Oudemansiella spp. Strain and culture method thereof

Abstract

The invention discloses an oudemansiella yunnanensis strain and a culture method thereof, and belongs to the technical field of edible fungus research. The preservation number of the Oudemansiella spp. Strain HK26 is CGMCC (China General Microbiological Culture Collection Center) NO.41674, the Oudemansiella spp. Strain HK26 is named as Oudemansiella spp. By taxonomy, the preservation date is November 28, 2024, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No.1 yard, Beichen West Road, Chaoyang District, Beijing. According to the invention, a space mutagenesis technology is utilized to improve the variety of the oudemansiella ovalis, and the strain HK26 with stable variation and good uniformity is screened out by continuous subculture experiment, variation stability and variation direction analysis, mycelium evaluation and fruiting agronomic character evaluation, so that the industrial application process of the oudemansiella ovalis is accelerated.

Description

Technical Field

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

[0002] There are two main methods of edible fungi production in my country: traditional edible fungi production and factory cultivation. Traditional edible fungi production requires a lot of manpower costs and is subject to seasonal restrictions. Factory production can achieve year-round production and standardized production of edible fungi, and has the advantages of high production efficiency, guaranteed product quality and safety, and low labor input. It is one of the most representative agricultural modernization development models. However, my country's factory mushroom varieties are relatively single, mainly concentrated in Flammulina velutipes, Pleurotus eryngii, Hokkaido mushrooms, Agaricus bisporus, and Shimeji mushrooms. They are highly homogenized and the profit margins are shrinking year by year. It is extremely necessary to carry out research and development of new varieties dedicated to factories to meet the public's demand for diversified edible fungi varieties while promoting the quality and efficiency of edible fungi factory enterprises.

[0003] Oudemansiella apalosarca belongs to the phylum Basidiomycota, family Physalacriceae, genus Oudemansiella, and was formerly known as light brown Oudemansiella and tropical Oudemansiella. Oudemansiella is delicious and rich in protein, carbohydrates, multiple vitamins and mineral elements. It is a rare edible mushroom suitable for sightseeing and picking and has health benefits such as anti-oxidation and inhibition of tumor cell proliferation. At present, the main cultivated Oudemansiella variety in China is Oudemansiella oosporei. Its cultivation process still has problems such as large workload of soil covering cultivation, long ripening time, and serious problem of repeated cropping, which is not suitable for factory cultivation. Oudemansiella apalosarca has significant advantages in mycelium growth rate, mushroom fruiting cycle, and no need for soil covering, and has prospects for factory cultivation. However, in its production process, the uniformity of fruiting bodies is still a problem to be solved. Therefore, in order to improve the uniformity of fruiting bodies, this study used mutation breeding technology to improve the variety of Odette mushroom.

[0004] Space-induced mutagenesis is an efficient breeding method that has applications in both plants and microorganisms. This technology uses returning spacecraft to send crop seeds into the space environment, and uses special conditions such as microgravity, high vacuum, cosmic radiation, and weak magnetic fields to induce mutations in plant seeds, tissue culture cells, or microbial strains, causing their genes to mutate. Similar to artificial mutagenesis, the advantages of space-induced mutagenesis are that the breeding cycle is short, the variation range is large, and there are no potential safety hazards brought about by genetic modification technology. The frequency, variation range, and specific variation probability of 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 Odenia sphaerocephala strain HK26 has at least one of the following characteristics:

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

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

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

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

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

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

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

[0014] (8) The stipe hardness of the Odetteria spp. strain HK26 was higher than that of the Odetteria spp. JZB2115055;

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

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

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

[0018] The third object of the present invention is to provide the protoplasts of the above-mentioned Odenia microcarpa strain HK26.

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

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

[0021] The sixth object of the present invention is to provide the application of the above-mentioned Odenia serrata strain HK26 in the breeding of Odenia serrata.

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

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

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

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

[0026] The invention utilizes spaceflight mutagenesis technology to improve the varieties of Agaricus serrata, analyzes the stability and direction of variation through continuous subculture experiments, screens out strains with stable variation and good uniformity through mycelium evaluation and mushroom agronomic trait evaluation, and accelerates the industrial application process of Agaricus serrata.

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

[0028] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[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 different successive generations of HK strains 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 Odenia tricholoma 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 of Jingpinjianjun 2015031 and is now 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 cryotube (Corning), cultured at 25°C until germination, weighed and sent for space transport, and the uncarried strain was 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 the PDA plate and recorded as the first generation, and preserved.

[0043] 1.2.2 Mutation stability test

[0044] After culturing 50 HK and CK strains of the first generation for 10 days, the bacteria were punched with a 6 mm borer in a sterile environment, and the bacterial blocks were inoculated into the center of a new PDA culture medium. After culturing at 25°C for 10 days, the next subculture was performed. This process was repeated until the tenth generation was reached.

[0045] 1.2.3 Antagonism assay

[0046] Under sterile conditions, the first, fifth and tenth generation HK strains and CK strains were inoculated into the same PDA comprehensive medium (1L potato glucose PDA comprehensive medium: 200g peeled potatoes, boiled and filtrated, 20g glucose, 20g agar, 3g potassium dihydrogen phosphate, 1.5g anhydrous magnesium sulfate, 5g peptone, 10mg vitamin B1, and deionized water to 1L) for confrontation culture. After 25 days of constant temperature culture at 25℃, the antagonistic differences between HK strains and CK strains at different subculture times were observed. HK strains that had antagonistic differences with CK after continuous subculture were considered to be stable variant strains. Subsequently, mushroom production experiments and agronomic trait determinations were carried out on the stable variant strains.

[0047] 1.2.4 Determination of mycelium growth rate

[0048] The growth rate of HK and CK strains after 1, 5, and 10 subcultures was determined by the cross method. The strains were cultured in PDA medium at 25°C for 2 days until mycelium germinated, and the growth rate was determined every 2 days. Three replicates were set for each strain.

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

[0050] The stable variant of the spaceflight strains obtained were evaluated by mushroom production experiments. HK strains and CK were activated and cultured on PDA plates for 14 days. When their mycelium growth was the most vigorous, they were transferred to the cultivation bag material treated with 121℃ high pressure sterilization for 120 minutes (cultivation material formula: 60% cottonseed hulls, 38% bran, 2% lime, culture material humidity of 65%, pH value of 7-8), and cultured at 25℃ and well ventilated conditions until the mycelium was full. After the mycelium was full, it was transferred to the artificial climate room with a temperature of 20℃ and a humidity of 90% for mushroom production. Each strain was inoculated with 10 bags. The single bag yield, single mushroom weight, cap diameter, thickness, hardness, and stipe length, diameter and hardness of each strain were counted to select excellent strains.

[0051] 1.2.6 Data processing and analysis

[0052] Excel 2021 software was used to process the experimental data, and IBM SPSS Statistics 19 software was used for deviation analysis and difference significance analysis. When p < 0.05, it indicated that there was a significant difference between the same indicators. OmicShare online software (https: / / www.omicshare.com / tools / ) was used for homogeneity analysis and violin plot drawing. Adobe Illustrator CS6 software was used for drawing.

[0053] 2 Results and analysis

[0054] 2.1 Analysis of intergenerational antagonism of HK strains after subculture of O. globosum

[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 were always antagonistic to 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 variation of strains. IBM SPSS Statistics19 software was used to analyze the significant difference between 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 five 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, and 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 both the 5th and 10th generations.

[0060] Based on a comprehensive analysis of the antagonistic and growth rate results, the strains that always showed antagonism during the subculture process were HK13, HK14, HK15, HK25, HK26, and HK28. Among them, except for the HK28 strain, the growth rate deviation changes of the other HK strains showed a gradually decreasing trend. Therefore, subsequent fruiting experiments were carried out on these 6 HK strains and their agronomic traits were evaluated.

[0061] 2.3 Evaluation of the Agronomic Traits of Stable Variant HK Strains

[0062] Table 2 Analysis of the Agronomic Traits of the Fruiting Bodies of HK Strains and CK

[0063]

[0064]

[0065] Note: When there was a significant difference in the agronomic traits between the HK strain and the CK strain (p < 0.01), it was marked as **; when 0.01 < p < 0.05, it was marked as *. Table 2 is the statistical result of cultivating the strains in the cultivation medium for 37 days.

[0066] Fruiting experiments were carried out on the 6 selected stable variant HK strains and CK, and the agronomic traits of their fruiting bodies were statistically analyzed. All 7 strains could successfully form fruiting bodies. In terms of the fruiting body formation time, HK13, HK14, and CK needed 42 days to form fruiting bodies, while the HK15, HK25, HK26, and HK28 strains only needed 37 days to form fruiting bodies. In terms of yield, the yield of HK26 was significantly higher than that of CK (p < 0.05), and the average yield per bag increased by 46.51% compared with the CK strain. The biological efficiency was consistent with the average yield result. Statistical comparison and analysis of the number of fruiting bodies formed found that, except for the HK15 strain, the number of fruiting bodies per bag of the other HK strains was higher than that of CK. A systematic analysis of the agronomic traits of individual fruiting bodies of Oudemansiella raphanipes was carried out (Table 2). The average single mushroom weight of HK26 was higher than that of CK, increasing by 4.06% compared with CK. Moreover, the weight deviation of individual fruiting bodies of HK26 and HK15 was less than that of CK, indicating that the uniformity of the fruiting bodies was better than that of CK. Analyzing their caps and stipes, 6 fruiting body data were collected from each mushroom bag. The cap diameters of all HK strains had no significant difference from those of CK. The stipe length of the HK26 strain was extremely significantly higher than that of CK (p < 0.01), being 1.25 times that of CK ( Figure 2), and HK26 had the smallest deviation in stipe length and good uniformity. In terms of hardness, the cap hardness of HK15 was significantly lower than that of CK, and HK13 had the smallest deviation in cap hardness. There was no significant difference in stipe hardness between each HK strain and CK, HK26 had the largest stipe hardness, and HK28 had the smallest stipe hardness deviation.

[0067] As a type of long root mushroom, the stipe length and uniformity of Agaricus serrata are important indicators for agronomic evaluation of fruiting bodies. The OmicShare online software was used to draw a violin plot of the stipe length of the fruiting bodies of HK strains 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 strain variation was studied by continuous subculture experiments combined with mycelial phenotype analysis. The results of the antagonistic experiment showed that only 12% of all strains had stable antagonistic variation, and 28% of the space strains gradually lost antagonism with the increase of subculture times. This indicates that there is a greater possibility of reversion mutation in binucleate strains after space travel, and it is extremely necessary to conduct continuous subculture experiments to screen stable variant strains. The agronomic traits of the fruiting bodies of the stable variant HK strains also varied to varying degrees. Overall, more than 60% of the HK strains had a mushroom fruiting time that was 5 days shorter than that of the CK strain, and the number of fruiting bodies was more than that of the CK. The agronomic traits of the fruiting bodies of the HK strains were analyzed using violin plots. The deviations between the agronomic traits of the fruiting bodies of different strains can present different shapes. Compared with traditional table analysis, the uniformity of each fruiting body can be analyzed more intuitively. This method of analyzing experimental data using violin plots can be widely used in omics data analysis. In this experiment, space mutagenesis combined with agronomic trait evaluation was used to screen out the excellent space-mutated strain HK26 of Odea trichoderma with stable mutation, shorter fruiting cycle, uniform fruiting body growth, and high yield, which has great prospects for factory application. In summary, the use of space mutagenesis breeding technology combined with agronomic trait evaluation is an effective way to breed high-quality edible fungi varieties.

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

2. The Odette mushroom strain HK26 according to claim 1, characterized in that: The Odette mushroom strain HK26 has at least one of the following characteristics: (1) There is antagonism between the Odenia sphaerocephala strain HK26 and the Odenia sphaerocephala JZB2115055; (2) The time it takes for the Odenia sphaerocephala strain HK26 to form fruiting bodies is shorter than that for the Odenia sphaerocephala strain JZB2115055; (3) The yield of O. gambiae strain HK26 was higher than that of O. gambiae JZB2115055; (4) The single mushroom weight of the Odetteria spp. strain HK26 was higher than that of the Odetteria spp. JZB2115055; (5) The fruiting body uniformity of the Odetteria spp. strain HK26 was better than that of the Odetteria spp. JZB2115055; (6) The stipe length of the Odenia sphaerocephala strain HK26 was significantly longer than that of the Odenia sphaerocephala strain JZB2115055; (7) The stipe length deviation of the Odetteria spp. strain HK26 is smaller than that of the Odetteria spp. JZB2115055; (8) The stipe hardness of the Odetteria spp. strain HK26 was higher than that of the Odetteria spp. JZB2115055; (9) The average stipe length of the Odenia sphaerocephala strain HK26 was significantly higher than that of the Odenia sphaerocephala strain JZB2115055; (10) The stipe uniformity of the Odetteria spp. strain HK26 was significantly better than that of the Odetteria spp. JZB2115055.

3. The fruiting body, mycelium and / or spore of the Odetteria tricholoma strain HK26 according to claim 1 or 2.

4. The protoplasts of the Odetteria tricholoma strain HK26 according to claim 1 or 2.

5. A mushroom stick containing the Odette mushroom strain HK26 according to claim 1 or 2.

6. Use of the Odenia sphaerocephala strain HK26 according to claim 1 or 2 in preparing Odenia sphaerocephala fruiting bodies and / or mycelium and / or spores.

7. Use of the Odetteria serrata strain HK26 according to claim 1 or 2 in Odetteria serrata breeding.

8. The method for cultivating the Odetteria tricholoma strain HK26 according to claim 1 or 2, characterized in that: The fruiting temperature of the Odette mushroom strain HK26 is 18-23°C.

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

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

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