Ganoderma sinensis spore germination culture medium and application thereof

Abstract

The invention relates to the technical field of microorganism culture, in particular to a ganoderma sinensis spore germination culture medium and application thereof.Ganoderma sinensis spores are collected through a fungus carrying bag or the culture medium, the optimal growth environment for ganoderma sinensis spore germination culture is obtained by optimizing the culture condition of the ganoderma sinensis spores, and the ganoderma sinensis spore germination culture medium is obtained by optimizing the culture medium formula and adding wood chip boiling liquid. A culture medium formula most suitable for ganoderma spore germination is obtained through raw material screening and orthogonal experiment optimization of boiling liquid, ganoderma spores can be effectively collected without reaching a sterile environment of a laboratory, the method is a practical and effective ganoderma sinensis spore collection and germination method, and a foundation is laid for promoting ganoderma sinensis new variety breeding research.

Description

Technical Field

[0001] The invention relates to the technical field of microbial culture, in particular to a spore germination culture medium of Ganoderma lucidum and application thereof. Background Art

[0002] When cultivating Ganoderma lucidum artificially, under suitable environmental conditions, it generally takes 25 to 30 days for the primordium of Ganoderma lucidum fruiting body to mature. Once the Ganoderma lucidum fruiting body matures, spores will be ejected and released from the ventral surface of the fruiting body one after another. When wild Ganoderma lucidum matures, the Ganoderma lucidum spores are ejected and scattered in the air, and cannot be collected, so it is impossible to have wild Ganoderma lucidum spore powder on the market. Compared with other edible and medicinal fungi, on the one hand, because the spores are basically no longer ejected after picking, on the other hand, because the size of the spores is relatively large and the fine spore wall is thick, the collection and germination of spores are difficult, which becomes a technical bottleneck for the selection of new varieties of Ganoderma lucidum such as hybrid breeding. At present, the method of collecting Ganoderma lucidum is usually a specific collection device in the laboratory, that is, the sterilized Ganoderma lucidum is inserted into the bracket of the spore collection device, which is generally formed by bending a glass rod or a thick iron wire. A culture dish bottom is placed under the bracket for collecting spores.

[0003] When collecting Ganoderma lucidum spores in the wild, due to the poor disinfection environment, it is difficult to collect Ganoderma lucidum spores, and the germination of spores will be inhibited, resulting in a low germination rate of Ganoderma lucidum spores. Similarly, we also found that different Ganoderma lucidum varieties have different physiological conditions and external structures, and it is inconvenient to unify the spore collection and germination methods. Therefore, we need to come up with a set of effective Ganoderma lucidum spore collection and germination methods for various aspects such as the field collection environment, collection conditions and culture medium optimization of specific Ganoderma lucidum varieties, laying the foundation for promoting the selection and breeding of new Ganoderma lucidum varieties. Summary of the invention

[0004] In view of the above, it is necessary to simplify the method of collecting Ganoderma lucidum spores, improve the spore germination rate of Ganoderma lucidum, and develop a set of practical and effective Ganoderma lucidum spore collection and germination methods based on various aspects such as field collection, collection conditions and culture medium optimization, so as to lay the foundation for promoting the research on the selection and breeding of new Ganoderma lucidum varieties.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The invention discloses a culture medium for spore germination of Ganoderma lucidum, which is composed of the following components: 1%-2% beef extract by mass, 0.5%-2.5% peptone by mass, 2%-6% agar by mass, 10% sawdust decoction by volume, and the balance is water.

[0007] Furthermore, the sawdust decoction liquid is pinewood liquid, pearwood liquid, maplewood liquid and / or pinewood liquid.

[0008] Further, the preparation method of the wood chip decoction is as follows: boil pine wood, pear wood, maple wood or castanopsis fargesii in water for 40 minutes respectively, then filter and preserve to obtain it.

[0009] Further, the volume ratio of the castanopsis fargesii liquid, pear wood liquid, maple wood liquid and pine wood liquid is 1 - 5:2 - 6:1 - 5:1 - 5.

[0010] Further, the culture medium consists of the following components: beef extract with a mass percentage of 1%, peptone with a mass percentage of 1.5%, agar with a mass percentage of 2%, and wood chip decoction with a volume percentage of 10%; among them, the wood chip decoction is obtained by mixing the castanopsis fargesii liquid, pear wood liquid, maple wood liquid and pine wood liquid according to a volume ratio of 3:4:3:3.

[0011] The present invention also includes a method for culturing Ganoderma sinense spores using the above culture medium. The method is as follows: dilute the just - ejected spores with sterile water into a suspension with a concentration of 2×10 5 cells / mL, and after stimulating in a water bath at 40°C for 40 minutes, take 0.1 mL and evenly coat it on the culture medium, and culture it at a constant temperature of 30°C in the dark; the pH value of the culture medium is 4.5.

[0012] Further, the Ganoderma sinense variety is Guizhi No. 8. The preservation number of this strain is CGMCC NO.41073. The taxonomic name of this strain is Ganoderma lucidum, the preservation number is CGMCC NO.41073, the preservation date is December 25, 2023, and the preservation institution is: China General Microbiological Culture Collection Center, and the preservation address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] Further, the method for collecting Ganoderma sinense spores is the cardboard box method.

[0014] Further, the specific operation of the cardboard box method is as follows: move the Ganoderma lucidum fruiting bodies cultivated in the greenhouse together with the fungus bags of the culture medium to the culture room. The selected fruiting bodies are required to be robust in growth, free from pests and diseases, the white growth ring at the edge of the mushroom cap has basically disappeared, and there is a small amount of spore release. First, gently wipe the surface of the fungus bag and the fruiting body with a small amount of 75% alcohol for surface disinfection treatment, then spray 2 times of sterile water and dry the excess water on the surface to increase the humidity; spray a small amount of 75% alcohol in a new cardboard box that has not been used, place the fungus bag containing the fruiting body and the culture medium in the cardboard box. The size specification of the cardboard box is: length 60 cm, width 30 cm, height 40 cm. Place a sterile petri dish under the fruiting body, and let it stand at 25 - 28°C until a layer of reddish - brown powder appears at the bottom of the petri dish, which is the spores. Take it out and store it in a 4°C refrigerator.

[0015] The present invention has the following beneficial effects:

[0016] 1. The present invention collects Ganoderma sinense spores using bacteria-carrying bags or culture media. By optimizing the culture conditions of Ganoderma sinense spores, the most suitable growth environment for the germination and cultivation of Ganoderma sinense spores is obtained. Through optimizing the culture medium formula and adding the extract of boiled sawdust, the most suitable culture medium formula for the germination of Ganoderma lucidum spores is obtained through raw material screening of the extract and orthogonal experiment optimization. It can effectively collect Ganoderma lucidum spores without reaching a sterile environment in the laboratory, and is a practical and effective method for Ganoderma sinense spore collection and germination, laying a foundation for promoting the research on the breeding of new varieties of Ganoderma sinense.

[0017] 2. In addition to containing the essential components required for the germination and growth of Ganoderma lucidum spores, the extract of boiled sawdust in this application also has the effect of inhibiting the growth of miscellaneous bacteria. No miscellaneous bacteria grow in the culture medium except for spore germination, greatly simplifying the steps of Ganoderma sinense spore germination and collection and significantly improving the work efficiency of operators. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a result diagram of the Ganoderma sinense basidiospore collection method.

[0019] Figure 2 It is a result diagram of the influence of the culture medium on the germination of Ganoderma sinense spores.

[0020] Figure 3 It is a result diagram of the influence of the culture temperature on the germination of Ganoderma sinense spores.

[0021] Figure 4 It is a result diagram of the influence of the culture medium pH on the germination of Ganoderma sinense spores.

[0022] Figure 5 It is a result diagram of the influence of the nitrogen source on the germination of Ganoderma sinense spores.

[0023] Figure 6 It is a result diagram of the influence of the carbon source on the germination of Ganoderma sinense spores.

[0024] Figure 7 It is a result diagram of the influence of the storage time on the germination of Ganoderma sinense spores.

[0025] Figure 8 It is the influence of the extract of boiled sawdust on the germination of Ganoderma sinense spores

[0026] Figure 9 It is a result diagram of the optimization experiment of the best spore germination culture conditions of Ganoderma sinense. DETAILED DESCRIPTION OF THE INVENTION

[0027] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following detailed description of the specific embodiments of the present invention will be provided in conjunction with the accompanying drawings. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0028] Example 1:

[0029] This example is about the method for collecting Ganoderma lucidum spores:

[0030] 1. Test strain: Guizhi No. 8 (alias: Ganoderma japonicum strain DZ020), the taxonomic name of this strain is Ganoderma lucidum, the preservation number is CGMCC NO. 41073, the preservation date is December 25, 2023, and the preservation institution is: General Microbiology Center of China Microbial Culture Collection Center, and the preservation address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0031] 2. Preparation of decoction: The method for preparing the decoction of sawdust of Ganoderma lucidum, pinewood, pearwood, maplewood, conewood, cyclobalanopsis glauca, or chestnut wood: Add 200 g of Ganoderma lucidum, pinewood, pearwood, maplewood, conewood, cyclobalanopsis glauca, or chestnut wood to 1.5 L of water respectively, boil for 40 min, filter and make up the volume to 0.5 L, and store at -20 °C for later use; obtain the corresponding decoctions of Ganoderma lucidum, pinewood, pearwood, maplewood, conewood, cyclobalanopsis glauca, or chestnut wood.

[0032] 3. Spore collection method:

[0033] ① Bell jar method (fruiting body): Move the Ganoderma lucidum fruiting bodies cultivated in the greenhouse to the culture room. The selected fruiting bodies are required to be robust, free from pests and diseases, the white growth ring at the edge of the mushroom cap has basically disappeared, and there is a small amount of spore release. Remove the stipe of the fruiting body, gently wipe the surface of the fruiting body with a small amount of 75% alcohol for surface disinfection treatment, then spray 2 times of sterile water and dry the excess water on the surface to increase humidity; insert the disinfected fruiting body onto the bracket of the spore collection device, place a sterile culture dish under the fruiting body, cover the fruiting body with a sterilized bell jar, and the size specifications of the bell jar are: diameter 18 cm, height 32 cm. Let it stand at 25 - 28 °C until a layer of reddish-brown powder appears at the bottom of the culture dish, which is the spore. Take it out and store it in a 4 °C refrigerator.

[0034] ② Bell jar method (fruiting body together with culture medium and fungus bag): Move the Ganoderma lucidum fruiting body grown in the greenhouse together with the fungus bag of the culture medium (15×30 cm) to the culture room. The selected fruiting body should be robust in growth, free from pests and diseases, with the white growth ring at the edge of the mushroom cap basically faded, and a small amount of spores released. First, gently wipe the surface of the fungus bag and the fruiting body with a small amount of 75% alcohol for surface disinfection, then spray with sterile water twice and dry the excess water on the surface to increase humidity; Place a sterile petri dish under the fruiting body, cover the fungus bag and the fruiting body with a sterilized bell jar. The size specifications of the bell jar are: diameter 18 cm, height 32 cm. Leave it static at 25 - 28 °C until a layer of reddish-brown powder appears at the bottom of the petri dish, which is the spore. Take it out and store it in a 4 °C refrigerator.

[0035] ③ Bagging method (fruiting body together with culture medium and fungus bag): Move the Ganoderma lucidum fruiting body grown in the greenhouse together with the fungus bag of the culture medium (15×30 cm) to the culture room. The selected fruiting body should be robust in growth, free from pests and diseases, with the white growth ring at the edge of the mushroom cap basically faded, and a small amount of spores released. First, gently wipe the surface of the fungus bag and the fruiting body with a small amount of 75% alcohol for surface disinfection, then spray with sterile water twice and dry the excess water on the surface to increase humidity; Cover the whole fruiting body part with a polypropylene sterile bag (17×33 cm) that has been autoclaved. Leave it static at 25 - 28 °C. When a small amount of reddish-brown powder appears in the polypropylene bag, it is the spore. Take it out, tie the bag mouth tightly and store it in a 4 °C refrigerator.

[0036] ④ Cardboard box method (fruiting body together with culture medium and fungus bag): Move the Ganoderma lucidum fruiting body grown in the greenhouse together with the fungus bag of the culture medium (15×30 cm) to the culture room. The selected fruiting body should be robust in growth, free from pests and diseases, with the white growth ring at the edge of the mushroom cap basically faded, and a small amount of spores released. First, gently wipe the surface of the fungus bag and the fruiting body with a small amount of 75% alcohol for surface disinfection, then spray with sterile water twice and dry the excess water on the surface to increase humidity; Spray a small amount of 75% alcohol in a new cardboard box that has not been used, place the fungus bag containing the fruiting body and the culture medium in the cardboard box. The size specifications of the cardboard box are: length 60 cm, width 30 cm, height 40 cm. Place a sterile petri dish under the fruiting body. Leave it static at 25 - 28 °C until a layer of reddish-brown powder appears at the bottom of the petri dish, which is the spore. Take it out and store it in a 4 °C refrigerator.

[0037] 4. Preparation of spore suspension:

[0038] Wash the Ganoderma lucidum spores collected by the above four methods with sterile water 2 - 3 times and dilute them. Observe the spore concentration with a hemocytometer under a microscope to be 2×10 7 cells / mL, and dilute it with sterile water to a spore suspension concentration of 10 6 cells / mL and 10 5per mL, water bath at 40 °C for 40 min, and set aside.

[0039] 5. Observation of spore germination:

[0040] Take 0.1 mL of the spore suspension with a concentration of 10 6 per mL collected by the above four methods respectively, and evenly coat it on the basic medium, and place it in a constant temperature incubator at 28 °C for dark cultivation. Set 30 replicates for each collection method, observe once every 1 - 2 days. When the spores germinate into colonies visible to the naked eye, count the average number of spore germination colonies on each plate (hereinafter referred to as the germination number), and record the contamination rate of each collection method at the same time to clarify the optimal spore collection method.

[0041] 6. Spore germination experiment:

[0042] After sterilization, streptomycin sulfate and kanamycin sulfate with a concentration of 50 mg / L were sequentially added to all the media, and then poured into petri dishes with a specification of 9 cm; an appropriate amount of Ganoderma lucidum spores collected by the optimal spore collection method was prepared into a spore suspension according to the method in item 4, coated and cultured according to the requirements of the following different spore germination experiments. Set 3 replicates for each treatment, and count the spore germination number according to the method in item 5.

[0043] (1) Screening of basic medium: Prepare the media ZM1 - ZM9 in Table 1, and screen the basic medium formula for the optimal germination of Ganoderma sinense spores according to the spore germination situation.

[0044] Table 1 Composition of the medium

[0045]

[0046] (2) Germination condition experiment:

[0047] ① Temperature experiment: After evenly coating the spore suspension on the basic medium ZM8 (pH natural), place it in a constant temperature incubator at 15 °C, 20 °C, 25 °C, 30 °C, 30 °C for dark cultivation; at the same time, take an appropriate amount of spore suspension, water bath and stimulate it at 40 °C for 40 min, then evenly coat it, and use the spore suspension without water bath stimulation as the control (CK), and place it in a constant temperature incubator at 28 °C for dark cultivation.

[0048] ② pH experiment: Adjust the pH value of the basic medium ZM8 to 6 gradients of 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 respectively, use the basic medium ZM8 (pH natural) as the control (CK), evenly coat the spore suspension on the medium, and place it in a constant temperature incubator at 28 °C for dark cultivation.

[0049] ③ Nitrogen source experiment: Replace peptone and beef extract in the basic medium ZM8 (pH natural) with 2% yeast extract, beef extract, peptone, potassium nitrate, soybean powder, and ammonium sulfate respectively to prepare media containing different nitrogen sources. Use the basic medium ZM8 as the control (CK). After evenly coating the spore suspension on the medium, place it in a constant temperature incubator at 28°C for dark cultivation.

[0050] ④ Carbon source experiment: Add 2% sucrose, lactose, maltose, trehalose, fructose, and glucose to the basic medium (pH natural) respectively to prepare media containing different carbon sources. Use the basic medium ZM8 as the control (CK). After evenly coating the spore suspension on the medium, place it in a constant temperature incubator at 28°C for dark cultivation.

[0051] ⑤ Spore storage time experiment: Prepare spore suspensions from spores stored for 1d, 5d, 10d, 15d, 30d, 45d, and 60d respectively, evenly coat them on the basic medium ZM8 (pH natural), and place them in a constant temperature incubator at 28°C for dark cultivation.

[0052] ⑥ Wood chip extract experiment: Add 10% wood chip extracts of pine, pear, maple, castanopsis, cyclobalanopsis, and chestnut to the basic medium ZM8 (pH natural) respectively to prepare media containing different wood chip extracts. Use the basic medium ZM8 as the control (CK). After evenly coating the spore suspension on the medium, place it in a constant temperature incubator at 28°C for dark cultivation, as shown in Table 15 specifically.

[0053] Table 2 Optimized medium formula

[0054]

[0055] The results obtained using the above experimental scheme are as follows:

[0056] I. Comparison of basidiospore collection methods is as specific as Figure 1 and shown in Table 3:

[0057] Table 3 Statistical results of basidiospore collection methods

[0058]

[0059] Note: Lowercase letters indicate significant differences (P < 0.05), the same below.

[0060] From Figure 1As can be seen from Table 3, the bell jar method (without handle) cannot collect spores. The results of the spore germination contamination of the three collection methods show that a total of 12 plates were contaminated by the bell jar method, with a contamination rate of 60%. Among them, 6 plates were contaminated by bacteria, 2 plates were contaminated by Penicillium viride, and 4 plates were contaminated by both bacteria and Penicillium viride; a total of 15 plates were contaminated by the bagging method, with a contamination rate of 75%. Among them, 9 plates were contaminated by bacteria, and 6 plates were contaminated by both bacteria and Penicillium viride; only 1 plate of the spores collected by the cardboard box method was contaminated by bacteria, with a contamination rate of only 5%. From the results of the germination situation, it can be seen that the average number of germinated colonies of the spores by the cardboard box method (hereinafter referred to as the germination number) was 46.6±12.8, which was significantly higher than 30.2±6.1 and 22.8±6.4 of the bell jar method and the bagging method. The bagging method had the highest contamination rate and the least germination number. The reason may be that the space of the polypropylene sterile bag is small, the temperature and humidity inside the bag are not easy to control, the fruiting body mildews, and the ejected spores have high humidity and may be contaminated by bacteria and molds; although the germination number of the bell jar method is moderate, the contamination rate is relatively high. Because the bell jar is made of glass and has poor air permeability, the humidity inside the bell jar is relatively high, and the Ganoderma sinense fruiting body is prone to mildew, and the ejected spores will rot or be contaminated by bacteria and Penicillium viride; while the cardboard box has good air permeability, the temperature and humidity are easy to control, and the ejected spores will not be contaminated by miscellaneous bacteria. Therefore, the best method for collecting spores of Ganoderma sinense in greenhouse cultivation is the cardboard box method.

[0061] II. Results of the germination experiment:

[0062] 1. Results of the basic medium screening experiment: As shown in Table 1, Table 4 and Figure 2 shown:

[0063] Table 4 Effects of media on the spore germination of Ganoderma sinense

[0064]

[0065] Combined with Table 1, Table 4 and Figure 2 it can be seen that the medium with the most spore germination was ZM8, with 91.7±4.0, followed by ZM7 and ZM9 with 55.7±3.1 and 54.3±5.5, indicating that the Ganoderma sinense spores had the best germination effect on the medium containing beef extract and peptone. The formula of ZM9 medium added 20% more potatoes than ZM8, indicating that potatoes inhibited the spore germination. There was a significant difference in the germination number between ZM8 and ZM7, ZM9; the germination numbers of the other 6 media were all less than 10, indicating that these media were not conducive to the spore germination of Ganoderma sinense. Therefore, ZM8 can be used as the basic medium for the Ganoderma sinense spore germination experiment.

[0066] 2. Effects of different germination conditions on the spore germination of Ganoderma sinense:

[0067] (1) The results of the effect of culture temperature on the spore germination of Ganoderma sinense are shown in Table 5 and Figure 3 shown:

[0068] Table 5 Effects of culture temperature on the spore germination of Ganoderma sinense

[0069]

[0070] As shown in Table 18 and Figure 3 it can be seen that the spores cannot germinate at 35°C. When the culture temperature is 15°C - 30°C, both the spore germination time and the germination number show an increasing trend with the increase of temperature. The spore germination number at 30°C reaches 110.7 ± 2.3, and the germination numbers at 15°C, 20°C, and 25°C are significantly lower than that at 30°C, and the mycelial growth at 15°C is the weakest. When the spore suspension is evenly coated after being stimulated by water bath at 40°C for 40 min and then cultured at 28°C, its germination number is as high as 285.0 ± 9.5, which is 227.6% higher than that without water bath treatment. The results show that the optimal germination temperature of Ganoderma sinense spores is 30°C, and the germination number of spores can be significantly increased after water bath treatment.

[0071] (2) The effects of the pH value of the medium on the spore germination of Ganoderma sinense are shown in Table 5 and Figure 4 as follows:

[0072] Table 5 Effects of culture temperature on the spore germination of Ganoderma sinense

[0073]

[0074] As shown in Table 5 and Figure 4 it can be seen that the spore germination number shows a decreasing trend with the increase of the pH value of the medium. The germination rate at pH 4.5 is the highest, which is 1.70%. The germination numbers at pH 5.0 and 6.0 are 185.7 ± 8.1 and 29.7 ± 6.0 respectively. There are significant differences in the germination numbers among pH 4.5, 5.0, and 6.0. Among them, the germination numbers at pH 4.5 and 5.0 are significantly higher than those of the basal medium (CK, natural pH). When the pH is greater than 7.0, the spores cannot germinate, indicating that acidic conditions promote the spore germination of Ganoderma sinense. When the pH of the medium is adjusted to less than 4.5, the concentration of agar needs to be increased to solidify the medium, but it will make the medium more viscous and inhibit the spore germination. Therefore, the optimal pH value of the spore germination medium of Ganoderma sinense is 4.5.

[0075] (3) The effects of nitrogen sources on the spore germination of Ganoderma sinense are shown in Table 6 and Figure 5 as follows:

[0076] Table 6 Effects of nitrogen sources on the spore germination of Ganoderma sinense

[0077]

[0078] As shown in Table 6 and Figure 5As shown in the figure, the highest number of germinated spores was observed on the basal medium (CK) supplemented with two nitrogen sources (beef extract and peptone) simultaneously, reaching 86.0 ± 4.6. The experimental results with a single nitrogen source showed that spores could not germinate on media with potassium nitrate or ammonium sulfate as the nitrogen source. The number of germinated spores was relatively high on the medium with beef extract as the nitrogen source, followed by peptone, indicating that beef extract had a greater impact on spore germination than peptone, while yeast extract, soybean powder, potassium nitrate, and ammonium sulfate were not suitable as nitrogen sources for spore germination.

[0079] (4) The results of the effect of carbon source on the germination of Ganoderma sinense spores are shown in Table 7 and Figure 6 as follows:

[0080] Table 7 Effect of carbon source on the germination of Ganoderma sinense spores

[0081]

[0082]

[0083] As shown in Table 7 and Figure 6 as follows, the number of germinated spores on the medium with maltose as the carbon source was 118.3 ± 6.4, significantly higher than that on the basal medium (CK). Although the number of germinated spores on lactose and fructose was higher than that on the basal medium, there was no significant difference among lactose, fructose, and the basal medium. The number of germinated spores on glucose and trehalose was lower than that on the basal medium, but there was also no significant difference among glucose, trehalose, and the basal medium. The number of germinated spores on the medium with sucrose as the carbon source was the lowest, at 65.3 ± 13.9, significantly lower than that on the basal medium, indicating that adding an appropriate amount of maltose, lactose, or fructose to the medium could promote spore germination, and the most suitable carbon source was maltose.

[0084] (5) The results of the effect of storage time on the germination of Ganoderma sinense spores are shown in Table 8 and Figure 7 as follows:

[0085] Effect of the extract of boiled sawdust on the germination of Ganoderma sinense spores Table 8 Effect of storage time on the germination of Ganoderma sinense spores

[0086]

[0087] As shown in Table 8 and Figure 7 as follows, the optimal germination conditions of Ganoderma sinense spores were used to verify the effect of spore storage time on the germination rate. Figure 7The results in Table 9 show that the germination number at a storage time of 1 day reached 98.3 ± 8.7, followed by 86.9 ± 10.5, 83.5 ± 13.3, and 83.5 ± 13.3 at storage times of 5 days, 10 days, and 15 days respectively. There was no significant difference in the germination number among the storage times of 5 days, 10 days, and 15 days. The germination numbers at storage times of 30 days, 45 days, and 60 days were 66.9 ± 14.0, 51.8 ± 15.7, and 36.6 ± 5.0 respectively, all lower than the germination rate within 15 days, and there were significant differences in the germination numbers among the storage times of 30 days, 45 days, and 60 days. This indicates that the longer the storage time of Ganoderma sinense spores, the lower the germination rate, and the storage time has a greater impact on spore germination.

[0088] (6) The influence of the decoction of wood chips on the germination of Ganoderma sinense spores. The results are shown in Table 9 and Figure 8 as follows:

[0089] Table 9 The influence of the decoction of wood chips on the germination of Ganoderma sinense spores

[0090]

[0091]

[0092] As shown in Table 9 and Figure 8 it can be seen that the spores could not germinate on the medium supplemented with the decoction of Cyclobalanopsis glauca and Castanea mollissima wood chips. The germination numbers on the medium supplemented with the decoction of Castanopsis fargesii and Pinus massoniana wood chips were 126.7 ± 6.4 and 117.7 ± 11.4 respectively, significantly higher than that of the basal medium (CK). The germination numbers on the medium supplemented with the decoction of Pyrus pyrifolia and Liquidambar formosana wood chips were also higher than that of the basal medium, but there was no significant difference among the three. This indicates that adding 10% of the decoction of Castanopsis fargesii, Pinus massoniana, Pyrus pyrifolia, and Liquidambar formosana wood chips to the basal medium can promote the germination of Ganoderma sinense spores, and the most suitable wood chip decoction for addition is that of Castanopsis fargesii.

[0093] (7) Optimization results of the best spore germination culture conditions

[0094] Combining the results of the basal medium screening experiment and the germination condition experiment, it can be known that ZM8 can be used as the basal medium for the germination of Ganoderma sinense spores. Add 2% maltose and 10% of the decoction of Castanopsis fargesii wood chips to the medium, adjust the pH value of the medium to 4.5, and dilute the freshly ejected spores with sterile water to a concentration of 2 × 10 5A suspension of [[ID=]], and after stimulating in a water bath at 40 °C for 40 min, take 0.1 mL and evenly coat it on the culture medium, and incubate it at a constant temperature of 30 °C in the dark, which can significantly increase the germination number of spores; and it can be seen from the experimental results that the beef extract and the extract of Castanopsis fissa wood have a greater impact on spore germination than maltose and peptone. To verify whether the concentration of beef extract and the extract of Castanopsis fissa wood affects the germination rate of Ganoderma sinense spores, 9 culture media with different concentrations of beef extract and the extract of Castanopsis fissa wood were prepared in this experiment, and compared with other reported spore germination culture media to screen the optimal culture medium for Ganoderma sinense spore germination. The results are shown in Table 10 and Figure 9 as shown below.

[0095] Table 10 Optimization experiment results of the best spore germination culture conditions

[0096]

[0097] As shown in Table 10 and Figure 9 as shown below, the germination rates of spores on the 3 reported control culture medium formulations are ranked from high to low as: control formulation 3 > control formulation 1 > control formulation 2, and the germination rate of control formulation 3 is 1.69%. The germination rates of spores on the 9 culture media ZM3-1 to ZM3-9 in this experiment are 3.32% - 3.88%. Among them, the culture medium with the highest germination rate is ZM8-8, followed by ZM8-9 and ZM8-5; when the concentration of beef extract is 1.0%, the spore germination rate increases with the increase of the concentration of the extract of Castanopsis fissa wood; when the concentrations of beef extract are 2.0% and 3.0%, the germination rate first increases and then decreases with the increase of the concentration of the extract of Castanopsis fissa wood; in addition, when the concentration of beef extract is 4.0%, the culture medium is viscous and spore germination is inhibited; therefore, the order of the promoting effect of beef extract concentration on spore germination is: 1.0% < 2.0% < 3.0%. It shows that adding 3% beef extract and 20% extract of Castanopsis fissa wood to the culture medium can maximize the promotion of spore germination, and the germination rate of Ganoderma sinense spores on the ZM8-8 culture medium is 129.39% higher than that of control formulation 3.

[0098] Example 2

[0099] Combined with the results of Example 1, it can be known that beef extract, peptone, Castanopsis fissa liquid, pear wood liquid, maple wood liquid and pine wood liquid all have a promoting effect on the germination of Ganoderma sinense spores. Therefore, combined with the above conditions, a spore germination culture medium for cultivating Ganoderma sinense spores and a germination culture medium for Ganoderma lucidum spores are prepared, keeping the mass percentage of agar at 6%, the extract of wood chips at 10%, and the pH at 4.0; the specific proportion of wood chips, and the mass percentages of beef extract and peptone are shown in Table 11 specifically, and the germination results of Ganoderma sinense spores are shown in Table 12:

[0100] Table 11 Orthogonal factor optimization experiment

[0101] Horizontal Beef extract (mass percentage) Peptone (mass percentage) Liquid from Castanopsis fargesii Franch. (volume ratio) Liquid from Pyrus pyrifolia Nakai (volume ratio) Liquid from Liquidambar formosana Hance (volume ratio) Liquid from Pinus massoniana Lamb. (volume ratio) 1 1.0％ 0.5％ 1 2 1 1 2 2.0％ 1.0％ 2 3 2 2 3 3.0％ 1.5％ 3 4 3 3 4 4.0％ 2.0％ 4 5 4 4 5 5.0％ 2.5％ 5 6 5 5

[0102] Table 12 Results of Orthogonal Experiment

[0103]

[0104] As can be seen from Table 12, beef extract, peptone, Castanopsis fargesii, Pyrus pyrifolia, Liquidambar formosana, and Pinus massoniana have different effects on the germination of Ganoderma sinense spores under different ratios. From the perspective of the ratio, when the content of beef extract is greater than 3%, the growth of Ganoderma sinense spores will be inhibited. When the content of beef extract is 1%-2%, the germination rate of Ganoderma sinense spores is greater than the optimal ratio of Example 1. However, the germination rates of Experiments 11-25 are less than the optimal ratios of Example 1 and Example 2, and the germination rate of some experimental levels even drops to 0%. This shows that when the content of beef extract is too high, the germination of Ganoderma sinense spores will be inhibited. Combining the experimental results, we believe that for Ganoderma sinense, the best germination medium formula is: beef extract with a mass percentage of 1%-2%, peptone with a mass percentage of 0.5%-2.5%, agar with a mass percentage of 2%, and the extract of wood chips with a volume percentage of 20%. Among them, the mass ratio of the extract of wood chips is: the volume ratio of Castanopsis fargesii liquid:Pyrus pyrifolia liquid:Liquidambar formosana liquid:Pinus massoniana liquid is 1-5:2-6:1-5:1-5. The optimal mass ratio in Table 12 is Experiment 3, that is: beef extract with a mass percentage of 1%, peptone with a mass percentage of 1.5%, agar with a mass percentage of 6%, and the extract of wood chips with a volume percentage of 10%. Among them, the volume ratio of the extract of wood chips is: the volume ratio of Castanopsis fargesii liquid:Pyrus pyrifolia liquid:Liquidambar formosana liquid:Pinus massoniana liquid is 3:4:3:3.

[0105] From the perspective of the range, the range of Ganoderma sinense spore germination rate: beef extract > Liquidambar formosana liquid > Pinus massoniana liquid > Pyrus pyrifolia liquid > peptone > Castanopsis fargesii liquid; this shows that for the germination rate of Ganoderma sinense spores, the most influential is beef extract, followed by Liquidambar formosana liquid and Pinus massoniana liquid, and the least influential is Castanopsis fargesii liquid.

[0106] Therefore, combining Example 1 and Example 2, we know that for the germination of Ganoderma lucidum spores, the best medium formula is: beef extract with a mass percentage of 1%, peptone with a mass percentage of 1.5%, agar with a mass percentage of 6%, and the extract of wood chips with a volume percentage of 10%. Among them, the volume ratio of the extract of wood chips is: the volume ratio of Castanopsis fargesii liquid:Pyrus pyrifolia liquid:Liquidambar formosana liquid:Pinus massoniana liquid is 3:4:3:3.

[0107] The best method for the germination of Ganoderma sinense spores is: dilute the freshly ejected spores with sterile water to a suspension with a concentration of 2×10 5 cells / mL, and after stimulating in a water bath at 40°C for 40 min, take 0.1 mL and evenly coat it on the medium, and culture it at a constant temperature of 30°C in the dark. The preferred method for collecting spores is the cardboard box method.

[0108] In summary, this application focuses on the research of the spore culture and collection methods of Ganoderma sinense, obtains a collection plan for Ganoderma sinense spores, and obtains a culture medium suitable for the germination of Ganoderma sinense spores by optimizing the culture medium components. It is a simple, rapid, and efficient method for collecting Ganoderma sinense spores and promoting spore germination, which can greatly improve the work efficiency of operators.

[0109] The above-described embodiments merely represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. A culture medium for germination of Ganoderma lucidum spores, characterized in that: The culture medium is composed of the following components: 1%-2% beef extract by mass, 0.5%-2.5% peptone by mass, 2%-6% agar by mass, 10% sawdust decoction by volume; and the remainder is water.

2. The Ganoderma lucidum spore germination medium according to claim 1, characterized in that: The sawdust decoction liquid is pinewood liquid, pearwood liquid, maplewood liquid and / or pinewood liquid.

3. The Ganoderma lucidum spore germination medium according to claim 1, characterized in that: The method for preparing the sawdust decoction is as follows: pine, pear, maple or cone wood is added into water, boiled for 40 minutes, filtered and stored to obtain the decoction.

4. The Ganoderma lucidum spore germination medium according to claim 2, characterized in that: The volume ratio of the pinewood sap, pearwood sap, maplewood sap and pinewood sap is 1-5:2-6:1-5:1-5.

5. The Ganoderma lucidum spore germination medium according to claim 1, characterized in that: The culture medium consists of the following components: 1% beef extract by mass, 1.5% peptone by mass, 2% agar by mass, and 10% sawdust decoction by volume; wherein the sawdust decoction is obtained by mixing vertebral wood liquid, pear wood liquid, maple wood liquid and pine wood liquid in a volume ratio of 3:4:3:

3.

6. A method for culturing Ganoderma lucidum spores using the culture medium according to any one of claims 1 to 5, characterized in that: The method is: dilute the newly ejected spores with sterile water to a concentration of 2×10 5 The suspension was prepared by stimulating the suspension in a water bath at 40°C for 40 minutes, then 0.1 mL was evenly spread on the culture medium and cultured at a constant temperature of 30°C in the dark; the pH value of the culture medium was 4.

5.

7. The method according to claim 6, characterized in that The Ganoderma lucidum variety is Guizhi No. 8, and the strain preservation number is CGMCC NO.41073.

8. The method according to claim 6, characterized in that The method for collecting Ganoderma lucidum spores is a carton method.

9. The method according to claim 8, characterized in that The specific operation of the carton method is as follows: the fruiting bodies of Ganoderma lucidum cultivated in the greenhouse are moved together with the fungus bags of the culture medium into the culture room. The selected fruiting bodies are required to be healthy and strong, free of pests and diseases, the white growth ring on the edge of the cap has basically disappeared, and a small amount of spores are released. First, a small amount of 75% alcohol is used to gently wipe the surface of the fungus bag and the fruiting body for surface disinfection, and then sterile water is sprayed twice and the excess water on the surface is wiped off to increase the humidity; a small amount of 75% alcohol is sprayed in a new carton that has not been used, and the fungus bag containing the fruiting body and the culture medium is placed in the carton. The size of the carton is: 60 cm long, 30 cm wide, and 40 cm high. A sterile culture dish is placed under the fruiting body, and it is left to stand at 25-28° C. until a layer of reddish-brown powder appears at the bottom of the culture dish, which is the spores, and then it is taken out and placed in a refrigerator at 4° C. for storage.

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