A strain of mycena purpureofusca and a culture method thereof

Abstract

The present invention discloses a strain of Oudemansiella apalosarca and a cultivation method thereof, belonging to the technical field of edible fungi research. The deposit number of the Oudemansiella apalosarca strain HK26 is CGMCC NO.41674, the taxonomic name is Oudemansiella apalosarca, the deposit date is November 28, 2024, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The present invention utilizes space mutagenesis technology to improve the variety of Oudemansiella apalosarca, analyzes the stability and direction of variation through continuous subculture experiments, and uses mycelium evaluation and mushroom agronomic trait evaluation to screen out the strain HK26 with stable variation and good uniformity, thereby accelerating the industrial application process of Oudemansiella apalosarca.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungi research, and in particular relates to a Agaricus globosum strain and a culture method thereof. Background Art

[0002] Edible fungi production in my country primarily involves traditional production and factory-based cultivation. Traditional production requires significant labor costs and is subject to seasonal restrictions. Factory-based production, however, allows for year-round and standardized production of edible fungi, offering advantages such as high production efficiency, guaranteed product quality and safety, and minimal labor input. It is one of the most representative models for agricultural modernization. However, the number of mushroom varieties cultivated in factory-based cultivation in my country is relatively limited, primarily focusing on enoki mushrooms, king oyster mushrooms, oyster mushrooms, button mushrooms, and shimeji mushrooms. This leads to significant homogeneity and shrinking profit margins. There is a critical need to develop new varieties specifically for factory-based cultivation to meet public demand for a diverse range of edible fungi while also promoting quality and efficiency improvements in factory-based cultivation.

[0003] Oudemansiella apalosarca, a species of mushroom belonging to the phylum Basidiomycota, family Physalacriceae, and genus Oudemansiella, was formerly known as light brown Oudemansiella and tropical Oudemansiella. Oudemansiella is delicious and rich in protein, carbohydrates, vitamins, and minerals. It is a rare edible mushroom suitable for sightseeing and harvesting, and boasts health benefits such as antioxidants and tumor cell proliferation inhibition. Currently, the main cultivated Oudemansiella species in China is Oudemansiella oosporei. Its cultivation process still suffers from the labor-intensive soil covering, long ripening time, and severe repeated cropping problems, making it unsuitable for industrial cultivation. Oudemansiella apalosarca exhibits significant advantages in terms of mycelial growth rate, fruiting cycle, and the lack of soil covering, making it a promising candidate for industrial cultivation. However, fruiting body uniformity remains an urgent issue during its production. Therefore, in order to improve the uniformity of fruiting bodies, this study used mutation breeding technology to improve the variety of Agaricus serrata.

[0004] Space-induced mutagenesis is a highly effective breeding method used in both plants and microorganisms. This technique involves sending crop seeds into the space environment via a returning spacecraft. Using the specialized conditions of microgravity, high vacuum, cosmic radiation, and weak magnetic fields, the technique induces genetic mutations in plant seeds, tissue culture cells, or microbial strains. Similar to artificial mutagenesis, space-induced mutagenesis offers advantages in a shorter breeding cycle, a greater range of mutations, and the absence of potential safety risks associated with genetic modification. The frequency, magnitude, and probability of specific mutations associated with space-induced mutagenesis are relatively high. Summary of the Invention

[0005] One of the purposes of the present invention is to provide an Oudemansiella apalosarca strain HK26, the Oudemansiella apalosarca strain HK26 has a preservation number of CGMCC NO.41674 and a taxonomic name of Oudemansiella apalosarca.

[0006] Preferably, the Odette mushroom strain HK26 has at least one of the following characteristics:

[0007] (1) There is antagonism between the Odenthetratus globulus strain HK26 and the Odenthetratus globulus JZB2115055;

[0008] (2) The time it takes for the Odenia globosum strain HK26 to form fruiting bodies is shorter than that of the Odenia globosum strain JZB2115055;

[0009] (3) The yield of O. gambiae strain HK26 was higher than that of O. gambiae JZB2115055;

[0010] (4) The weight of a single mushroom of the Odenma spp. strain HK26 was higher than that of the Odenma spp. JZB2115055;

[0011] (5) The fruiting body uniformity of the Odenia spp. strain HK26 was better than that of the Odenia spp. JZB2115055;

[0012] (6) The stipe length of the Odensis globulus strain HK26 was significantly longer than that of the Odensis globulus strain JZB2115055;

[0013] (7) The stipe length deviation of the Odenthetratus globulus strain HK26 is smaller than that of the Odenthetratus globulus JZB2115055;

[0014] (8) The stipe hardness of the Odensis globulus strain HK26 is higher than that of the Odensis globulus strain JZB2115055;

[0015] (9) The average stipe length of the Odensis globulus strain HK26 was significantly higher than that of the Odensis globulus strain JZB2115055;

[0016] (10) The stipe uniformity of the Agaricus serrata strain HK26 was significantly better than that of the Agaricus serrata JZB2115055.

[0017] A second object of the present invention is to provide the fruiting bodies, mycelium and / or spores of the above-mentioned Odetteria globosum strain HK26.

[0018] A third object of the present invention is to provide the protoplasts of the above-mentioned Odetteria globosum strain HK26.

[0019] A fourth object of the present invention is to provide a mushroom log containing the above-mentioned Odetteria globosum strain HK26.

[0020] A fifth object of the present invention is to provide a use of the above-mentioned Odenia globosum strain HK26 in preparing Odenia globosum fruiting bodies and / or mycelium and / or spores.

[0021] A sixth object of the present invention is to provide the use of the above-mentioned Agaricus serrata strain HK26 in Agaricus serrata breeding.

[0022] A seventh object of the present invention is to provide a method for cultivating the above-mentioned Odetteria serrata strain HK26, wherein the fruiting temperature of the Odetteria serrata strain HK26 is 18-23°C.

[0023] Preferably, the culture temperature of the Agaricus globosum strain HK26 after fruiting is 22-28°C.

[0024] More preferably, the fruiting time of the Odette mushroom strain HK26 is 37-42 days after inoculation.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention utilizes space-induced mutagenesis technology to improve the varieties of Agaricus serrata, analyzes the stability and direction of mutation through continuous subculture experiments, and screens strains with stable mutations and good uniformity through mycelium evaluation and mushroom fruiting agronomic trait evaluation, thereby accelerating the industrial application process of Agaricus serrata.

[0027] Description of biological preservation of A. serrata HK26 (ADMHK26):

[0028] Depository: General Microbiology Center of China Culture Collection Administration of Microorganisms;

[0029] Deposit number: CGMCC NO.41674;

[0030] Deposit date: November 28, 2024;

[0031] Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0032] Taxonomic name: Oudemansiella apalosarca. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The growth rates of the different successive generations of HK and CK strains in Example 1, where A: 1st generation, B: 5th generation, and C: 10th generation.

[0034] Figure 2 This is a diagram of the fruiting bodies of each strain in Example 1.

[0035] Figure 3 This is an analysis of the stipe length of the fruiting bodies of the stable variant HK and CK strains in Example 1. DETAILED DESCRIPTION

[0036] Example 1

[0037] 1 Materials and Methods

[0038] 1.1 Test materials

[0039] The JZB2115055 strain of Agaricus sphaerocephala is a new edible fungus variety bred by our team with independent intellectual property rights. It has been identified as a new edible fungus variety in Beijing with the identification number Jingpinjianjun 2015031 and is currently preserved in the Beijing Edible Fungus Germplasm Resource Bank.

[0040] 1.2 Experimental methods

[0041] 1.2.1 Aerial mutagenesis breeding and strain screening

[0042] The JZB2115055 strain was activated in a PDA (potato dextrose medium 1L: potato dextrose agar powder 39g) plate, and the activated strain was transferred to a 2ml cryopreservation tube (Corning), cultured at 25°C until germination, weighed, and sent for space transport. The uncarried strain was used as the control strain (CK). The carried strain was carried on the Shenzhou XII spacecraft on June 17, 2021, entered the Chinese space station, stayed in the space station for 92 days, and returned on September 17, 2021. The returned carried strain was subjected to tip separation, and a total of 50 carried-on space strains (HK) were isolated. The HK strain was inoculated into a PDA plate and recorded as the first generation, and preserved.

[0043] 1.2.2 Mutation stability test

[0044] After culturing 50 first-generation HK and CK strains for 10 days, use a 6mm borer under a sterile environment to punch holes. The bacterial plugs are then inoculated into the center of new PDA culture medium and incubated at 25°C for 10 days before the next subculture. This process is repeated until the tenth generation is reached.

[0045] 1.2.3 Determination of antagonism experiment

[0046] Under sterile conditions, the first, fifth, and tenth generation HK and CK strains were inoculated onto the same PDA comprehensive medium (1 L potato dextrose PDA comprehensive medium: 200 g of boiled peeled potatoes, filtrate, 20 g glucose, 20 g agar, 3 g potassium dihydrogen phosphate, 1.5 g anhydrous magnesium sulfate, 5 g peptone, 10 mg vitamin B1, and diluted to 1 L with deionized water). These strains were cultured in parallel at 25°C for 25 days. Antagonistic differences between HK and CK strains at different subculture times were observed. HK strains that showed antagonistic differences with CK after consecutive subcultures were considered stable variants. Subsequently, fruiting experiments and agronomic trait assessments were performed on these stable variants.

[0047] 1.2.4 Determination of mycelial growth rate

[0048] The growth rate of HK and CK strains after 1, 5, and 10 subcultures was measured using the cross-cross method. Cultures were performed on PDA medium at 25°C for 2 days until mycelial germination, with growth rate measurements taken every 2 days. Three replicates were set for each strain.

[0049] 1.2.5 Analysis of agronomic traits of the fruiting bodies of Agaricus globosum strains induced by space flight

[0050] The stable variants of the aerospace strains were evaluated for fruiting. The HK and CK strains were activated and cultured on PDA plates for 14 days. When their mycelial growth reached peak activity, they were transferred to culture bags (composition: 60% cottonseed hulls, 38% bran, 2% lime, 65% moisture, pH 7-8) that had been autoclaved at 121°C for 120 minutes. The bags were incubated at 25°C with good ventilation until full mycelium growth. After full mycelium growth, the bags were transferred to a climatic chamber at 20°C and 90% humidity for fruiting. Ten bags were inoculated for each strain. The yield per bag, weight per mushroom, cap diameter, thickness, and hardness, and stipe length, diameter, and hardness were calculated for each strain to select superior strains.

[0051] 1.2.6 Data Processing and Analysis

[0052] Experimental data were processed using Excel 2021, and analysis of variance and significance was performed using IBM SPSS Statistics 19. A p < 0.05 indicated significant differences between the same indicators. Homogeneity analysis and violin plots were performed using OmicShare online software (https: / / www.omicshare.com / tools / ). Drawings were created using Adobe Illustrator CS6.

[0053] 2 Results and Analysis

[0054] 2.1 Analysis of intergenerational antagonism of HK strains after subculture of Odetteria glomerata

[0055] Biological antagonism is the competitive exclusion, interference and inhibition between organisms. Antagonism tests can quickly distinguish the differences between two strains. During the 1st, 5th and 10th generation cultivation, there were 6 strains that always had antagonism with CK (Table 1), namely HK13, HK14, HK15, HK25, HK26 and HK28. There were 22 strains that never had antagonistic reactions with CK. There were 14 HK strains that had antagonistic reactions in the 1st or 5th generation but not in the 10th generation, and the antagonism gradually disappeared. There were 0 HK strains that had antagonistic reactions in the 1st or 5th generation but not in the 10th generation, and the antagonism gradually accumulated. There were 6 strains that had antagonistic reactions with CK only in the 5th generation.

[0056] Table 1 Analysis of intergenerational antagonism between HK strain and CK after subculture

[0057]

[0058] Mycelial growth rate of 2.2HK strain

[0059] Mycelial growth rate is one of the indicators for intuitively judging the occurrence of strain variation. IBM SPSS Statistics19 software was used to analyze the significant differences in the growth rates of the 1st, 5th, and 10th generation HK strains and CK ( Figure 1 During the subculture process, the growth rates of the Aerospace strain and the CK strain fluctuated. In the first generation, only the growth rates of the two HK strains, HK26 and HK42, were significantly different from those of the CK strain ( Figure 1 A), of which HK42 grew significantly faster than CK, while HK26 grew significantly slower than CK. After 5 consecutive subcultures, a total of 8 HK strains showed significant differences from CK, and their growth rates were significantly higher than CK ( Figure 1 B). After 10 consecutive subcultures, the growth rate of HK44 was significantly higher than that of CK, while the growth rates of the three HK strains were significantly lower than that of CK ( Figure 1 C), among which HK44 was significantly higher than CK in the 5th and 10th generations.

[0060] Comprehensive analysis of antagonism and growth rate results revealed that strains HK13, HK14, HK15, HK25, HK26, and HK28 consistently exhibited antagonism during subculture. With the exception of HK28, the growth rate deviations of the remaining HK strains all showed a decreasing trend. Therefore, subsequent fruiting experiments and agronomic trait evaluations were conducted on these six HK strains.

[0061] 2.3 Evaluation of agronomic traits of stable variant HK strains

[0062] Table 2 Fruiting Body Agronomic characteristics analysis of HK and CK strains

[0063]

[0064]

[0065] Note: The agronomic traits of HK and CK strains showed significant differences when p < 0.01, marked as **, 0.01 <p<0.05时标注为*,表2是将菌株栽培于栽培料中培养37d的统计结果。

[0066] Six stable variant HK strains were screened and tested against CK strains for fruiting, and their fruiting body agronomic traits were statistically analyzed. All seven strains successfully formed fruiting bodies. HK13, HK14, and CK required 42 days to fruit, while HK15, HK25, HK26, and HK28 only took 37 days to fruit. In terms of yield, HK26 significantly increased its yield (p < 0.05), with an average yield per bag increase of 46.51% compared to CK strains. Its biological efficiency was consistent with the average yield. A statistical comparison of the number of fruiting bodies formed revealed that, with the exception of HK15, all other HK strains had a higher number of fruiting bodies per bag than CK. A systematic analysis of the agronomic traits of individual fruiting bodies of Agaricus serrata was conducted (Table 2). The average weight of HK26 fruiting bodies was higher than that of CK, increasing by 4.06%. The individual fruiting body weights of HK26 and HK15 exhibited less variation than those of CK, indicating superior fruiting body uniformity. The caps and stipes of the HK26 strains were analyzed. Six fruiting bodies were collected from each bag. The cap diameters of all HK strains were not significantly different from those of CK. The stipe length of HK26 strain was significantly higher than that of CK (p<0.01), which was 1.25 times that of CK ( Figure 2), and HK26 had the smallest deviation in stipe length, demonstrating good uniformity. In terms of hardness, HK15 had significantly lower cap hardness than CK, while HK13 had the smallest deviation. There were no significant differences in stipe hardness between the HK strains and CK, with HK26 having the greatest stipe hardness and HK28 having the smallest deviation.

[0067] As a type of long root mushroom, the stipe length and uniformity of the Odette mushroom are important indicators for agronomic evaluation of the fruiting body. The OmicShare online software was used to draw a violin plot of the stipe length of the fruiting bodies of the HK strain and CK. The flatter the shape of the violin plot, the smaller the difference between the fruiting bodies. On the contrary, the narrower and longer the shape, the greater the difference between the fruiting bodies. The violin plots were analyzed, and the results showed that the average stipe length and median of HK26 were significantly higher than those of CK. In the violin plot, the stipe length of HK26 was flat, indicating that the length of each fruiting body was relatively uniform. The stipe length distribution of CK was narrow and long, indicating that the stipe uniformity of HK26 was significantly better than that of CK ( Figure 3 ), and the stipe hardness of HK26 was also higher than that of CK (Table 2).

[0068] 3 Conclusion

[0069] In this study, the stability of space-borne strain variation was investigated through continuous subculture experiments combined with mycelial phenotypic analysis. Antagonism experiments revealed that only 12% of all strains exhibited stable antagonistic variations, while 28% of space-borne strains gradually lost antagonism with increasing subculture times. This suggests that binucleate strains harboring space travel are highly susceptible to reversion mutations, necessitating continuous subculture experiments to screen for stable variants. Stable HK strains also exhibited varying degrees of variation in the agronomic traits of their fruiting bodies. Overall, over 60% of HK strains exhibited a 5-day reduction in fruiting time compared to CK strains, and exhibited greater fruiting body numbers. Violin plots were used to analyze the agronomic traits of HK fruiting bodies, revealing that the deviations between the agronomic traits of different strains exhibited distinct patterns. This allows for a more intuitive analysis of the uniformity of individual fruiting bodies compared to traditional tabular analysis. Analyzing experimental data using violin plots can be widely applied to omics data analysis. This experiment, through space-induced mutagenesis combined with agronomic trait evaluation, identified a superior space-induced variant of the Odette mushroom strain HK26, characterized by stable mutations, a shorter fruiting cycle, uniform fruiting body growth, and high yield. This strain has great potential for industrial application. In summary, utilizing space-induced mutagenesis breeding technology combined with agronomic trait evaluation is an effective approach for selecting high-quality edible fungi varieties.

[0070] 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 strain of Agaricus serrata HK26, characterized in that The deposit number of the Odenia serrata strain HK26 is CGMCC NO. 41674, and its taxonomic name is Odenia serrata Oudemansiella apalosarca .

2. The fruiting body, mycelium and / or spore of the Odette mushroom strain HK26 according to claim 1.

3. The protoplast of the Agaricus serrata strain HK26 described in claim 1.

4. A mushroom log containing the Odette Agaricus globosum strain HK26 according to claim 1.

5. Use of the Odetteria serrata strain HK26 according to claim 1 in preparing Odetteria serrata fruiting bodies and / or mycelia and / or spores.

6. Use of the Agaricus serrata strain HK26 according to claim 1 in Agaricus serrata breeding.

7. The method for cultivating the Odette mushroom strain HK26 according to claim 1, characterized in that: The fruiting temperature of the Agaricus globosum strain HK26 is 18-23°C.

8. The culture method according to claim 7, characterized in that The culture temperature of the Odette mushroom strain HK26 after fruiting is 22-28°C.

9. The culture method according to claim 7, characterized in that The fruiting time of the Odette mushroom strain HK26 is 37-42 days after inoculation.

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