Dictyophora rubrovolvata cooking-free liquid culture medium as well as preparation method and application thereof

Abstract

The invention relates to the technical field of edible fungus cultivation, in particular to a dictyophora rubrovolvata cooking-free liquid culture medium and a preparation method and application thereof. The formula of the culture medium consists of the following components: soybean meal, corn flour, sorghum flour, peptone, glucose, brown sugar, monopotassium phosphate, magnesium sulfate and distilled water. The preparation method of the culture medium comprises the following steps: weighing the components according to the formula, adding a proper amount of distilled water to dissolve and fix the volume, and then sub-packaging, sterilizing and cooling to obtain the culture medium. The formula and the preparation method of the dictyophora rubrovolvata cooking-free liquid culture medium are simple, and the production efficiency can be improved while the production cost is reduced. Meanwhile, the culture medium can meet the nutritional requirements of dictyophora rubrovolvata strains, the culture time for preparing secondary strains can be shortened, and after the prepared secondary strains are inoculated into fungus bags, the strain germination speed is high, the germination rate is high, the hypha growth speed is high, and the bag filling time is short. Technical support is provided for efficient cultivation of dictyophora rubrovolvata, and the method has wide market application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of edible fungus cultivation, and in particular to a boiling-free liquid culture medium for red-stalked bamboo fungus, a preparation method thereof, and an application thereof. Background Art

[0002] Dictyophora rubrovolvata, classified as a member of the genus Dictyophora in the Basidiomycota, Gasteromycetes, Phallales, Phallaceae family, and the genus Dictyophora, is a rare edible fungus used for both medicinal and edible purposes, earning the title "Queen of Fungi." Dictyophora rubrovolvata is known for its soft, fragrant flavor, and superior quality among known bamboo fungus varieties, and market demand is increasing. Therefore, there is an urgent need to develop efficient cultivation models for Dictyophora rubrovolvata to support the sustainable development of the industry. The development of liquid culture media for cultivating Dictyophora rubrovolvata has become a research hotspot in the field.

[0003] In the prior art, the patent application with publication number CN118872541A, "A culture medium and method for rapidly cultivating liquid strains of red-stemmed bamboo fungus," discloses a culture medium for cultivating liquid strains of red-stemmed bamboo fungus. The culture medium can quickly cultivate the strains to be cultivated into liquid strains in only about ten days, shortening the cultivation time by more than 10%. However, in the preparation of the culture medium, dry bamboo leaves and water need to be evenly mixed, heated and boiled, and solid-liquid separation is performed to prepare an extract. The operation is relatively complicated, and the cultivation time for preparing secondary strains is relatively long. The patent application with publication number CN118020570A, "A method for producing red-stemmed bamboo fungus bags based on liquid non-boiling culture medium," discloses a non-boiling liquid culture medium that can be completely dissolved and used only with room temperature water, eliminating the tedious steps of steaming and filtering, and scientifically and rationally optimizing the production process. The cultivation of the secondary liquid strains of red-stemmed bamboo fungus can be completed in an average of 12 days. However, the germination time and average full bag time of the secondary strain after inoculation into the bag are relatively long. In summary, the following problems still exist in the prior art: (1) In the preparation of culture medium, it is often necessary to prepare the extract through the process of boiling, extraction and filtration, which is cumbersome and takes a long time to process, greatly reducing the production efficiency; (2) The composition of the culture medium is complex and the production cost is high; (3) The nutrition of the culture medium cannot fully meet the growth of the red-suspension bamboo fungus, resulting in a long culture time for preparing liquid seeds. After the prepared liquid seeds are applied to the mushroom bags, the germination time and the filling time of the bags are both long.

[0004] Based on the above problems, this study provides a nutrient-complete, no-cooking liquid culture medium that can meet the growth of Dictyophora rubra. It aims to simplify the production process, effectively shorten the culture time of Dictyophora rubra liquid seeds, and at the same time improve the utilization value of agricultural product waste, laying the foundation for the efficient cultivation of Dictyophora rubra. Summary of the Invention

[0005] The invention aims to provide a boiling-free liquid culture medium of Dictyophora rubra and a preparation method thereof.

[0006] Another object of the present invention is to provide the use of the red-sprung bamboo fungus no-cooking liquid culture medium in preparing red-sprung bamboo fungus secondary varieties.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The red stem bamboo fungus no-cooking liquid culture medium of the present invention is composed of the following components per liter: 10-40g of soybean meal powder, 5-30g of corn flour, 5-30g of sorghum flour, 1-15g of peptone, 5-25g of glucose, 1-15g of brown sugar, 1-10g of potassium dihydrogen phosphate, 1-5g of magnesium sulfate, and the balance is distilled water.

[0009] Preferably, each liter of the red-tubed bamboo fungus no-cooking liquid culture medium of the present invention is composed of the following components: 15-35g of soybean meal powder, 10-25g of corn flour, 10-25g of sorghum flour, 5-15g of peptone, 10-20g of glucose, 5-15g of brown sugar, 2-8g of potassium dihydrogen phosphate, 1-3g of magnesium sulfate, and the balance is distilled water.

[0010] Further preferably, each liter of the red-tubed bamboo fungus no-boil liquid culture medium of the present invention is composed of the following components: 20-30g of soybean meal powder, 10-20g of corn flour, 10-20g of sorghum flour, 5-10g of peptone, 10-15g of glucose, 5-10g of brown sugar, 3-5g of potassium dihydrogen phosphate, 1-2g of magnesium sulfate, and the balance is distilled water.

[0011] The preparation method of the red stem bamboo fungus boiling-free liquid culture medium of the present invention is specifically as follows: after weighing each component according to the formula, adding distilled water to dissolve and fix the volume, and then packaging, sterilizing and cooling to obtain the liquid culture medium.

[0012] The invention discloses an application of the boiling-free liquid culture medium of Dictyophora rubrae in preparing secondary seeds of Dictyophora rubrae.

[0013] Preferably, the method for preparing the secondary species of Dictyophora rubra of the present invention comprises the following steps:

[0014] S1 Preparation of Dictyophora rubra mother stock:

[0015] After activating the red stem bamboo fungus, the fungus is inoculated into a solid culture medium for cultivation to obtain the red stem bamboo fungus mother culture; wherein the solid culture medium is prepared by adding 1.5% to 2% agar to a no-boil liquid culture medium;

[0016] S2 Preparation of the first-class red stem bamboo fungus:

[0017] Prepare a boil-free liquid culture medium, and dispense 150 mL of each into a 250 mL container. After sterilization and cooling, inoculate the mother culture prepared in step S1. After inoculation, culture in a constant temperature shaker for 10 to 20 days to obtain the first-grade red-stalked bamboo fungus.

[0018] S3 Preparation of Red-stalked Dictyophora secondary species:

[0019] The first-grade red stem bamboo fungus seed prepared in step S2 is transferred to a fermentation tank filled with a boil-free liquid culture medium, and cultured at a constant temperature in the dark for 2 to 6 days to obtain the second-grade red stem bamboo fungus seed.

[0020] Further preferably, in the method for preparing the secondary seeds of Dictyophora rubra of the present invention, the inoculation amount in step S2 is: 4 to 6 pieces of mother seeds with a diameter of 0.5 cm are inoculated into every 150 mL of liquid culture medium.

[0021] Further preferably, in the method for preparing the secondary species of Dictyophora rubra of the present invention, the constant temperature shaking incubation in step S2 is specifically: culturing for 15 days in a constant temperature shaking incubator at 26° C. and a rotation speed of 140 r / min.

[0022] Further preferably, in the method for preparing the secondary strain of Dictyophora rubra of the present invention, the transfer in step S3 is specifically as follows: the primary strain prepared in step S2 is inoculated into a fermentation tank containing a boil-free liquid culture medium at a volume ratio of the strain amount to the culture medium of 1:150.

[0023] Further preferably, in the method for preparing the secondary species of Dictyophora rubra of the present invention, the constant temperature cultivation under dark conditions in step S3 is specifically: cultivation at 26° C. in the dark for 4 days.

[0024] Beneficial effects of the present invention:

[0025] 1. The present invention provides a boiling-free liquid culture medium for red stem bamboo fungus, which has a simple formula and low production cost and can also improve the utilization value of agricultural product waste.

[0026] 2. The liquid culture medium of Dictyophora rubra provided by the present invention eliminates the process of steaming and filtering to prepare the extract in the preparation of conventional culture medium, thereby greatly improving production efficiency.

[0027] 3. The boiling-free liquid culture medium for red-sprung bamboo fungus provided by the present invention has comprehensive nutrition and can meet the growth of red-sprung bamboo fungus bodies. The red-sprung bamboo fungus secondary seed can be obtained in as short as 12 days after inoculation of the mother seed, shortening the culture time of the secondary seed.

[0028] 4. The secondary seed prepared using the boiling-free red bamboo fungus liquid culture medium provided by the present invention was inoculated into the fungus bag. The fungus began to germinate in 48 hours, with a germination rate of up to 100%, and the mycelium grew rapidly. It was determined that the average mycelium growth rate could reach 2.27 mm / d, and the average bag-filling time was 79 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a secondary variety of Dictyophora rubra obtained under different culture medium formulas;

[0030] Figure 2 It is a no-boil liquid culture medium prepared based on formula 5;

[0031] Figure 3 It is a secondary species of red stem bamboo fungus prepared based on formula 5;

[0032] Figure 4 The method is to inoculate the fungus bag of the second-grade red bamboo fungus obtained based on recipe 5. DETAILED DESCRIPTION

[0033] The following is a detailed description of the technical solution of the present invention in conjunction with specific embodiments. The following embodiments are only for explanation and illustration, and do not constitute a limitation of the technical solution of the present invention.

[0034] Example 1

[0035] Formula for the no-cooking liquid culture medium of Dictyophora rubra:

[0036] 20g soy flour, 10g corn flour, 10g sorghum flour, 5g peptone, 10g glucose, 5g brown sugar, 3g potassium dihydrogen phosphate, 1g magnesium sulfate.

[0037] Preparation method of boiling-free liquid culture medium of Dictyophora rubra:

[0038] After weighing each component according to the formula, add appropriate amount of distilled water to dissolve and make up to 1L, put 150mL of each portion into a 250mL container, sterilize and cool.

[0039] Example 2

[0040] Formula for the no-cooking liquid culture medium of Dictyophora rubra:

[0041] 30g soy flour, 20g corn flour, 20g sorghum flour, 10g peptone, 15g glucose, 10g brown sugar, 5g potassium dihydrogen phosphate, 2g magnesium sulfate.

[0042] Preparation method of boiling-free liquid culture medium of Dictyophora rubra:

[0043] After weighing each component according to the formula, add appropriate amount of distilled water to dissolve and make up to 1L, put 150mL of each portion into a 250mL container, sterilize and cool.

[0044] Example 3

[0045] Formula for the no-cooking liquid culture medium of Dictyophora rubra:

[0046] 10g soy flour, 5g corn flour, 5g sorghum flour, 1g peptone, 5g glucose, 1g brown sugar, 1g potassium dihydrogen phosphate, 1g magnesium sulfate.

[0047] Preparation method of boiling-free liquid culture medium of Dictyophora rubra:

[0048] After weighing each component according to the formula, add appropriate amount of distilled water to dissolve and make up to 1L, put 150mL of each portion into a 250mL container, sterilize and cool.

[0049] Example 4

[0050] Formula for the no-cooking liquid culture medium of Dictyophora rubra:

[0051] 40g soy flour, 30g corn flour, 30g sorghum flour, 15g peptone, 25g glucose, 15g brown sugar, 10g potassium dihydrogen phosphate, 5g magnesium sulfate.

[0052] Preparation method of boiling-free liquid culture medium of Dictyophora rubra:

[0053] After weighing each component according to the formula, add appropriate amount of distilled water to dissolve and make up to 1L, put 150mL of each portion into a 250mL container, sterilize and cool.

[0054] Example 5

[0055] Formula for the no-cooking liquid culture medium of Dictyophora rubra:

[0056] 15g soy flour, 10g corn flour, 10g sorghum flour, 5g peptone, 10g glucose, 5g brown sugar, 2g potassium dihydrogen phosphate, 1g magnesium sulfate.

[0057] Preparation method of boiling-free liquid culture medium of Dictyophora rubra:

[0058] After weighing each component according to the formula, add appropriate amount of distilled water to dissolve and make up to 1L, put 150mL of each portion into a 250mL container, sterilize and cool.

[0059] Example 6

[0060] Formula for the no-cooking liquid culture medium of Dictyophora rubra:

[0061] 35g soy flour, 25g corn flour, 25g sorghum flour, 15g peptone, 20g glucose, 15g brown sugar, 8g potassium dihydrogen phosphate, 3g magnesium sulfate.

[0062] Preparation method of boiling-free liquid culture medium of Dictyophora rubra:

[0063] After weighing each component according to the formula, add appropriate amount of distilled water to dissolve and make up to 1L, put 150mL of each portion into a 250mL conical flask, sterilize and cool.

[0064] Example 7

[0065] The boiling-free liquid culture medium of Dictyophora rubra is used to prepare the secondary varieties of Dictyophora rubra. The preparation method is as follows:

[0066] S1 Preparation of Dictyophora rubra mother stock:

[0067] After activating the red-sprung bamboo fungus, the solid culture medium was inoculated and cultured to obtain the red-sprung bamboo fungus mother culture; wherein the solid culture medium was prepared by adding agar to a boil-free liquid culture medium, and its formula composition was: 35g soybean meal powder, 25g corn flour, 25g sorghum flour, 15g peptone, 20g glucose, 15g brown sugar, 8g potassium dihydrogen phosphate, 3g magnesium sulfate, and 20g agar;

[0068] S2 Preparation of the first-class red stem bamboo fungus:

[0069] Prepare a boil-free liquid culture medium, dispense 150 mL of each into 250 mL containers, sterilize and cool, inoculate five pieces of the mother culture prepared in step S1 with a diameter of 0.5 cm, and culture in a constant temperature shaker at 26°C and 140 rpm for 15 days to obtain the first-grade red stem cultivar.

[0070] S3 Preparation of Red-stalked Dictyophora secondary species:

[0071] The first-level red stem bamboo fungus obtained in step S2 was transferred to a fermentation tank containing a boil-free liquid culture medium at a volume ratio of the culture volume to the culture medium of 1:150, and cultured at a constant temperature of 26° C. in the dark for 4 days to obtain the second-level red stem bamboo fungus.

[0072] Example 8

[0073] The boiling-free liquid culture medium of Dictyophora rubra is used to prepare the secondary varieties of Dictyophora rubra. The preparation method is as follows:

[0074] S1 Preparation of Dictyophora rubra mother stock:

[0075] After activating the red-sprung bamboo fungus, the solid culture medium was inoculated and cultured to obtain the red-sprung bamboo fungus mother culture; wherein the solid culture medium was prepared by adding agar to a boil-free liquid culture medium, and its formula composition was: 35g soybean meal powder, 25g corn flour, 25g sorghum flour, 15g peptone, 20g glucose, 15g brown sugar, 8g potassium dihydrogen phosphate, 3g magnesium sulfate, and 20g agar;

[0076] S2 Preparation of the first-class red stem bamboo fungus:

[0077] Prepare a boil-free liquid culture medium, dispense 150 mL of each into 250 mL containers, sterilize and cool, inoculate five pieces of the mother culture prepared in step S1 with a diameter of 0.5 cm, and culture in a constant temperature shaker at 26°C and 140 rpm for 10 days to obtain the first-grade red stem cultivar.

[0078] S3 Preparation of Red-stalked Dictyophora secondary species:

[0079] The first-level red stem bamboo fungus obtained in step S2 was transferred to a fermentation tank containing a boil-free liquid culture medium at a volume ratio of the culture volume to the culture medium of 1:150, and cultured at a constant temperature of 26° C. in the dark for 2 days to obtain the second-level red stem bamboo fungus.

[0080] In order to further verify the reliability of the present invention and screen out the best solution, the inventors conducted a series of experiments, as follows:

[0081] 1. Materials

[0082] 1.1 Test strains

[0083] The test strain was the red-top bamboo fungus Wansun No. 2, which was deposited in Guizhou Bailu Modern Agriculture Technology Co., Ltd.

[0084] 1.2 Test materials

[0085] Potatoes, glucose, pine needles, corn flour, soybean meal, sorghum flour, peptone, brown sugar, potassium dihydrogen phosphate, magnesium sulfate, gypsum powder, sawdust, wheat bran, sorghum hulls.

[0086] 2. Test methods

[0087] 2.1 Liquid culture medium formulation

[0088] The liquid culture medium formula is shown in Table 1.

[0089] Table 1 Liquid culture medium formula

[0090]

[0091] 2.2 Mushroom bag formula

[0092] Formula of red bamboo fungus bag: sawdust (82% to 85%), wheat bran (5% to 8%), sorghum husk (5% to 8%), potassium dihydrogen phosphate (0.5% to 0.8%), magnesium sulfate (0.3% to 0.5%), gypsum powder (1% to 1.5%), and water content (65% to 68%).

[0093] 2.3 Experimental design

[0094] (1) Preparation of liquid culture medium:

[0095] First, accurately weigh each component according to the formulation in Table 1 under "2.1." Dissolve the components in distilled water and dilute to 1 L. Then, aliquot and sterilize the mixture. After cooling, liquid culture media with different formulations are obtained. (Formulas 1-3 require boiling the potatoes / pine needles / wood chips before filtering and distilling to 1 L with distilled water. Subsequent steps are the same as for the no-boiling formulations.) The target liquid culture medium for liquid culture medium formulation screening is 150 mL / bottle, with 10 bottles per formulation.

[0096] (2) Preparation of red stem bracteata secondary species

[0097] After activating the red-sprung bamboo fungus, the strain was inoculated into a solid culture medium for culture (the solid culture medium was prepared by adding 1.5% to 2% agar to a boil-free liquid culture medium) to obtain a red-sprung bamboo fungus mother strain; the prepared liquid culture medium was divided into 250 mL conical flasks at 150 mL each, sterilized, and cooled. Five pieces of the mother strain prepared in the above step with a diameter of 0.5 cm were inoculated on a clean bench. After the inoculation, the strain was cultured in a constant temperature shaker at 26° C. at a speed of 140 r / min for 15 days to obtain a red-sprung bamboo fungus first-class strain; the red-sprung bamboo fungus first-class strain prepared in the above step was transferred to a fermenter containing a boil-free liquid culture medium at a volume ratio of strain amount to culture medium of 1:150, and cultured at a constant temperature of 26° C. in the dark for 4 days.

[0098] (3) Preparation of red bamboo fungus sticks

[0099] Prepare red-stemmed bamboo fungus bags according to the recipe under "2.2". The target mushroom stick weight is 1.3±0.05kg / stick, and a total of 100 sticks are prepared. After preparation, inoculate red-stemmed bamboo fungus liquid inoculum at an inoculum volume of 30mL / stick.

[0100] 3. Data Collection

[0101] The criteria for judging the growth of mycelium are: not long (-), very weak (+), weak and sparse (++), strong and dense (+++), strong, thick and dense (++++).

[0102] Calculation of germination rate: germination rate (%) = number of cultivated germination frames / total number of cultivated frames.

[0103] 4. Test results

[0104] 4.1 Screening of liquid culture medium formula for Dictyophora rubra

[0105] The secondary varieties of Dictyophora rubra obtained under different culture medium formulas are as follows Figure 1 The growth comparison is shown in Table 2. The growth comparison of mycelium after the secondary strains obtained based on different culture media were inoculated into the fungus bag is shown in Table 3. The boiling-free liquid culture medium prepared based on formula 5 is shown in Table 3. Figure 2 As shown, the red stem bamboo fungus secondary species prepared based on formula 5 is as follows Figure 3 As shown, the fungus bag after inoculation of the red bamboo fungus secondary species obtained based on formula 5 is as follows Figure 4 shown.

[0106] Table 2 Comparison of the growth of secondary varieties of Dictyophora rubra obtained under different culture medium formulas

[0107]

[0108] Table 3 Comparison of mycelial growth after secondary strains obtained from different culture media were inoculated into fungus bags

[0109]

[0110] The results show that the liquid culture medium prepared by formula 5 is yellow-brown in color; the secondary culture liquid of red-stemmed bamboo fungus prepared based on formula 5 is light yellow, the color of the fungus ball is milky white, and the diameter is about 1.93 mm; after the secondary culture of red-stemmed bamboo fungus prepared based on formula 5 is inoculated into the fungus bag, the fungus bag begins to germinate 48 hours later, with a germination rate of up to 100%, and the mycelium grows fast. It has been measured that the average mycelium growth rate can reach 2.27 mm / d, and the average bag-filling time is 79 days.

[0111] Although the present invention has been described in detail above using general descriptions, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A boiling-free liquid culture medium for Dictyophora rubra, characterized in that: The red stem bamboo fungus no-cooking liquid culture medium is composed of the following components per liter: 10-40g of soybean meal powder, 5-30g of corn flour, 5-30g of sorghum flour, 1-15g of peptone, 5-25g of glucose, 1-15g of brown sugar, 1-10g of potassium dihydrogen phosphate, 1-5g of magnesium sulfate, and the balance is distilled water.

2. The boiling-free liquid culture medium of Dictyophora rubra according to claim 1, characterized in that The red stem bamboo fungus no-boil liquid culture medium is composed of the following components per liter: 15-35g of soybean meal powder, 10-25g of corn flour, 10-25g of sorghum flour, 5-15g of peptone, 10-20g of glucose, 5-15g of brown sugar, 2-8g of potassium dihydrogen phosphate, 1-3g of magnesium sulfate, and the balance is distilled water.

3. The boiling-free liquid culture medium of Dictyophora rubra according to claim 2, characterized in that The red stem bamboo fungus no-cooking liquid culture medium is composed of the following components per liter: 20-30g of soybean meal powder, 10-20g of corn flour, 10-20g of sorghum flour, 5-10g of peptone, 10-15g of glucose, 5-10g of brown sugar, 3-5g of potassium dihydrogen phosphate, 1-2g of magnesium sulfate, and the balance is distilled water.

4. The method for preparing the boiling-free liquid culture medium of Dictyophora rubra according to any one of claims 1 to 3, wherein: The preparation method specifically comprises the following steps: weighing each component according to the formula, adding distilled water to dissolve and adjust the volume, and then performing subpackaging, sterilizing, and cooling to obtain the product.

5. Use of the boiling-free liquid culture medium of Dictyophora rubra according to any one of claims 1 to 3 in preparing Dictyophora rubra secondary varieties.

6. The use according to claim 5, characterized in that The preparation method of the red-suspensa bamboo fungus secondary species comprises the following steps: S1 Preparation of Dictyophora rubra mother stock: After activating the red stem bamboo fungus, the fungus is inoculated into a solid culture medium for cultivation to obtain the red stem bamboo fungus mother culture; wherein the solid culture medium is prepared by adding 1.5% to 2% agar to a no-boil liquid culture medium; S2 Preparation of the first-class red stem bamboo fungus: Prepare a boil-free liquid culture medium, and dispense 150 mL of each into a 250 mL container. After sterilization and cooling, inoculate the mother culture prepared in step S1. After inoculation, culture in a constant temperature shaker for 10 to 20 days to obtain the first-grade red-stalked bamboo fungus. S3 Preparation of Red-stalked Dictyophora secondary species: The first-grade red stem bamboo fungus seed prepared in step S2 is transferred to a fermentation tank filled with a boil-free liquid culture medium, and cultured at a constant temperature in the dark for 2 to 6 days to obtain the second-grade red stem bamboo fungus seed.

7. The use according to claim 6, characterized in that In the method for preparing the secondary seeds of Dictyophora rubra, the inoculation amount in step S2 is: 4 to 6 pieces of mother seeds with a diameter of 0.5 cm are inoculated into every 150 mL of liquid culture medium.

8. The use according to claim 6, characterized in that In the preparation method of the second-level species of Dictyophora rubra, the constant temperature shaking incubation in step S2 is specifically: culturing for 15 days in a constant temperature shaking incubator at 26° C. and a rotation speed of 140 r / min.

9. The use according to claim 6, characterized in that In the method for preparing the secondary strain of Dictyophora rubra, the transfer in step S3 is specifically as follows: the primary strain prepared in step S2 is inoculated into a fermentation tank filled with a boil-free liquid culture medium at a volume ratio of strain amount to culture medium of 1:

150.

10. The use according to claim 6, characterized in that The constant temperature cultivation under dark conditions in step S3 specifically includes: cultivating at 26° C. in the dark for 4 days.

Images