation temperature of the fruiting body of the Pholiota nameko strain HK6 is room temperature.

[0018] The application further provides application of the Pholiota nameko strain HK6 in breeding of Pholiota nameko.

[0019] The application further provides fruiting bodies, mycelia and / or spores of the Pholiota nameko strain HK6.

[0020] The application further provides protoplasts of the Pholiota nameko strain HK6.

[0021] The application further provides a fungus stick containing the Pholiota nameko strain HK6.

[0022] The application further provides application of the Pholiota nameko strain HK6 in preparation of fruiting bodies and / or mycelia and / or spores of Pholiota nameko.

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

[0024] The application screens a Pholiota nameko strain, the biological efficiency of which reaches 82%, the stem length of which is long, and the Pholiota nameko strain HK6 is suitable for factory production, the spaceflight mutagenesis technology enriches the germplasm resources of Pholiota nameko, and the suitable factory production Pholiota nameko strain is obtained, thereby providing a new idea for breeding of new varieties of edible fungi.

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

[0026] Depository: General Microbiology Center of China Culture Collection Administration of Microorganisms;

[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 showing the antagonistic reaction between the 10th generation of the HS5 mutagenized 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 mutagenesis strain in Example 1 are shown.

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

[0034] Example 1

[0035] 1 Materials and Methods

[0036] 1.1 Test strains

[0037] The test strain, Pholiota adiposa HS5 (CK strain), was derived from a wild strain isolated from willow trees in Zizhuyuan Park, Haidian District, Beijing. This strain, selected through tissue isolation, identification, and comparative testing, has been certified as a Beijing Crop Variety (identification number: Jingpinjianjun 2013007). It is currently under the Edible Fungi Research Laboratory, Institute of Plant Protection, Beijing Academy of Agricultural and Forestry Sciences (accession number: BIPP21160005).

[0038] 1.2 Test culture

[0039] 1.2.1 Comprehensive PDA medium: Boil 200 g of potatoes, filter the filtrate through gauze, add 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 dilute 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% moisture 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 Green Forest 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 Space-induced mutagenesis of Pholiota adiposa strain HS5

[0044] The CK strain was inoculated into a sterile cryovial containing 1 mL of comprehensive PDA medium and cultured in a dark incubator at 25°C for 3 days before being aboard the Shenzhou XII spacecraft for space mutagenesis. The Shenzhou XII spacecraft's launch orbit is 200 km (perigee) × 348 km (apogee), with an orbital inclination of 41.3°. The spacecraft will operate from June 17 to September 17, 2021, for a total of 92 days. The tip of the returned strain was isolated, with only 1-2 cells from the anterior segment of the hyphae. This yielded 50 Pholiota adiposa HS5 space strains (HK strains) designated HK 1 to HK 50.

[0045] 1.5 Test method

[0046] 1.5.1 Subculture

[0047] To investigate the genetic stability of mutations induced by mutagenesis, 50 isolated strains of HK and CK strains were designated as the first generation and cultured at 25°C. When the hyphae tips of the first generation reached two-thirds of the plate, they were punched with a sterilized 6 mm borer. The resulting cake was inoculated into the center of a 20 mL polystyrene-based diatomaceous earth (PDA) plate, designated the second generation. Subsequent generations were 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 inoculate using the four-point inoculation method. Inoculate the 1st, 5th, and 10th generation HK strains and the 10th generation CK strain on the same integrated PDA plate and culture at a constant temperature of 25°C. Observe whether there is an antagonistic line between the strains and the degree of antagonism about 16 days after inoculation, and take photos and record them.

[0050] 1.5.3 Determination of growth rate

[0051] The growth rate of the 1st, 5th, and 10th generation strains in subculture was measured using the cross-streaking method. After mycelial germination, a cross was drawn on the back of the plate. Graduation marks were made at the growing tip every two days for a total of four crosses. Measurements were made using an ABS digital vernier caliper. Three replicate plates were set for each strain.

[0052] 1.5.4 Mushroom Fruiting Experiment

[0053] Using the CK as a control, a mutagenic strain was selected that exhibited stable antagonism against the CK strain and consistently grew faster than the CK. Mycelium from the petri dishes was inoculated into sterilized fruiting bags (170mm x 330mm polyethylene bags). A 1.2.2 Pholiota adiposa cultivation medium formulation was used, with a natural pH, a fruiting temperature of 25.5°C (no temperature change during subsequent cultivation), a humidity of 154.2%, a carbon dioxide concentration of 532 ppm, and a light intensity of 0.3 kL. Ten replicates were set for each strain. During the fruiting experiment, temperature and humidity were adjusted appropriately according to the environment, and ventilation was ensured. During the fruiting process, when the mycelium had fully grown into the bag, the bag was scratched. After about a week to allow the mycelium to recover, the bag was opened and covered with newspaper, which was then watered. When measuring agronomic traits, five Pholiota cap fruiting bodies were randomly measured in each bag of mushrooms. The cap diameter (mm), cap thickness (mm), stipe length (mm), and stipe diameter (mm) were measured with a vernier caliper. The hardness of the cap and stipe was measured using a hardness meter (FR-5102). 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 / cultivation dry material weight * 100%

[0055] 1.6 Data Measurement and Processing

[0056] The experimental data were statistically analyzed and sorted using Excel 2019, and significance analysis and variance analysis were performed using IBM SPSS Statistics 25 software. Single-factor test was used, and the significance categories were: * indicates significant difference, 0.01 <P<0.05;**表明有显著差异,0.001<P<0.01;***表明有极显著差异,P<0.001,使用Origin2022软件绘图。

[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 CK strains and HK strains, it was found that 10 of the first-generation HS5 mutant strains had an antagonistic effect with the CK strain, with a mutation rate of 20%; after 10 subcultures, 5 HS5 mutant strains remained stable during the subculture process, that is, they were still antagonistic to CK, with a mutation rate of 10%. The mutation rate showed a significant downward trend after the subculture of the strains, which indicates that some of the mutant strains have undergone reverse mutations. Table 1 shows the space-age mutant strains that still have an antagonistic effect with CK after 10 generations of subculture. Figure 1 More intuitively, the mutagenized strains in Table 1 exhibited clear antagonistic lines with CK after multiple subcultures. These strains were antagonistic to CK, with no antagonism between generations, indicating that their genomic mutations, following space travel, were stable and heritable.

[0060] Table 1 Antagonism analysis among generations of HS5 space-induced mutagenesis strains after subculture

[0061]

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

[0063] 2.2 Analysis of mycelial growth rate

[0064] The 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 can be preliminarily determined whether the growth characteristics of the strain have mutated. The mycelial 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 growth rate significantly faster than that of 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 strain 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 mutagenized strains. This indicates that after spaceflight mutagenesis, HS5 is more likely to mutate in the direction of increased growth rate.

[0065] 2.3 Mushroom production experiment

[0066] Five mutant strains (HK1, HK4, HK6, HK15, and HK37) that showed a stable antagonistic reaction with CK and consistently grew faster than CK were selected for fruiting trials. Using CK as a control, strains with superior traits were screened by comparing yield, fruiting body agronomic characteristics, and biological efficiency. According to fruiting data (Table 2), the CK had a bag-full time of 61 days, while the mutant strain HK6 had a bag-full time of 54 days.

[0067] HK6 showed the fastest growth rate on the substitute feed. The six strains grew primordia in 63 to 73 days, with CK reaching 73 days. In terms of the number of bodies per bag, CK averaged 24, while HK1 had the highest number, 29, 20.83% higher than CK. The other mutant strains also had higher body counts per bag than CK. The average yield per bag of the mutant strains was higher than that of CK, with HK6 having the highest yield, 12.18% higher than CK. The average yields of HK1, HK15, and HK37 were also higher than CK by 9.31%, 9.79%, and 10.76%, respectively. In terms of biological efficiency, CK had a biological efficiency of 73%, while HK6 had the highest, reaching 82%. HK1, HK15, and HK37 also achieved biological efficiencies exceeding 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. Therefore, the agronomic traits of the mutant strain HK6 should be focused on.

[0068] Agronomic traits (Table 3) show that CK had the largest cap diameter, at 28.04 mm, while the cap diameter of the mutant strain HK6 was 19.91 mm, making HK6 smaller than CK. HK6 also had a stipe length of 62.89 mm, the longest of the six fruiting strains. HK6's stipe diameter was smaller than that of CK and HK4, but larger than that of HK37, HK1, and HK15. Its cap hardness was greater than that of CK and HK4, but smaller than that of HK15, HK37, and HK1. HK6's stipe hardness was smaller than that of CK, but the difference was not significant. Stipe length is the most important metric for breeding high-yielding Pholiota cap mushrooms. Based on Tables 2 and 3, we screened the mutant strain HK6, which has a long stipe, a short fruiting time, and high yield and biological efficiency.

[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 collected after 64 days for the space species and 73 days for the CK.

[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 species.

[0075] 3 Conclusion

[0076] Using space mutagenesis, the Pholiota adiposa strain HS5 was aboard the Shenzhou XII launch vehicle for space mutagenesis. The mutated strains were isolated and purified using a tip-based method to obtain 50 strains. These 50 strains and the CK strains were subjected to 10 subculture tests and evaluated based on antagonism, growth rate significance, fruiting tests, and agronomic traits. The resulting strain, HK6, was selected. The resulting Pholiota adiposa strain exhibits a biological efficiency exceeding 80% and exhibits a short fruiting time, long stipes, a small cap, and moderate stipe hardness, making it suitable for industrial production. In summary, the HK6 strain was identified as a mutant strain with a long stipe, high yield, and high biological efficiency.

[0077] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the 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 fall within the scope of protection determined by the claims of the present invention.

Claims

1. A Pholiota adiposa 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 cultivation method of the Pholiota adiposa strain HK6 according to claim 1, characterized in that: The Pholiota adiposa strain HK6 was fruited at 22-28°C.

3. The cultivation method according to claim 2, wherein The culture temperature of the Pholiota adiposa strain HK6 during the fruiting body stage is room temperature.

4. Use of the Pholiota adiposa strain HK6 according to claim 1 in Pholiota adiposa breeding. The fruiting body, mycelium and / or spore of the Pholiota adiposa strain HK6 according to claim 1 . The protoplasts of the Pholiota adiposa strain HK6 according to claim 1 . A mushroom log containing the Pholiota adiposa strain HK6 according to claim 1 .

8. Use of the Pholiota adiposa strain HK6 according to claim 1 in preparing Pholiota adiposa fruiting bodies and / or Pholiota adiposa mycelia and / or Pholiota adiposa spores.

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