A method for culturing Ganoderma curvicuspis

Abstract

The present invention discloses a method for culturing wild Ganoderma curtisii. Using a puncher, 7-mm mycelial blocks are obtained by punching at the same radius on a petri dish covered with mycelia of wild Ganoderma curtisii strains, and then inoculated on a culture medium petri dish and cultured in the dark in a constant temperature incubator at 15-30 °C. Compared with the prior art, the optimal culture conditions for the wild Ganoderma curtisii of the present invention are: carbon source starch, nitrogen source yeast powder, pH 9.0, temperature 30 °C. Under such culture conditions, the wild Ganoderma curtisii grows fastest with the best growth vigor and prefers to grow in a slightly alkaline environment; it provides basic research data for the further development and utilization of Ganoderma curtisii resources and also provides basic data reference for the research on the biological characteristics and domestication cultivation of related species of this genus.

Description

Technical Field

[0001] The present invention relates to the technical field of edible mushroom cultivation, in particular to a method for cultivating wild Ganoderma flexipes. Background Art

[0002] Ganoderma flexipes was first reported as a new species by Patouillard in 1907 based on specimens collected from Tonkin, Vietnam. Scholars such as Steyeart, Ryvarden, Zhao Jiding, Zhang Xiaoqing, Wang & Wu have studied this species. Monographs such as "Illustrated Atlas of Macrofungal Resources in China" and "Illustrated Atlas of Macrofungi in Yinggeling, Hainan" have all described it. Its main morphological characteristics are that the fruiting body is annual, corky to woody; the pileus is nearly spatulate to nearly circular, small, with a yellowish red-brown to red-brown surface, having a lacquered luster, and the margin is obtuse or truncate; it has a dorsal or median stipe, curved, with the same color as the pileus on the surface; the context is light yellowish brown to brown, the shape of the cortical cells is relatively regular, and the basidiospores are elliptical with relatively fine ornamentation. It grows singly on underground rotten wood in broad-leaved forests and bamboo forests in summer and autumn, causing white rot of the wood. Usually, in the wild, Ganoderma flexipes has a relatively high similarity to young Ganoderma sinense, but the color of the pileus of Ganoderma flexipes is relatively lighter, not reaching the state of purplish red or even nearly purplish black, and usually, its pileus is also smaller when mature, with little difference from when it is young.

[0003] Currently, the domestic research and development on Ganoderma fungi mainly focus on species such as Ganoderma lingzhi and Ganoderma sinense, while Ganoderma flexipes, as one of the dominant species in tropical and subtropical regions, has relatively few reported studies. Therefore, carrying out relevant research and development on Ganoderma flexipes has important scientific significance and industrial value. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies existing in the prior art and provide a method for cultivating wild Ganoderma flexipes.

[0005] To achieve the above purpose, the present invention is implemented according to the following technical scheme:

[0006] A method for cultivating wild Ganoderma flexipes, comprising the following steps:

[0007] S1. Prepare a culture medium, and the culture medium is: 20 g of carbon source, 2 g of nitrogen source, 1.5 g of KH2PO4, 20 g of agar, 1000 mL of distilled water. The carbon source is one of starch, glucose, sucrose, lactose, fructose, mannitol, and maltose, and the nitrogen source is one of peptone, yeast powder, ammonium nitrate, sodium nitrate, ammonium chloride, urea, and potassium nitrate. The pH of the culture medium is 5 - 9;

[0008] S2. Use a punch to punch out 7-mm mycelial blocks from the same radius of a petri dish covered with the mycelium of the wild Ganoderma flexipes strain, inoculate them on the culture medium petri dish, and culture them in the dark in a constant temperature incubator at 15 - 30°C.

[0009] Preferably, the carbon source is starch.

[0010] Preferably, the nitrogen source is yeast powder.

[0011] Preferably, the pH of the culture medium is 9.

[0012] Preferably, the culture temperature is 30°C.

[0013] Preferably, in step S1, the pH of the culture medium is adjusted with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide.

[0014] Compared with the prior art, the optimal culture conditions for the wild Ganoderma flexipes of the present invention are: carbon source starch, nitrogen source yeast powder, pH 9.0, temperature 30°C. Under these culture conditions, the wild Ganoderma flexipes grows the fastest, has the best growth trend, and prefers to grow in a slightly alkaline environment; it provides basic research data for the further development and utilization of Ganoderma flexipes resources, and also provides basic data reference for the research on the biological characteristics and domestication cultivation of related species in this genus. Specific Embodiments

[0015] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further details the present invention with reference to embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0016] The wild fruiting bodies of Ganoderma flexipes were collected from Hainan Jianfengling National Nature Reserve. After tissue isolation and purification, pure culture strains were obtained, which were identified and preserved by the Herbarium of Fungal Collections, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences (FCATAS), and the strain number is FCATAS3; this strain was comprehensively identified as Ganoderma flexipes through macroscopic morphology and microscopic structure characteristics. Through ITS sequence analysis and homology comparison using Blast in Gen Bank, the maximum homology with Ganoderma flexipes in the genus Ganoderma is as high as 99.84%. Combined with macroscopic morphology and microscopic structure, it is identified as Ganoderma flexipes, and its ITS base sequence is:

[0017]

[0018] A method for culturing wild Ganoderma flexipes in this embodiment specifically includes the following steps:

[0019] S1. Prepare a culture medium, and the culture medium is: 20 g of starch, 2 g of yeast powder, 1.5 g of KH2PO4, 20 g of agar, 1000 mL of distilled water, and adjust the pH of the culture medium to 9 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide;

[0020] S2. Use a punch to punch out 7-mm mycelial blocks from the same radius of the plate covered with the mycelium of the wild Ganoderma curtisii strain, inoculate them on the culture medium plate, and culture them in the dark in a constant temperature incubator at 30 °C.

[0021] Under the cultivation with the above method and culture medium, the wild Ganoderma curtisii grows the fastest, has the best growth vigor, and prefers to grow in a slightly alkaline environment.

[0022] Furthermore, in order to verify the feasibility of the above culture medium cultivation, the following study on the biological characteristics of the mycelium was carried out.

[0023] First, determine the carbon source basal medium: 2 g of peptone, 2 g of MgSO4, 1.5 g of KH2PO4, 20 g of agar, 1000 mL of distilled water, and the pH is natural.

[0024] Nitrogen source basal medium: 20 g of glucose, 2 g of MgSO4, 1.5 g of KH2PO4, 20 g of agar, 1000 mL of distilled water, and the pH is natural.

[0025] Effect of different carbon sources on mycelial growth

[0026] Using the carbon source basal medium as the blank control, 7 carbon sources (starch, glucose, sucrose, lactose, fructose, mannitol, maltose) are respectively set as the treatment groups, and the concentration of each is 20 g / L. Use a punch to punch out 7-mm mycelial blocks from the same radius of the plate covered with the mycelium, and inoculate them on the 7 carbon source culture medium plates and culture them in the dark in a constant temperature incubator at 25 °C. Each treatment has 4 replicates. After inoculation, observe and record the mycelial growth vigor every 24 h, measure the colony diameter by the cross method until one group of mycelia fills the plate, then stop the experiment, and calculate the daily average growth rate of the mycelia.

[0027] Effect of different nitrogen sources on mycelial growth

[0028] Using the nitrogen source basal medium as the blank control, 7 nitrogen sources (peptone, yeast powder, ammonium nitrate, sodium nitrate, ammonium chloride, urea, potassium nitrate) are respectively set as the treatment groups, and the concentration of each is 2 g / L.

[0029] Effect of different pH values on mycelial growth

[0030] Adjust the pH value of the PDA medium to 5.0, 6.0, 7.0, 8.0, and 9.0 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide, and inoculate 7-mm mycelial blocks on the PDA plate medium. Inoculate 7-mm mycelial blocks on it and culture them in the dark at 25°C.

[0031] Effect of different temperatures on mycelial growth

[0032] Punch out 7-mm mycelial blocks and inoculate them on the PDA plate medium. Place them in a constant temperature incubator at 15°C, 20°C, 25°C, and 30°C respectively and culture them in the dark.

[0033] Taking the mycelial growth rate and mycelial growth vigor as evaluation indicators, select the optimal 3 groups of levels determined by the above 4 single-factor test results of carbon source, nitrogen source, pH, and temperature as the orthogonal test factors, and obtain the best combination according to the L9(34) orthogonal test without interaction. The results are as follows:

[0034] Effect of carbon source on mycelial growth

[0035] As can be seen from Table 1, the tested strains can grow on 7 carbon source media, indicating that Ganoderma curtisii has a relatively wide range of carbon source requirements. The average mycelial growth rate from fast to slow is: starch > glucose > sucrose > fructose > maltose > lactose > mannitol > blank control; among them, the mycelial growth rate on the blank control medium is the slowest and the growth vigor is the worst; on the medium with starch as the carbon source, the mycelial growth rate is the fastest, the mycelia are the densest, thickest, and the growth condition is good; considering the mycelial growth rate and growth vigor comprehensively, starch is the most suitable carbon source for the mycelial growth of Ganoderma curtisii.

[0036] Table 1

[0037]

[0038] Note: Analyzed by Duncan's multiple range test method, different letters in the same column indicate significant differences (p < 0.05, n = 3).

[0039] Lowercase letters represent comparisons at the 0.05 level with significant differences; uppercase letters represent comparisons at the 0.01 level with extremely significant differences.

[0040] “+” indicates sparse and weak mycelia; “++” indicates dense mycelia and average growth vigor; “+++” indicates dense and thick mycelia.

[0041] **: The data are the average value and standard deviation of 4 times, the same below.

[0042] Effect of nitrogen source on mycelial growth

[0043] As can be seen from Table 2, the tested strains can all grow on 7 nitrogen source media; the average mycelial growth rate from fast to slow is as follows: yeast powder > potassium nitrate > ammonium nitrate > peptone > ammonium chloride > blank > sodium nitrate > urea; when yeast powder is used as the nitrogen source, the mycelial growth rate is the fastest and the growth vigor is the best, showing a significant difference from the blank control; when sodium nitrate and urea are used as the nitrogen source, there is no significant difference from the blank control, indicating that sodium nitrate and urea have little effect on the mycelial growth rate; considering the mycelial growth rate and growth vigor comprehensively, yeast powder is the most suitable nitrogen source for the mycelial growth of Ganoderma curvicorne.

[0044] Table 2

[0045]

[0046] Effect of pH on mycelial growth

[0047] As can be seen from Table 3, Ganoderma curvicorne can grow at different pH gradients; considering the mycelial growth vigor and growth rate comprehensively, the mycelial growth of Ganoderma curvicorne is the best at pH 9.0; the mycelial growth in other pH gradient media is poor and the difference is not significant; thus, the most suitable pH for the mycelial growth of Ganoderma curvicorne is 9.0.

[0048] Table 3

[0049]

[0050] Effect of temperature on mycelial growth

[0051] As can be seen from Table 4, Ganoderma curvicorne can grow at 4 temperatures, and the average mycelial growth rate from fast to slow is as follows: 30°C > 32°C > 25°C > 20°C > 15°C; considering the mycelial growth vigor and growth rate comprehensively, the most suitable temperature for the mycelial growth of Ganoderma curvicorne is 30°C.

[0052] Table 4

[0053]

[0054]

[0055] Orthogonal experiment

[0056] The single-factor experiment did not consider the influence of the interaction between factors and its results cannot be used as the final conclusion. Therefore, an orthogonal experiment was conducted on 4 factors: carbon source, nitrogen source, pH, and temperature. Three optimal level treatments of each factor were selected from the single-factor experiment to form an orthogonal analysis table (Table 5) of 4 factors and 3 levels, in order to select the most suitable medium combination and lay a foundation for the domestication and cultivation of Ganoderma curvicorne.

[0057] Table 5

[0058]

[0059] As can be seen from Table 6, through the analysis of the mycelial growth rate of Ganoderma flexipes based on the four factors respectively, it is found that when the carbon source is starch, the nitrogen source is yeast powder, the pH is 9.0, and the temperature is 30 °C, it is most suitable for mycelial growth, which is consistent with the single-factor experiment. The order of the significant differences of the four growth factors on Ganoderma flexipes is: pH > nitrogen source > temperature > carbon source, which is consistent with the result of the intuitive analysis.

[0060] Table 6

[0061]

[0062] The results of the single-factor experiment on the growth factors of Ganoderma flexipes and the subsequent orthogonal experiment show that the optimal culture conditions for Ganoderma flexipes are: carbon source starch (20 g / L), nitrogen source yeast powder (2 g / L), pH 9.0, and temperature 30 °C. According to relevant reports, glucose is the optimal carbon source for most macrofungi in terms of carbon source selection. However, in this study, starch is the best carbon source for Ganoderma flexipes. In terms of nitrogen source utilization, yeast powder is an organic nitrogen source, and most of its nitrogen source substances exist in the form of easily absorbable protein degradation products. The degradation products, especially amino acids, can be directly absorbed by the mycelium through transamination, serving as a readily available nitrogen source and being more easily utilized by the mycelium. It is found in this invention that the mycelium of Ganoderma flexipes grows fastest and shows good growth at a pH value of 9.0, indicating that it prefers to grow in a slightly alkaline environment. This is inconsistent with the research by Sérgio Luiz Moreira Neto et al. which shows that most polypores are suitable to grow in an acidic pH environment. In this invention, the mycelium grows fastest and shows the best growth at 30 °C, with a neat colony edge and white mycelium. At 35 °C, the growth on the plate slows down, and the inoculation block does not grow at 40 °C. Based on the analysis of the collection time and climate conditions of the tested strains, Ganoderma flexipes should be a high-temperature type of fungus. The orthogonal experiment considered the interaction effects among various factors. In this experiment, the order of the significant differences of the four factors on the mycelial growth of Ganoderma flexipes is pH > nitrogen source > temperature > carbon source. pH has a greater impact on the growth quality of the mycelium compared to the other three factors. When multiple factors act together, the growth of the mycelium will tend to be influenced more by the factor with a greater impact, and it will show a certain adaptability to the factor with a smaller impact. It is recommended to first meet the optimal pH for culturing Ganoderma flexipes, followed by considering the nitrogen source, and finally the growth temperature and carbon source.

[0063] The technical solution of the present invention is not limited to the restrictions of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A method for cultivating Ganoderma curtisii, characterized in that, Culturing with a wild Ganoderma gibbosum strain includes the following steps: S1. Prepare a culture medium, which is: 20 g of carbon source, 2 g of nitrogen source, 1.5 g of KH2PO4, 20 g of agar, 1000 mL of distilled water. The carbon source is one of starch, glucose, sucrose, lactose, fructose, mannitol, and maltose. The nitrogen source is one of peptone, yeast powder, ammonium nitrate, sodium nitrate, ammonium chloride, urea, and potassium nitrate. The pH of the culture medium is 9; S2. Use a puncher to punch out 7-mm mycelium blocks from the same radius of a plate covered with the mycelium of the wild Ganoderma gibbosum strain, inoculate them on the culture medium plate, and culture them in the dark in a constant temperature incubator at 15-30°C.

2. The method for culturing wild Ganoderma curvicaule according to claim 1, characterized in that: The carbon source is starch.

3. The method for culturing wild Ganoderma curtisii according to claim 1, wherein: The nitrogen source is yeast powder.

4. The method for culturing wild Ganoderma curvicaule according to claim 1, wherein: The culture temperature is 30°C.

5. The method for cultivating wild Ganoderma curtisii according to claim 1, characterized in that: In step S1, adjust the pH of the culture medium with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide.

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