Lentinus edodes strain Le2538 suitable for plate culture and efficient fruiting method of lentinus edodes strain Le2538

Abstract

The invention relates to the field of edible mushroom strain breeding and cultivation, and provides a shiitake mushroom strain Le2538 suitable for plate culture, the preservation number of the strain is GDMCC No: 67456, the strain is preserved in Guangdong Microbial Culture Collection Center, and the life history from mycelium to sporocarp can be stably completed on a PDA culture medium. Through directional domestication, a model strain Le2538 capable of stably and efficiently completing sporocarp morphogenesis on a PDA flat plate is successfully obtained, the problem that most of shiitake strains are retarded in reproductive development under laboratory conditions and cannot form effective phenotypes for quantitative analysis is solved, and a matched punching type mycelium stimulation and low-temperature induction combined method has the advantages that the method is simple and convenient to implement, and the yield is high. According to the method, stable fruiting within 30-40 days can be achieved in a controlled laboratory environment, an experimental system for completely observing the whole development process of the mushrooms from mycelia to sporocarp on a flat plate is successfully established, only conventional laboratory equipment and a cheap PDA culture medium are needed in the whole process, operation is easy and convenient, a large amount of manpower and material resources are saved, and the screening flux is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of edible fungi strain selection and cultivation, specifically to a shiitake mushroom strain Le2538 suitable for plate culture and its efficient fruiting method. Background Technology

[0002] As the world's leading edible fungus in both consumption and production, the commercial cultivation of shiitake mushrooms heavily relies on stable strains with excellent agronomic traits and heterogeneous lignin-cellulose composite substrates. This cultivation method is not only time-consuming and resource-intensive, but also suffers from problems such as low screening throughput, high signal-to-noise ratio of phenotypic data, difficulty in observing key developmental events, and difficulty in tracing the genetic basis of terminal agronomic traits due to complex factors such as batch-to-batch variations in cultivation substrate formulations and variable fruiting environments. These problems severely restrict the systematic analysis of the genetic mechanisms of shiitake mushrooms and further improvement in breeding efficiency.

[0003] In basic research, potato dextrose agar (PDA) is the standard substrate for pure culture of fungi, physiological and biochemical analysis, and strain preservation. However, the vast majority of shiitake mushroom cultivation strains exhibit reproductive arrest on PDA, with highly random primordia formation and difficulty in ensuring the integrity of fruiting body development, making it impossible to form effective phenotypes for quantitative analysis. Therefore, industry and academia have long lacked a technical platform that enables rapid, accurate, repeatable observation and quantitative analysis of key reproductive and developmental traits in shiitake mushrooms under controlled and standardized laboratory conditions.

[0004] Developing a model shiitake mushroom strain that can adapt to plate culture and produce fruiting stably, along with its corresponding efficient and standardized fruiting method, is of vital scientific and industrial significance for accelerating the basic research process of shiitake mushrooms, innovating the genetic breeding system, and improving the precision of cultivation practices. Summary of the Invention

[0005] To address the shortcomings of existing technologies, a model shiitake mushroom strain that can adapt to plate culture and produce fruiting stably, along with its corresponding efficient and standardized fruiting method, is of vital scientific and industrial significance for accelerating the basic research process of shiitake mushrooms, innovating the genetic breeding system, and improving the precision of cultivation practices.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: a shiitake mushroom strain Le2538 suitable for plate culture, the strain having the accession number GDMCC No:67456, is deposited at the Guangdong Provincial Microbial Culture Collection Center, and can stably complete the life cycle from mycelium to fruiting body on PDA medium.

[0007] Preferably, the fruiting cycle of the strain is 30 to 40 days, with 2 to 5 fruiting fruits per plate, and the cap, stipe, gills, basidia and basidiospores of the fruiting bodies are intact.

[0008] A highly efficient fruiting method for shiitake mushroom strain Le2538 includes the following steps:

[0009] Step 1: Preparation of PDA culture medium;

[0010] Step 2: Inoculation and culture;

[0011] Step 3: Punch-type scratching;

[0012] Step 4: Low-temperature induction;

[0013] Step 5: Mushroom cultivation.

[0014] Preferably, the PDA culture medium is prepared as follows:

[0015] Cut 200g of fresh, unsprouted, peeled potatoes into chunks, add 1000mL of distilled water and boil for 20-30 minutes. After filtering, add 20g of glucose and 20g of agar strips to the filtrate, heat to dissolve, and then add distilled water to bring the volume to 1000mL. Sterilize by high-pressure steam at 121℃ for 30 minutes, cool to 50-60℃ and pour into sterile plates. Let stand to solidify. The agar strips should be cut into small pieces and added to the filtrate.

[0016] Preferably, the amount of PDA culture medium poured into a 90mm diameter sterile plate in step one is 10-15mL.

[0017] Preferably, the inoculation and culture in step two are as follows:

[0018] Use a 5mm diameter punch to make holes in a PDA plate covered with Le2538 mycelium. Inoculate the mycelium block into the center of the PDA plate prepared in step one and incubate in the dark at 23-25℃ for 7-10 days. During this period, seal the inoculated plate with sterile sealing film and maintain a constant temperature.

[0019] Preferably, in step three, the perforation-type mycelial scratching is carried out in a clean bench by using a 5mm diameter sterile punch to select 5 to 30 locations on the surface of the culture plate and making holes in a uniformly distributed manner. Some mycelial blocks are removed and some are retained in situ. After replacing the sealing film, the plate is cultured in the dark at 23 to 25°C for 1 to 3 days.

[0020] Preferably, in step four, the low-temperature induction involves placing the plate in the dark at 10–15°C for 2–7 days.

[0021] Preferably, the fruiting culture in step five is as follows:

[0022] The plates induced by low temperature were placed in the dark at 18-20℃ until fruiting bodies formed. The criteria for judging the formation of fruiting bodies were the integrity of the cap, stipe, gills, basidia, and basidiospores.

[0023] This invention provides a shiitake mushroom strain Le2538 suitable for plate culture and a method for efficient fruiting.

[0024] It has the following beneficial effects:

[0025] 1. This invention successfully obtained the model strain Le2538, which can stably and efficiently complete the fruiting body morphology on PDA plates through targeted domestication, solving the problem that most shiitake mushroom strains are unable to form an effective phenotype that can be used for quantitative analysis due to reproductive developmental arrest under laboratory conditions.

[0026] 2. The perforation-type mycelium-scraping combined with low-temperature induction method of this invention can achieve stable fruiting within 30-40 days in a controlled laboratory environment, successfully establishing an experimental system for observing the entire development process of shiitake mushrooms from mycelium to fruiting body on plates. This system shortens the observation period of traditional cultivation from 3-4 months to about 1 month, significantly improving research efficiency;

[0027] 3. Under standard PDA culture medium and stable, controllable laboratory conditions, this invention eliminates batch-to-batch variations in cultivation substrates and interference from environmental changes, significantly reducing background noise in phenotypic data. The fruiting bodies form completely and have normal morphology, making precise observation, quantitative analysis, and genetic tracing of key developmental traits possible, providing an ideal platform for elucidating the genetic basis of important traits in shiitake mushrooms.

[0028] 4. The entire process of this invention only requires conventional laboratory equipment and inexpensive PDA culture medium. It is easy to operate and does not rely on complex, heterogeneous woody cultivation substrates and large-scale mushroom-growing facilities, saving a lot of manpower and material resources and greatly improving screening throughput.

[0029] 5. The system of this invention is not only applicable to basic research such as developmental biology and genetics of shiitake mushrooms, but can also be used as a high-throughput functional screening platform. It can be widely used in applied research fields such as mutant library evaluation, gene function verification, environmental factor response analysis, and rapid initial screening of cultivation auxiliary materials. It is of great significance to promote the genetic breeding innovation and industrial technology progress of shiitake mushrooms and even edible fungi. Attached Figure Description

[0030] Figure 1 These are the 92 candidate strains involved in the PDA culture medium acclimatization test in this invention;

[0031] Figure 2 This invention compares the number of mushrooms produced on plates after perforation-type mycelium scratching treatment and without treatment of the Le2538 shiitake mushroom strain, revealing the fruiting-promoting effect of this key step;

[0032] Figure 3 This invention describes the continuous development process of strain Le2538 from primordium to mature fruiting body on PDA medium.

[0033] Figure 4 This is the Le2538 fruiting body successfully cultivated on PDA medium according to the present invention, including its complete macroscopic morphology and microscopic structural details. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Please see the appendix Figure 1 - Appendix Figure 4 As one aspect of this invention, this invention provides a shiitake mushroom strain Le2538 suitable for plate culture. This strain, with accession number GDMCC No:67456, is deposited at the Guangdong Provincial Microbial Culture Collection Center. It can stably complete the entire life cycle from mycelium to fruiting body on PDA medium, with a fruiting cycle of 30–40 days and 2–5 fruiting bodies per plate. The fruiting bodies have intact cap, stipe, gills, basidia, and basidiospore structures. The domestication and selection of this strain are as follows:

[0036] Using 63 single-hybrid progeny strains, 1 wild-type strain, 2 commercially available strain components, and 26 cultivated strains as materials, a three-generation directional domestication and fruiting stability screening experiment was conducted on PDA medium. The specific steps are as follows:

[0037] First, all strains underwent a first round of fruiting culture on PDA medium. The first-generation domesticated strains were obtained from the successfully fruiting bodies through tissue isolation. Subsequently, the first-generation domesticated strains underwent a second round of fruiting culture on PDA medium, and the second-generation domesticated strains were again obtained through tissue isolation. Finally, the above process was repeated to obtain the third-generation domesticated strains, which were then used to screen for candidate strains that could stably form fruiting bodies on PDA medium.

[0038] Throughout the acclimatization process, the culture conditions remained consistent: each strain was inoculated onto PDA plates and cultured in the dark at 23–25°C; once yellow pigment appeared on the surface of the mycelium, the culture was transferred to a dark environment at 18–20°C for continued cultivation until fruiting bodies formed. After fruiting, tissue isolation was performed using aseptic techniques in a clean bench: the fruiting bodies were longitudinally dissected with a sterile scalpel, and tissue blocks were excised at the junction of the stipe and cap with sterile forceps, then transferred to fresh PDA medium to complete the isolation and subculture of the first-generation strains.

[0039] Table 1. Changes in strain number at each stage

[0040]

[0041] Four third-generation domesticated strains were ultimately obtained. Among them, strain Le2538 had the shortest fruiting cycle of 40–50 days and exhibited stable and reproducible fruiting traits across three consecutive generations, thus it was selected as the preferred strain for subsequent research on the PDA medium method. This strain was obtained by our team through single-cross hybridization of Huxiang 922 and Xiaoxiang mushroom.

[0042] As another aspect of the present invention, the present invention provides a highly efficient fruiting method suitable for shiitake mushroom strain Le2538, comprising the following steps:

[0043] Step 1: Preparation of PDA culture medium

[0044] Select fresh, unsprouted potatoes, wash and peel them, weigh out 200g, and cut them into approximately 1m pieces. 3 Cut the potatoes into small pieces. Place the potato pieces in 1000mL of distilled water and boil for 20-30 minutes until the potato pieces are soft but not mushy. Filter using 4-8 layers of gauze and collect the filtrate. Add 20g of glucose and 20g of chopped agar strips to the filtrate, and heat while stirring until the agar strips are completely dissolved. Add distilled water to bring the volume to 1000mL. Dispense the culture medium into 500mL Erlenmeyer flasks and seal them with breathable sealing film. After autoclaving at 121℃ for 30 minutes, cool to 50-60℃, and pour 10-15mL of the medium onto a 90mm diameter sterile agar plate in a laminar flow hood. Allow to solidify.

[0045] Step 2: Inoculation and Culture

[0046] Inside a clean bench, use a 5mm diameter punch to make holes in a PDA plate covered with Le2538 mycelium, and use a sterile inoculation needle to transfer the mycelium to the center of the solidified PDA plate. After sealing the plate, invert it and place it in a 23-25℃ incubator in the dark for 7-10 days, observing mycelial growth daily.

[0047] Step 3: Punching and Scratching

[0048] In a clean bench, after removing the original sealing film, use a 5mm diameter sterile punch to make 5-30 holes evenly distributed on the surface of the culture plate. Some mycelial blocks are removed, while others are left in situ. Replace the sealing film to avoid contamination. Then, place the plate in a constant temperature incubator at 23-25℃ and incubate in the dark for 1-3 days to promote the recovery of the mycelium on the culture medium surface after mechanical damage.

[0049] Step 4: Low Temperature Induction

[0050] The above plates were placed in the dark at 10-15℃ for 2-7 days to induce mycelial growth at low temperature.

[0051] Step 5: Fruiting Cultivation

[0052] The plates induced by low temperature were placed in the dark at 18-20℃ until fruiting bodies formed. The criteria for judging the formation of fruiting bodies were the integrity of the cap, stipe, gills, basidia, and basidiospores.

[0053] Using the above method, after 30–40 days of cultivation, all test plates formed fruiting bodies, with 2–5 fruits per plate. Morphological observation confirmed that the cap, stipe, gills, basidia, and basidiospores of the fruiting bodies were very complete.

[0054] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A shiitake mushroom strain Le2538 suitable for plate culture, characterized in that, The strain, with accession number GDMCC No:67456, is deposited at the Guangdong Provincial Microbial Culture Collection Center and can stably complete its life cycle from mycelium to fruiting body on PDA medium.

2. The shiitake mushroom strain Le2538 suitable for plate culture according to claim 1, characterized in that, The fruiting cycle of the strain is 30-40 days, with 2-5 fruiting fruits per plate, and the cap, stipe, gills, basidia, and basidiospores of the fruiting bodies are intact.

3. A highly efficient fruiting method applicable to the shiitake mushroom strain Le2538 of claim 2, characterized in that, Includes the following steps: Step 1: Preparation of PDA culture medium; Step 2: Inoculation and culture; Step 3: Punch-type scratching; Step 4: Low-temperature induction; Step 5: Mushroom cultivation.

4. The efficient fruiting method for shiitake mushroom strain Le2538 according to claim 3, characterized in that, The PDA culture medium was prepared as follows: Cut 200g of fresh, unsprouted, peeled potatoes into chunks, add 1000mL of distilled water and boil for 20-30 minutes. After filtering, add 20g of glucose and 20g of agar strips to the filtrate, heat to dissolve, and then add distilled water to bring the volume to 1000mL. Sterilize by high-pressure steam at 121℃ for 30 minutes, cool to 50-60℃ and pour into sterile plates. Let stand to solidify. The agar strips should be cut into small pieces and added to the filtrate.

5. The efficient fruiting method for shiitake mushroom strain Le2538 suitable for plate culture according to claim 3, characterized in that, In step one, the amount of PDA culture medium poured into a 90mm diameter sterile plate is 10-15 mL.

6. The efficient fruiting method for shiitake mushroom strain Le2538 suitable for plate culture according to claim 3, characterized in that, The inoculation and culture in step two are as follows: Use a 5mm diameter punch to make holes in a PDA plate covered with Le2538 mycelium. Inoculate the mycelium block into the center of the PDA plate prepared in step one and incubate in the dark at 23-25℃ for 7-10 days. During this period, seal the inoculated plate with sterile sealing film and maintain a constant temperature.

7. The efficient fruiting method for shiitake mushroom strain Le2538 suitable for plate culture according to claim 3, characterized in that, In step three, the perforation-type mycelial scratching is carried out in a clean bench. Using a 5mm diameter sterile puncher, 5 to 30 holes are selected on the surface of the culture plate and perforated in an evenly distributed manner. Some mycelial blocks are removed and some are retained in situ. After replacing the sealing film, the plate is cultured in the dark at 23 to 25°C for 1 to 3 days.

8. The efficient fruiting method for shiitake mushroom strain Le2538 suitable for plate culture according to claim 3, characterized in that, In step four, the low-temperature induction involves placing the plate in the dark at 10–15°C for 2–7 days.

9. The efficient fruiting method for shiitake mushroom strain Le2538 suitable for plate culture according to claim 3, characterized in that, The fruiting culture in step five is as follows: The plates induced by low temperature were placed in the dark at 18-20℃ until fruiting bodies formed. The criteria for judging the formation of fruiting bodies were the integrity of the cap, stipe, gills, basidia, and basidiospores.

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