Method for breeding by culturing sporocarp under pure culture condition of boletus sinensis

Abstract

The invention discloses a method for breeding by culturing sporocarp under pure culture conditions of boletus sinensis, and belongs to the technical field of edible mushroom breeding and cultivation. The method comprises the following steps: activating the boletus sinensis strain JSJ-Bx1, inoculating the boletus sinensis strain JSJ-Bx1 into a culture medium for dark culture, then performing low-temperature induction culture on an activated flat plate which is nearly overgrown with hyphae, changing the hyphae from white to golden yellow and generating hypha kinking, then transferring the hypha flat plate into a fruiting room for culture, and after obvious primordium differentiation is seen, inoculating the hyphae into a culture medium for culture, thereby obtaining the boletus sinensis strain JSJ-Bx1. Continuously performing induced culture to develop into mature sporocarp; sporocarp basidiospores are collected, diluted and coated in a B1 culture medium for germination culture, and monospora hyphae and polyspora hyphae are obtained and serve as stock seeds for breeding the boletus sinensis. According to the method, the mature sporocarp of the boletus sinensis can be directly and rapidly cultured in the agar culture medium, the harvested basidiospore can normally germinate and grow, and the whole life history of the boletus sinensis can be completed under the pure culture condition.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi breeding and cultivation technology in the bio-agriculture industry, specifically involving a method for rapidly obtaining and breeding *Boletus sinensis* fruiting bodies directly on agar medium. Background Technology

[0002] Chinese Boletus saprophyticus Buchwaldoboletus xylophilus (Petch) Both&B. Ortiz, belonging to the family Boletaceae, subfamily Chalciporoideae, genus *Boletus*. Buchwaldoboletus It is a typical tropical and subtropical rare bolete. It is a non-ectomycorrhizal fungus with saprophytic ability, is edible, and has high economic value; however, its wild resources are extremely scarce.

[0003] To address the scarcity of cultivated strains of *Boletus saprophyticus*, domestic institutions and individuals have conducted research and exploration on its artificial cultivation and propagation. Typical examples include Chinese Patent CN113348963A, which discloses an artificial cultivation method for *Boletus saprophyticus*; Chinese Patent CN120591113A, which discloses a mother culture medium for *Boletus saprophyticus* and its preparation method and application; and the "Biological Characteristics and Cultivation of *Boletus saprophyticus*" published in the *Acta Mycosystema Sinica* (https: / / doi.org / 10.13346 / j.mycosystema.2502 27). However, these methods generally involve cumbersome processes such as primary strain cultivation, inoculation with spawn bags, mycelial culture, and casing for fruiting. The cultivation time is long and the cost is high, becoming an obstacle in the breeding and propagation of *Boletus saprophyticus*. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing breeding technologies and provide a method for cultivating fruiting bodies of Boletus saprophyticus under pure culture conditions and breeding them efficiently, thereby significantly shortening the breeding cycle of Boletus saprophyticus and achieving efficient "fruiting body regeneration" of Boletus saprophyticus.

[0005] The technical solution adopted in this invention is as follows: Chinese saprophytic bolete ( Buchwaldoboletus xylophilus The method for breeding fruiting bodies under pure culture conditions includes the following steps: S1. The *Boletus chinensis* strain JSJ-Bx1 was activated and inoculated into B1 medium and cultured in the dark for 5-7 days at a temperature of 30-35℃. The raw material ratio of the B1 medium was: 20 g glucose, 2 g yeast extract, 1.5 g ferrous sulfate, 0.3 g 1 / 2 MS medium, 20 g agar, and 1 L water. S2. The activated plates that are nearly covered with mycelium are subjected to low-temperature induction culture at a temperature of 20-30℃ and in the dark for 5-7 days. The mycelium changes from white to golden yellow and mycelial knots occur. S3. Transfer the mycelial plates cultured in step S2 to the fruiting room for primordia induction culture. The culture temperature T is 26℃≤T<30℃, the relative humidity is 10~90%, and the light conditions are 12 h / 12 ​​h light and dark alternation culture. After 7~10 days of culture, after seeing obvious primordia differentiation, continue the induction culture to develop into mature fruiting bodies. S4. Collect the basidiospores from the obtained mature fruiting bodies under aseptic conditions, and dilute the basidiospores 10... 2 ~10 5 Spread onto B1 medium and germinate at 28-30℃. The hyphae germinating from a single colony are considered pseudomonosporal hyphae, and the hyphae germinating from multiple colonies are considered polysporal hyphae. The pseudomonosporal hyphae and polysporal hyphae are used as the original strain for the propagation of Boletus sinensis.

[0006] Preferably, in step S2 above, the culture temperature for inducing low-temperature culture of plates that are nearly covered with mycelia is 26~28℃.

[0007] Preferably, in step S3 above, the primordium induction culture temperature is 28°C and the relative humidity is 70%.

[0008] Preferably, in step S4 above, the basidiospores are diluted 10... 3 Then it was spread onto B1 medium for germination culture.

[0009] The method for breeding by culturing fruiting bodies under pure culture conditions as described in this invention can directly and rapidly cultivate mature fruiting bodies of Boletus sinensis in agar medium, and the harvested basidiospores can germinate and grow normally, realizing the completion of the entire life cycle of Boletus sinensis under pure culture conditions.

[0010] The method of this invention can rapidly and efficiently obtain physiologically active basidiospores of Boletus sinensis, accelerating the propagation speed of Boletus sinensis species and significantly shortening its breeding cycle; it provides a new approach for the breeding of Boletus sinensis and new ideas for the breeding of fungi in the Boletaceae family. Attached Figure Description

[0011] Figure 1 The effect of culture temperature on primordium induction of Boletus sinensis JSJ-Bx1 was shown; Figure 2 The effect of relative humidity on the induction of primordia of Boletus sinensis JSJ-Bx1 was shown. Figure 3The fruiting body development process of *Boletus chinensis* JSJ-Bx1 under pure culture conditions; Figure 4 Basidiospores and their germination in mature fruiting bodies of Boletus sinensis JSJ-Bx1 under pure culture conditions; Figure 5 Fruiting conditions of monosporous and multisporous mycelia of Boletus sinensis JSJ-Bx1; Figure 6 The image shows the germination of monosporous and multisporous mycelia and basidiospores of *Boletus sinensis* JSJ-Bx1 under pure culture conditions. Detailed Implementation

[0012] To make the objectives, technical solutions, and effects of this invention clearer, the following detailed description of the invention is provided in conjunction with specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0013] (1) Preparation of mother culture medium for Boletus saprophyticus B1 Weigh out 20 g of glucose, 2 g of yeast extract, 1.5 g of ferrous sulfate, 0.3 g of 1 / 2 MS medium, 20 g of agar, and 1 L of water according to the raw material mass ratio, put them into a container, and stir well; heat the container and boil the liquid in the container over low heat to form a homogeneous mixed solution, which is the nutrient solution; sterilize the nutrient solution after bottling to obtain the liquid culture medium; dispense the liquid culture medium into disposable petri dishes with a diameter of 9 cm, with each dish containing 20 mL of culture medium, to obtain the B1 mother culture medium of Boletus saprophyticus.

[0014] (2) Activation culture of Boletus sinensis JSJ-Bx1 After activation, the *Boletus spp.* strain JSJ-Bx1 was inoculated into B1 medium and cultured in the dark for 7 days at 30℃. Induction culture was continued until the *Boletus spp.* mycelium of *Boletus spp.* strain JSJ-Bx1 completely covered the plate. Figure 3 As shown in Image A, the *Boletus spp.* strain JSJ-Bx1 is the strain of *Boletus spp.* mother culture disclosed in Chinese Patent CN120591113B. As can be seen from the image, the *Boletus spp.* mycelium is white and dense, covering the entire plate, with neat colony edges, indicating a high-quality mother culture.

[0015] (3) Screening of primordium induction temperature for *Boletus chinensis* JSJ-Bx1 Select mycelial plates with consistent growth status and place them in five temperature gradients (22℃, 24℃, 26℃, 28℃, and 30℃) for primordium induction under alternating light and dark conditions for 12 h / 12 ​​h with relative humidity of 50-70%. Six replicates were performed for each temperature treatment. Figure 1 The effect of culture temperature on the induction of primordia of Boletus sinensis JSJ-Bx1 was shown. Figure 1 In the image, images A, B, C, D, and E show the primordium induction results at culture temperatures of 22℃, 24℃, 26℃, 28℃, and 30℃, respectively. From... Figure 1 As can be seen, *Boletus chinensis* JSJ-Bx1 can induce primordia in the experimental temperature range of 24℃ and above, but the occurrence of primordia is significantly affected by temperature. Specifically, when the culture temperature is below 24℃, it is difficult to induce primordia or primordia do not occur. Figure 1 (Image A); Appropriate low-temperature stimulation (24~26℃) will induce yellowing of the hyphae, but this phenomenon differs from the golden-yellow coloration of hyphae as they transition from the vegetative growth stage to the reproductive growth stage. It is more likely a phenotypic variation in response to low-temperature stimulation. Figure 1 (See images B and C). Furthermore, moderate low-temperature treatment resulted in later primordia development and a lower incidence rate. The highest primordia development rate was observed at a culture temperature of 28℃, with a short primordia development cycle, a large number of primordia, and a high fruiting body rate. Figure 1 (Image D) When the culture temperature is 30℃, the mycelium exhibits exudation, the number of primordia is small, and no fruiting bodies develop. Figure 1 (See image E). Therefore, temperature has a significant impact on primordium formation under pure culture conditions of *Boletus sinensis*, and the optimal primordium induction temperature is 28℃.

[0016] (4) Screening of relative humidity by inducing primordia of Boletus sinensis JSJ-Bx1 Select mycelial plates with consistent growth status and place them in five relative humidity gradients (10%, 30%, 50%, 70%, and 90%) for primordium induction under alternating light and dark conditions for 12 h / 12 ​​h at a temperature of 26–28 °C. Six replicates were performed for each relative humidity treatment. Figure 2 The effect of relative humidity on the induction of primordia of Boletus sinensis JSJ-Bx1 was shown. Figure 2 In the image, images A, B, C, D, and E show the primordium induction results at relative humidity levels of 10%, 30%, 50%, 70%, and 90%, respectively. From... Figure 2As can be seen, *Boletus spp.* JSJ-Bx1 can induce primordia under all culture conditions except 90% relative humidity, but the occurrence of primordia varies significantly under different relative humidity levels. Specifically, when the relative humidity is between 10% and 70%, the primordia occurrence cycle is negatively correlated with relative humidity; while the primordia occurrence rate, quantity, and size are positively correlated with relative humidity. Furthermore, primordia cannot be induced at 90% relative humidity, likely because the high relative humidity leads to severe water accumulation in the culture dish, causing the mycelia to lose vitality and fail to form primordia under prolonged submerged conditions. Therefore, relative humidity has a relatively small impact on primordia occurrence under pure culture conditions of *Boletus spp.*, and the optimal relative humidity for primordia induction is 70%.

[0017] (5) The fruiting body development process of Boletus sinensis JSJ-Bx1 under optimized culture conditions The mycelial plates that showed obvious primordia differentiation were further induced to develop into mature fruiting bodies. The primordia induction culture was carried out at a temperature of 28℃, a relative humidity of 70%, and a light condition of 12 h / 12 ​​h light-dark alternation. Figure 3 The development process of the fruiting body of Boletus sinensis JSJ-Bx1 under pure culture conditions.

[0018] from Figure 3 As can be seen from this, the fruiting body development process of *Boletus sinensis* JSJ-Bx1 under pure culture conditions can be divided into the hyphal twisting stage (… Figure 3 Image B shows the hyphae twisted into clumps, 0.5 mm in size, during the primordia development stage. Figure 3 Image C shows mycelial tissue formation with exudation, measuring 1.5 mm in size, and early stipe development. Figure 3 Image D shows a cluster of tissue with a protruding tip and exhibiting exudation, measuring 2 mm in size; the stipe is in the middle stage of development (…). Figure 3 Image E shows a stipe structure already present, measuring 3 mm in size; the stipe development is in its later stages (…). Figure 3 Image F shows a fully differentiated stipe structure, 7-10 mm in length, and the early stage of fruiting body development. Figure 3 Image G shows the differentiation of the cap structure, 13-15 mm in length, and the fruiting body maturation stage ( Figure 3 Image H shows the differentiation of a distinct cap structure, gradually growing to the size shown in Images I and J (with regular pores and prominent basidiospores, measuring 15-20 mm), exhibiting seven developmental stages. This indicates that *Boletus sinensis* JSJ-Bx1 can directly utilize the nutrients in agar medium to obtain mature fruiting bodies.

[0019] (6) Observation of basidiospores of mature fruiting bodies and their germination under pure culture conditions of Boletus sinensis JSJ-Bx1 Figure 4 Basidiospores and their germination in mature fruiting bodies of Boletus sinensis JSJ-Bx1 under pure culture conditions.

[0020] from Figure 3 As shown in image J, the mature Boletus sinensis JSJ-Bx1, under pure culture conditions, possesses tissue structures such as pores and hymenium. After placing a certain amount of sterile water on the surface of the pores and preparing a slide for observation under an optical microscope, basidiospores can be clearly observed. Figure 4 Image A (observation results under a 40X optical microscope) and Image B (observation results under a 100X optical microscope).

[0021] Basidiospores were collected under aseptic conditions and diluted 10-10 3 Then, it was evenly spread on B1 medium and placed in a 30℃ constant temperature incubator for dark incubation. After 3 days of incubation, basidiospore germination and growth could be observed, and the produced basidiospores were physiologically active and could germinate and grow normally. Figure 4 Image C shows the germination state of basidiospores.

[0022] (7) Fruiting of pseudomonosporal and multisporal mycelia of Boletus sinensis JSJ-Bx1 The present invention provides a method for rapidly obtaining fruiting bodies of Boletus sinensis on agar medium, which further determines the fruiting characteristics of pseudomonosporoid mycelia and multisporoid mycelia, and verifies this method in conjunction with fruiting conditions under cultivation.

[0023] Figure 5 Fruiting conditions of pseudomonosporoid mycelia and multisporoid mycelia of Boletus sinensis JSJ-Bx1. Figure 5In the image, images A, B, and C show the primordia differentiation of wild-type *Boletus chinensis* JSJ-Bx1 mycelium, pseudomonospora mycelium, and multispora mycelium under pure culture conditions, respectively. Images D, E, and F show the fruiting of wild-type *Boletus chinensis* JSJ-Bx1 mycelium, pseudomonospora mycelium, and multispora mycelium under cultivation conditions, respectively. The cultivation conditions involved inoculating five pieces of the propagated spawn into a liquid culture medium (20 g glucose, 2 g yeast extract, 1.5 g ferrous sulfate, 0.3 g 1 / 2 MS medium, and 1 L water) using a 0.65 cm diameter punch, and then incubating them in the dark at 30℃ and 150 r / min for 7 days. d; Inoculate the cultured liquid into a cultivation substrate mainly composed of sawdust (20% wheat grains, 10% corn flour, 10% red soil, 8% wheat bran, 1% gypsum powder, 1% glucose, 0.1% KH2SO4, 0.1% FeSO4) and incubate at 30℃ in the dark; After the mycelium has fully colonized the cultivation bottle, mix red soil and peat moss in equal volume ratios to obtain a casing material with a moisture content of 55% and a casing thickness of 3 cm; After casing, place the bottle at 26~28℃, relative humidity of 80%~90%, light intensity of 300~500 lx, and CO2 concentration of 1000~1500 mL / m³. 3 Fruiting bodies are induced to grow in the fruiting room.

[0024] from Figure 5 As can be seen, the mycelia, pseudomonospora mycelia, and multispora mycelia of wild-type Boletus chinensis JSJ-Bx1 can all be induced to differentiate into primordia under pure culture conditions; under cultivation conditions, the mycelia, pseudomonospora mycelia, and multispora mycelia of wild-type Boletus chinensis JSJ-Bx1 can also develop into mature fruiting bodies normally.

[0025] Figure 6 The image shows the fruiting bodies of *Boletus sinensis* under pure culture conditions, including monosporous and multisporous mycelia and their basidiospore germination. Figure 6 Image A shows fruiting from *Pseudomonas aeruginosa* mycelium; Image B shows fruiting from *Pseudomonas aeruginosa* mycelium; Image C shows the germination of basidiospores collected from fruiting bodies cultured from *Pseudomonas aeruginosa* mycelium; and Image D shows the germination of basidiospores collected from fruiting bodies cultured from *Pseudomonas aeruginosa* mycelium. From... Figure 6 It can be seen that when the fruiting bodies of pseudomonosporal and multisporal hyphae matured and opened under pure culture conditions, their basidiospores were collected and diluted 10⁻⁶. 3 It was then spread onto B1 mother culture medium and germinated at 28°C, where it also germinated and grew.

[0026] The above results indicate that *Boletus chinensis* JSJ-Bx1 can rapidly produce mature fruiting bodies directly on agar medium, and its basidiospores can germinate and grow normally, demonstrating that *Boletus chinensis* can complete its entire life cycle under pure culture conditions. This breeding method can rapidly verify that both monosporous and polysporous mycelia possess good fruiting characteristics and can complete species propagation under pure culture conditions, significantly shortening the breeding cycle of *Boletus chinensis*. Example 1

[0027] Chinese saprophytic bolete ( Buchwaldoboletus xylophilus The method for breeding fruiting bodies under pure culture conditions is as follows: According to the raw material ratio of B1 culture medium, accurately weigh 20 g of glucose, 2 g of yeast extract, 1.5 g of ferrous sulfate, 0.3 g of 1 / 2 MS medium, 20 g of agar, and 1 L of water, put them into a container, and stir well; heat the container and boil the liquid in the container over low heat to form a homogeneous mixed solution, which is the nutrient solution; sterilize the nutrient solution after bottling to obtain liquid culture medium; dispense the liquid culture medium into disposable petri dishes with a diameter of 9 cm, with each dish containing 20 mL of culture medium, to obtain the B1 mother culture medium of Boletus saprophyticus.

[0028] The activated *Boletus chinensis* strain JSJ-Bx1 was inoculated into B1 medium and cultured in the dark at 30°C for 7 days until mycelia covered the entire plate. Subsequently, it was placed in a 26°C incubator for 6 days of low-temperature induction culture until mycelial knots were observed. The mycelia from the above plate were then transferred to a fruiting house under light conditions of 28°C, 70% relative humidity, and alternating light and dark conditions (12 h / 12 ​​h). Once primordia differentiation and further development into fruiting bodies were observed, the culture continued until the fruiting bodies matured (this stage of culture lasted 9 days). When the pores matured and opened, the basidiospores were collected and diluted 10... 3 The samples were then spread onto B1 medium, and their germination and growth were observed. Results are shown below. Figure 3 and Figure 4 .

[0029] Figure 3 The development process of the fruiting body of Boletus sinensis under pure culture conditions is shown.

[0030] Figure 4 The basidiospores of the mature fruiting body of Boletus sinensis JSJ-Bx1 and their germination under pure culture conditions are shown.

[0031] As can be seen from the figure, using the technical method described in this invention, the mycelium of *Boletus spp.* can quickly cover the plate, and can rapidly induce primordia to develop into mature fruiting bodies. The collected basidiospores can germinate and grow normally; indicating that *Boletus spp.* JSJ-Bx1 can complete its entire life cycle under pure culture conditions.

[0032] In the method of this invention, the culture time, culture temperature, and basidiospore dilution of each step can be appropriately adjusted to achieve the expected technical effect, enabling *Boletus chinensis* JSJ-Bx1 to complete its entire life cycle under pure culture conditions. Further experimental verification showed that after activating the *Boletus chinensis* strain JSJ-Bx1, it was inoculated into B1 medium and cultured in the dark at 30-35℃ for 5-7 days until the mycelium basically covered the plate. Further induction culture at 20-30℃ for 5-7 days after the mycelium covered the plate resulted in mycelial knotting. Transferring the plate with the knotted mycelium to a temperature above 26℃ but not exceeding 30℃, a relative humidity of 10-90%, and alternating light and dark conditions of 12 h / 12 ​​h resulted in primordia differentiation and further development into fruiting bodies. When the fruiting bodies matured and opened, the basidiospores were collected and diluted 10... 2 ~10 5 Afterwards, the seeds were spread onto B1 mother culture medium and germinated at 28-30℃, and fruiting bodies were obtained. Example 2

[0033] The method for breeding *Boletus chinensis* by culturing fruiting bodies under pure culture conditions is as follows: Prepare the same mother culture medium for *Boletus chinensis* B1 as in Example 1.

[0034] The pseudomonospora and multispora mycelia of the *Boletus chinensis* strain JSJ-Bx1 were inoculated into B1 medium and cultured in the dark at 30°C for 5-7 days. After the mycelia covered the entire plate, they were placed in a 26°C incubator for low-temperature induction culture for 5-7 days until mycelial knots were observed. The mycelia from the above plates were then transferred to a fruiting room under light conditions of 28°C, 70% relative humidity, and alternating light and dark conditions of 12 h / 12 ​​h. It was observed that both pseudomonospora and multispora mycelia could induce primordia under pure culture conditions. The results are shown in [Figure 1]. Figure 5 Images A, B, and C in the image depict, respectively, the induction of primordia from wild-type *Boletus sinensis* JSJ-Bx1 mycelia, pseudomonospora mycelia, and multispora mycelia under pure culture conditions. Basidiospores from the fruiting bodies cultured under pure culture conditions were collected and diluted 10⁻⁶ when the mycelia and multispora mycelia matured and opened. 3After being spread onto B1 mother culture medium, they were germinated and cultured at 28℃. All of them germinated and grew, and the results are shown in the figure. Figure 6 Images A, B, C, and D in the image represent, in order, the fruiting process of pseudomonosporal mycelium, the fruiting process of multisporal mycelium, the germination of basidiospores collected from fruiting bodies cultured from pseudomonosporal mycelium, and the germination of basidiospores collected from fruiting bodies cultured from multisporal mycelium.

[0035] Furthermore, under cultivation conditions, both monosporous and multisporous mycelia can normally produce fruit and develop into complete fruiting bodies, as shown in the results. Figure 5 Images D, E, and F show the fruiting results of wild-type Boletus sinensis JSJ-Bx1 mycelium, pseudomonospora mycelium, and multispora mycelium under cultivation conditions.

[0036] This indicates that the technical method of the present invention can be applied to determine the fruiting characteristics of monospore and polyspore mycelia of Boletus sinensis, and both types of mycelia can produce fruit and complete their entire life cycle under pure culture conditions.

[0037] In the method of this invention, the culture time, culture temperature, and basidiospore dilution of each step can be appropriately adjusted to achieve the expected technical effect, enabling the entire life cycle of the *Boletus spp.* strain JSJ-Bx1 (simoleon and multispore germination hyphae) to be completed under pure culture conditions. Further experimental verification showed that after activating the *Boletus spp.* strain JSJ-Bx1 (simoleon and multispore germination hyphae), inoculating them into B1 medium, they can be cultured in the dark at a constant temperature of 30-35℃ for 5-7 days until the hyphae basically cover the plate. Further induction culture at 20-30℃ for 5-7 days after the hyphae have fully grown can be observed, resulting in hyphal knotting. Continuing to transfer the plates with the observed hyphal knotting to a temperature above 26℃ but not exceeding 30℃, a relative humidity of 10-90%, and alternating light and dark conditions of 12 h / 12 ​​h, primordia differentiation and further development into fruiting bodies can be observed in the plates. When the fruiting bodies mature and open their pores, collect their basidiospores and dilute them 10 times. 2 ~10 5 Afterwards, the seeds were spread onto B1 mother culture medium and germinated at 28-30℃, and fruiting bodies were obtained.

[0038] This invention marks the first successful acquisition of mature fruiting bodies of *Boletus spp.* under pure culture conditions, with the collection of basidiospores demonstrating their normal germination and growth. Compared to traditional breeding techniques such as systematic selection and hybridization, this invention is the first to directly utilize the nutrients in agar medium to meet the needs of Boletaceae fungi to complete their entire life cycle under pure culture conditions. It eliminates a series of cumbersome processes, including primary culture, inoculation of spawn bags, mycelial culture, and casing for fruiting, resulting in rapid and efficient acquisition of mature fruiting bodies and significantly shortening the breeding cycle. Furthermore, this invention allows for the rapid and effective identification of single-spore and multi-spore mycelia with fruiting characteristics, accelerating species reproduction and providing a new approach for the breeding of *Boletus spp.* and new ideas for the breeding of Boletaceae fungi.

Claims

1. Chinese saprophytic bolete ( Buchwaldoboletus xylophilus A method for breeding fruiting bodies under pure culture conditions, characterized in that, Includes the following steps: S1. The *Boletus chinensis* strain JSJ-Bx1 was activated and inoculated into B1 medium and cultured in the dark for 5-7 days at a temperature of 30-35℃. The raw material ratio of the B1 medium was: 20 g glucose, 2 g yeast extract, 1.5 g ferrous sulfate, 0.3 g 1 / 2 MS medium, 20 g agar, and 1 L water. S2. The activated plates that are nearly covered with mycelium are subjected to low-temperature induction culture at a temperature of 20-30℃ and in the dark for 5-7 days. The mycelium changes from white to golden yellow and mycelial knots occur. S3. Transfer the mycelial plates cultured in step S2 to the fruiting room for primordia induction culture. The culture temperature T is 26℃≤T<30℃, the relative humidity is 10~90%, and the light conditions are 12 h / 12 ​​h light and dark alternation culture. After 7~10 days of culture, after seeing obvious primordia differentiation, continue the induction culture to develop into mature fruiting bodies. S4. Collect the basidiospores from the obtained mature fruiting bodies under aseptic conditions, and dilute the basidiospores 10... 2 ~10 5 Spread onto B1 medium and germinate at 28-30℃. The hyphae germinating from a single colony are considered pseudomonosporal hyphae, and the hyphae germinating from multiple colonies are considered polysporal hyphae. The pseudomonosporal hyphae and polysporal hyphae are used as the original strain for the propagation of Boletus sinensis.

2. The *Boletus chinensis* saprophyticus according to claim 1 (… Buchwaldoboletus xylophilus A method for breeding fruiting bodies under pure culture conditions, characterized in that, In step S2 above, the culture temperature for inducing low-temperature culture of plates that are nearly covered with mycelium is 26~28℃.

3. The *Boletus chinensis* saprophyticus according to claim 1 (… Buchwaldoboletus xylophilus A method for breeding fruiting bodies under pure culture conditions, characterized in that, In step S3 above, the primordium induction culture temperature is 28℃ and the relative humidity is 70%.

4. The *Boletus chinensis* saprophyticus according to claim 1 (… Buchwaldoboletus xylophilus A method for breeding fruiting bodies under pure culture conditions, characterized in that, In step S4 above, the basidiospores are diluted 10... 3 Then it was spread onto B1 medium for germination culture.

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