Pholiota nameko strain pian 31, its cultivation method and application

Abstract

The present application relates to the technical field of edible mushroom breeding, and specifically discloses a pholiota nameko strain smooth 31, a cultivation method and application thereof. Compared with traditional main cultivars, the pholiota nameko strain smooth 31 provided by the present application has the characteristics of uniform and consistent fruiting, high survival rate of primordium, and short time interval between fruiting and intertidal, and has high yield, good mushroom quality, high biological conversion rate, and short cultivation period. The pholiota nameko strain is of high quality, has strong environmental adaptability, is suitable for large-scale planting in regions with suitable climate conditions nationwide, and is also suitable for factory intelligent cultivation.

Description

Technical Field

[0001] This invention relates to the field of edible fungi breeding technology, and in particular to a slippery mushroom strain, smooth 31, its cultivation method and application. Background Technology

[0002] Nameko mushrooms, an important low-temperature edible fungus, are highly favored by the market for their unique flavor and rich nutrition. However, in long-term production practice, due to factors such as repeated transfers, environmental stress, and viral infections, existing main cultivated nameko mushroom varieties generally suffer from severe aging and degeneration of the mycelium. This not only leads to a decline in mycelial vitality and a slowdown in growth rate, but also directly causes a series of production bottlenecks: First, poor resistance to stress and disease, resulting in a high contamination rate during cultivation; second, a significant reduction in the survival rate of primordia in commercial mushroom fruiting bodies; third, a decline in yield and quality, with reduced yield per unit area and fruiting bodies often exhibiting problems such as thin caps and poor color; fourth, insufficient mycelial after-ripening or inconsistent physiological maturity leads to a longer fruiting flush interval and extended fruiting cycle, greatly reducing facility turnover rate and economic benefits. Currently, conventional rejuvenation methods are insufficient to fundamentally restore spore vitality. Therefore, there is an urgent need to provide a new nameko mushroom strain to meet the production needs of modern facility agriculture. Summary of the Invention

[0003] In response to the problems caused by the aging and degeneration of existing nameko mushroom varieties, such as poor resistance to pathogens, low survival rate of mushroom buds, low yield, poor quality, long interval between mushroom flushes, and long fruiting cycle, this invention provides a nameko mushroom strain, Pinghua 31, along with its cultivation method and application.

[0004] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a Pholiotia nameko strain, which was deposited on June 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42867.

[0005] Compared with traditional nameko mushroom varieties, the nameko strain Pinghua 31 provided by this invention has the characteristics of uniform fruiting, high survival rate of fruiting body primordia, and short interval between fruiting and flushing. At the same time, this strain has high yield, good mushroom quality, high biological conversion rate, and short cultivation cycle, making it a high-quality nameko mushroom strain. Moreover, it has strong environmental adaptability and is suitable for large-scale promotion and cultivation in areas with suitable climate conditions throughout the country. It is also suitable for industrialized intelligent cultivation applications.

[0006] This invention provides a protoplast prepared from the mycelium of the above-mentioned Pleurotus ostreatus strain Smooth 31.

[0007] The present invention provides spores produced by the above-mentioned slippery 31 strain of nameko mushroom.

[0008] The present invention provides a mycelium produced by the above-mentioned Nameko mushroom strain smooth 31.

[0009] The present invention provides a fruiting body produced by the above-mentioned nameko mushroom strain smooth 31.

[0010] The present invention provides a nameko mushroom spawn comprising the above-mentioned nameko strain smooth 31.

[0011] The application of the Nameko mushroom strain Smooth 31 provided by this invention in Nameko mushroom breeding.

[0012] This invention provides the application of the above-mentioned Nameko strain Smooth 31 in the preparation of Nameko fruiting bodies, Nameko mycelium, Nameko protoplasts or Nameko spores.

[0013] The present invention provides a cultivation method for the above-mentioned nameko mushroom strain smooth 31, comprising the following steps: inoculating the preserved strain or fruiting body of the nameko mushroom strain smooth 31 into a mother culture medium to obtain mother mycelium; inoculating the mother mycelium into a primary culture medium for expansion culture to obtain primary mycelium; and then inoculating the primary mycelium into a culture bag medium for culture bag cultivation and fruiting management.

[0014] Preferably, the formula of the mother culture medium includes: potato 180g / L~220g / L, glucose 15g / L~25g / L, broadleaf tree sawdust 8g / L~10g / L, wheat bran 4g / L~6g / L, agar 18g / L~22g / L, and natural pH.

[0015] Preferably, the original culture medium comprises the following components in parts by weight: 80-84 parts sawdust, 15-18 parts wheat bran, 0.5-1.5 parts gypsum, 0.2-0.5 parts lime, 0.5-1.5 parts brown sugar, 120-150 parts water, and natural pH.

[0016] Preferably, the culture medium for the mushroom bags comprises the following components in parts by weight: 81-85 parts sawdust, 15-18 parts wheat bran, 0.5-1.5 parts gypsum, 0.2-0.5 parts lime, 120-150 parts water, and natural pH.

[0017] Preferably, the culture temperature of the mother mycelium and the original mycelium is 15℃~22℃.

[0018] Preferably, the culture temperature of the mushroom bags is 15℃~22℃.

[0019] Preferably, the relative humidity of the air during the cultivation of the mother mycelium and the original mycelium is 55%~60%.

[0020] Preferably, the relative humidity of the air in the culture bag is 55%~60%.

[0021] For example, during the cultivation of the mother culture mycelium, the original culture mycelium, and the culture bags, the carbon dioxide concentration needs to be controlled below 4000 ppm, and direct sunlight should be avoided.

[0022] The Nameko mushroom strain 31 provided by this invention has strong environmental adaptability and is suitable for large-scale cultivation. Attached Figure Description

[0023] Figure 1 Morphological diagram of the fruiting body of strain 31 provided by the present invention; Figure 2 The maximum likelihood phylogenetic tree diagram constructed based on the ITS sequence of strain 31 provided by this invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] Unless otherwise specified, the raw materials and reagents used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0026] Example 1 This embodiment provides relevant materials for the selection and breeding of the nameko mushroom strain smooth 31.

[0027] Parental strains: Commonly used nameko mushroom varieties in northern regions, including nameko SC3, Zaozhuang, C3-1, and Ri19; strains were obtained from liquid nitrogen preservation by Pingquan Xicai Applied Fungi Technology Development Co., Ltd.

[0028] Culture medium formulation The mother culture medium consists of the following formula: 200 g / L potato, 20 g / L glucose, 10 g / L broadleaf tree sawdust, 5 g / L wheat bran, 20 g / L agar, and natural pH.

[0029] The original culture medium consists of the following components in parts by weight: 80 parts sawdust, 15 parts wheat bran, 1 part gypsum, 0.5 parts lime, 1 part brown sugar, 130 parts water, and natural pH.

[0030] The culture medium for the mushroom bags consists of the following components in parts by weight: 85 parts sawdust, 15 parts wheat bran, 1 part gypsum, 0.5 parts lime, 130 parts water, and natural pH.

[0031] Example 2 This embodiment provides the breeding process of the nameko mushroom strain smooth 31.

[0032] On a clean bench, equal amounts of mycelium from test tubes of the parent strains of *Pleurotus ostreatus* SC3, Zaozhuang, C3-1, and Ri19, along with the culture medium, were scooped into sterilized mortars. Sterilized quartz sand (30 wt% of the total culture medium) was added and the mixture was ground until small pieces were formed. Then, mannitol solution (250 mL, 50 g) was added to the mortar, and the mixture was ground again until it reached a thin porridge consistency, with a particle size ≤0.5 mm. After grinding, the porridge-like mixture was placed on a sterilized blank agar plate, shaken to level the surface, and the thickness was controlled to 8 mm. The plate was then sealed and secured with a plastic sealing strip. The plate was placed in a microwave oven at 2450 MHz and 700 W for 10 seconds. The microwaved plate was then placed in a biochemical incubator for mycelial culture, with the culture temperature controlled at 20°C. After 15-16 days of incubation... After 8 days of cultivation, white mycelial spots appeared on the surface of the plate medium. These white mycelial spots were picked out and transferred to the center of the mother culture medium plate for continued mycelial cultivation. The cultivation temperature was set at 20℃, the relative humidity at 55%, the carbon dioxide concentration below 4000ppm, and the light intensity below 1000lux for propagation. After the mycelium germinated, the plate was cultivated for another 5 days for three subcultures, and then screened and discarded. In the first subculture, after 4 days, inoculum blocks that did not germinate or germinated slowly were discarded. In the second subculture, after 8 days, plates with angular or fan-shaped changes, uneven edge growth, or slow mycelial growth were discarded. In the third subculture, when the mycelium was about to cover the petri dish, plates with uneven mycelial edges, abnormal color, discoloration, or aging of the inoculum point (yellowing, discoloration, patches, exudation of yellow liquid, shrinkage, etc.) were discarded.

[0033] Preliminary screening was conducted using sensory indicators such as the density, color, growth rate, and morphological characteristics of the mycelial growth of the fusion hybrid strains. Twenty strains were obtained. When the mycelia covered the plate medium (90 mm), they were transferred to a new plate medium for mycelial culture, resulting in 20 test strains.

[0034] Example 3 This embodiment provides the breeding process of the nameko mushroom strain smooth 31.

[0035] ① Antagonistic screening of hybrid fusion strains and parent strains The 20 different strains obtained from the initial screening were subjected to antagonism tests with the parent strains Pleurotus ostreatus SC3, Zaozhuang, C3-1 and Ri19. The presence of antagonistic lines between the hybrid offspring and the parent strains in the plates was used to determine whether the hybrid offspring were different from the parent strains, and thus to determine whether the hybrid strains were successfully hybridized. Those that were different were used as the experimental materials for the next step. Antagonism tests revealed that 13 out of 20 strains showed clear antagonistic lines with their parents.

[0036] ② Antagonism test between hybrid fusion products Antagonism tests were conducted among the fusion strains that successfully underwent hybridization, ultimately identifying only 7 new strains.

[0037] ③ Locking-type combined microscopic examination of hybrid fusions Microscopic examination of the seven identified hybrid fusions yielded 50 fields of view, with clamp fusion rates exceeding 75%, all meeting the requirements for viability.

[0038] Example 4 This embodiment provides the propagation and culture of the nameko mushroom strain smooth 31.

[0039] Seven new strains HZGZJ1~HZGZJ7 were identified and cultivated. The mycelia were inoculated into the mother culture medium and cultured at 20℃ to obtain the mother culture mycelia. The mother culture mycelium was inoculated into the original culture medium and cultured at a temperature of 20℃ and a humidity of 65%. When the mycelium covered the original culture medium, the original culture mycelium was obtained. The original mycelium was inoculated into the culture medium of the cultivated spawn and cultured at a temperature of 20℃ and a humidity of 65%. When the mycelium covered the culture medium of the cultivated spawn, the culture medium of the cultivated spawn was obtained. Polyethylene folded bags with dimensions of 17cm×60cm×0.005cm were used for inoculation. 1000 bags were used for each strain, and each bag contained 1100g of mushroom substrate. The bags were sterilized at 100℃ under normal pressure for 24 hours. When the temperature of the substrate in the center of the cultivation bag was below 20℃, mycelium of the cultivation spawn was inoculated. The bags were then cultured in a mycelium incubation room with a relative humidity of less than 65% and a temperature of 20℃. The mycelium incubation period was set at 90-100 days. When a light yellow color change layer appeared on the surface of the bags and the signal mushrooms appeared, the bags were placed on shelves for fruiting management. Fruiting management after shelf placement was carried out according to conventional methods. Parent strains of *Pleurotus ostreatus*, namely SC3, Zaozhuang, C3-1, and Ri19, were set as controls.

[0040] The mycelium growth and fruiting of different *Pleurotus ostreatus* strains were monitored, and the monitoring results are shown in Table 1. Table 1 As shown in Table 1, among the obtained hybrid strains, HZGZJ7 has a short mycelial incubation time, low infection rate, and strong resistance to pathogens. At the same time, its fruiting rate and yield per bag are better than other hybrid strains and the four parent strains, showing obvious hybrid vigor.

[0041] For each strain, 30 fruiting bodies were randomly selected. Each fruiting body contained a complete stipe and cap. The characteristics of the fruiting bodies and the weight of a single mushroom were monitored for each strain. 100 mushroom logs were randomly selected to calculate the biological conversion rate, and the average value was calculated. The results are shown in Table 2. Table 2 As shown in Table 2, the fruiting bodies of the new hybrid strain HZGZJ7 are larger and thicker than those of the parent strains, with a moderate cap-to-stem ratio and a yellowish-brown color, which better meets current market demands. It is suitable for drying, fresh sales, and pickling. In terms of biological conversion rate, the HZGZJ7 strain has a higher conversion rate of 112.1%, which is higher than that of the four parent strains, resulting in a significant increase in yield and fully demonstrating the hybrid vigor.

[0042] The survival rate of fruiting body primordia is the ratio of the number of primordia differentiating into fruiting bodies to the number of primordia forming. One hundred mycelial logs were randomly selected, and the survival rate of fruiting bodies on each log was counted and the average value was calculated. The survival rate of fruiting body primordia of strain HZGZJ7 was monitored, and the results are shown in Table 3. Table 3 Note: Under normal circumstances, one fruiting cycle of nameko mushroom cultivation can produce four flushes of mushrooms.

[0043] During the growth and development of *Pleurotus ostreatus* fruiting body primordia, varying degrees of mortality occur. The proportion of these mortalitys directly impacts the overall yield; a higher survival rate results in a higher yield. For both the HZGZJ7 strain and its parent strain, the survival rate of fruiting bodies gradually decreases with each fruiting flush.

[0044] Table 3 shows that the average survival rate of the fruiting body primordia of strain HZGZJ7 was still 78.5%, which is high and demonstrates the strong yield-increasing advantage of this strain.

[0045] The interval between fruiting flushes of hybrid strain HZGZJ7 and its parent strain was monitored, and the monitoring results are shown in Table 4. Table 4 After the first flush of nameko mushrooms is harvested, a certain interval is needed for mycelial growth before the next flush of mushrooms can emerge. The length of this interval affects the length of the fruiting period. The interval refers to the time from the end of the harvest of the previous flush to the formation of the mushroom buds in the next flush. The HZGZJ7 strain took only 25 days for the three intervals, which is 27 days less than the 52 days of the parent strain SC3, 21 days less than the 46 days of the parent strain Zaozhuang, 30 days less than the 55 days of the parent strain C3-1, and 16 days less than the 41 days of the parent strain Ri19, showing a significant reduction in time.

[0046] Microscopic examination of the cord-like structures of strains HZGZJ7, SC3, Zaozhuang, C3-1, and Ri19 showed that the probability of cord-like structures appearing in 50 fields of view was 97% for strain HZGZJ7, 84% for strain SC3, 81% for strain C3-1, 79.6% for Zaozhuang, and 87.8% for Ri19.

[0047] The hybrid strain HZGZJ7 has a mycelial growth cycle of 105 days, a first flush harvest time of 116 days, and a total flush transition period of 25 days. The fruiting rate reaches 99%, and the average survival rate of primordia in the fourth flush reaches 78.5%. The infection rate is only 0.7%. The cap thickness of the HZGZJ7 strain is 8-12 mm, the cap diameter is 18-28 mm, the stipe length is 22-32 mm, the single mushroom weight reaches 18-21 g, and the bioconversion rate reaches 112.1%. Compared with the traditional SC3, Zaozhuang, C3-1, and Ri19 strains, the new hybrid strain HZGZJ7 (named Pinghua 31) possesses excellent qualities such as a short cultivation cycle, low infection rate, high yield, significantly improved bioconversion rate, and high fruit set, showing great potential for development and promotion.

[0048] Molecular biological identification and preservation of strain 31 This invention uses the CTAB method to extract total DNA from the bacterial strain. A mixed reagent is used: upstream primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and downstream primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR amplification system (25 μL) consists of: 10 μL of 2×Mix, 1 μL of each primer, 7 μL of ddH2O, and 1 μL of DNA template. The PCR amplification program is: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 20 s, 55℃ annealing for 20 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min. The PCR product is sequenced, and the ITS sequence obtained is SEQ ID NO.1.

[0049] The specific details of SEQ ID NO.1 are as follows: .

[0050] Based on the ITS sequence, a phylogenetic tree was constructed using the maximum likelihood (ML) method, and the branch reliability was assessed by 1000 replicate samplings using the bootstrap method. BLAST homology search results showed that the ITS sequence fragment of the tested strain, Pinghua 31, had high homology with species of the genus *Pholiota* in the NCBI database, with 100% coverage and identity, and a highest score of 737. All alignment results belonged to members of the genus *Pholiota* within the family Pholiotaceae. Figure 2The provided phylogenetic tree shows that Smooth 31 is located within the Pholiota cluster, forming the same cluster as known Pholiota strains (such as Pholiota adiposa and Pholiota limonella), and exhibiting high support branches (spread support ≥90%), indicating stable evolutionary relationships and close phylogenetic relationships. Based on the combined results of BLAST alignment and phylogenetic tree analysis, Smooth 31 is definitively identified as a member of the genus Pholiota, belonging to the family Strophariaceae and the genus Lepidoptera. This conclusion is consistent with strain identification results based on morphological and ITS sequence analysis.

[0051] Comprehensive comparative analysis and phylogenetic tree topology analysis revealed that strain 'Smooth 31' forms a tightly clustered monophyletic branch with multiple known species within the genus *Pholiota*, consistent with its morphological classification (Basidiomycota, Agaricales, Agaricales, Psilocybeaceae, *Pholiota*). It did not exhibit cross-clustering with other genera within the same family (such as *Stropharia* and *Psilocybe*). Furthermore, the BLAST search results only matched sequences from the genus *Pholiota*, further validating the strain's genus-level classification. All matched sequences covered the full length of the query sequence, with an E-value of 0.0, indicating extremely high reliability of the alignment results. Based on the above molecular systematic evidence and morphological characteristics, 'Smooth 31' was confirmed as a fungus belonging to the genus *Pholiota*.

[0052] Based on the above classification, identification and molecular phylogenetic studies of the ITS sequence, it was determined that the smooth 31 strain of the present invention belongs to Pholiotia nameko in taxonomy. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42867, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on June 9, 2026.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of *Pleurotus ostreatus* strain, smooth 31, characterized in that, Its accession number is CGMCC No.42867.

2. A protoplast prepared from the mycelium of the slippery 31 strain of mushroom as described in claim 1.

3. A type of spore produced by the slippery 31 strain of the mushroom nameko as described in claim 1.

4. A mycelium produced by the smooth 31 strain of *Pleurotus ostreatus* as described in claim 1.

5. A fruiting body produced by the slippery 31 strain of nameko mushroom according to claim 1.

6. A Nameko mushroom log comprising the Nameko strain Smooth 31 as described in claim 1.

7. The application of the Nameko mushroom strain Smooth 31 as described in claim 1 in Nameko mushroom breeding.

8. The use of the Nameko strain Smooth 31 as described in claim 1 in the preparation of Nameko fruiting bodies, Nameko mycelium, Nameko protoplasts or Nameko spores.

9. A method for cultivating the *Pleurotus ostreatus* strain smooth 31 according to claim 1, characterized in that, Includes the following steps: The preserved strain or fruiting body of the *Pleurotus ostreatus* strain 31 was inoculated onto a mother culture medium to obtain mother mycelium; the mother mycelium was inoculated onto a primary culture medium to expand the culture and obtain primary mycelium; and the primary mycelium was then inoculated onto a culture bag for culture and fruiting management.

10. The cultivation method of the nameko mushroom strain smooth 31 as described in claim 9, characterized in that, The formula of the mother culture medium includes: potato 180g / L~220g / L, glucose 15g / L~25g / L, broadleaf tree sawdust 8g / L~10g / L, wheat bran 4g / L~6g / L, agar 18g / L~22g / L, and natural pH. The original culture medium comprises the following components in parts by weight: 80-84 parts sawdust, 15-18 parts wheat bran, 0.5-1.5 parts gypsum, 0.2-0.5 parts lime, 0.5-1.5 parts brown sugar, 120-150 parts water, and natural pH. The culture medium for the mushroom bags comprises the following components in parts by weight: 81-85 parts sawdust, 15-18 parts wheat bran, 0.5-1.5 parts gypsum, 0.2-0.5 parts lime, 120-150 parts water, and natural pH.

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