A strain of Ganoderma lucidum

Abstract

The present invention relates to a strain of Ganoderma lucidum, belonging to the field of microbial technology. The strain of Ganoderma lucidum of the present invention is classified and named as Ganoderma lucidum, and its Latin name is Ganoderma lucidum 41GL‑4; the strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on October 15, 2024, with the deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the deposit number is: CGMCC No. 41573. The Ganoderma lucidum strain of the present invention has a late formation time of the fruiting body cap, a large fruiting body, a smooth upper surface of the cap, and a slightly blunt edge. The Ganoderma lucidum strain of the present invention has a pleasant color, and the powder production cycle and powder production of the fruiting body are significantly better than those of strain 32GL‑26, and the average powder production per strain is about 1.5 times that of strain 32GL‑26.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a ganoderma lucidum strain. Background Art

[0002] Lingzhi is widely distributed in tropical and subtropical regions such as Asia, America and Africa. In my country, it is distributed in Northeast China, Guizhou, Sichuan, Yunnan and other regions. Lingzhi is a white-rot fungus of the genus Ganoderma. It is a traditional medicinal fungus that has been included in the pharmacopoeia and has multiple pharmacological activities such as regulating immune function, anti-oxidation, anti-tumor, and liver protection.

[0003] It has been widely used in my country for more than 2,000 years, but people did not start cultivating it until 1937. With people's increasing attention to the big health industry, Ganoderma lucidum was included in the catalogue of Chinese medicine and food in 2023. The development and utilization of Ganoderma lucidum and its products have received people's attention.

[0004] As one of the four major Ganoderma lucidum producing areas in China, Changbai Mountain is the only area that maintains the traditional log-covered soil cultivation model. This cultivation model has high requirements on strain resistance and yield. At present, the main Ganoderma lucidum varieties cultivated in the domestic market are only those numbered 32GL-26, 22GL-23, 11GL-16, 22GL-9 and 41GL-14. Therefore, it is urgent to develop a new strain of high-quality Ganoderma lucidum suitable for the Changbai Mountain area. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a new Ganoderma lucidum strain. The Ganoderma lucidum strain of the present invention has large fruiting bodies, pure color and shape, bitter taste, and high Ganoderma lucidum spore powder yield, which will be more favored by Ganoderma lucidum growers.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A Ganoderma lucidum strain, the strain is classified and named Ganoderma lucidum, and its Latin name is Ganoderma lucidum41GL-4; the strain was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on October 15, 2024, with the deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the deposit number is: CGMCC No. 41573.

[0008] The beneficial effects of the present invention are:

[0009] The ganoderma strain of the present invention is classified and named as ganoderma lucidum, and its Latin name is Ganoderma lucidum 41GL-4. It has the characteristics of late fruiting body cap formation time, large fruiting body, smooth upper surface of cap, slightly blunt edge, irregular cap shape (often multiple kidney shapes combined together), thick cap, long stipe, bitter fruiting body, high yield of spore powder and fruiting body.

[0010] The Ganoderma lucidum strain of the present invention has a pleasant color (orange-red to reddish brown cap, purple-brown stipe), and its fruiting body has a significantly better powder production cycle and powder production than the strain 32GL-26, with an average single-strain powder production of about 1.5 times that of the strain 32GL-26. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0012] Figure 1 This is a plate confrontation picture of the strain 41GL-4 of the present invention and the existing typical strains of Ganoderma lucidum 41GL-14 and 22GL-9.

[0013] Figure 2 This is a plate confrontation picture of the strain 41GL-4 of the present invention and the existing typical Ganoderma lucidum strains 11GL-16 and 22GL-23.

[0014] Figure 3 This is a plate confrontation picture of the strain 41GL-4 of the present invention and the existing typical Ganoderma lucidum strains 32GL-26 and 22GL-23.

[0015] Figure 4 This is a graph showing the results of UPGMA cluster analysis based on SRAP between the strain 41GL-4 of the present invention and existing typical strains of Ganoderma lucidum. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0017] Example 1 Sequence Identification of the Strain 41GL-4 of the Present Invention

[0018] The sequence identification of strain 41GL-4 of the present invention was entrusted to Beijing Huada Gene Technology Co., Ltd., and included the following process: Extraction of the genome of strain 41GL-4: Extraction was performed according to the instructions of the fungal genomic DNA extraction kit (M5 FungalGenomic DNA Kit) of Beijing Polymer Biotechnology Co., Ltd. The obtained DNA solution was stored at -20°C for future use.

[0019] The PCR amplification system was as follows: total volume 25 μL, including: genomic template 7 μL, ITS primer ITS1 0.5 μL, ITS4 0.5 μL, 2×M5 Hiper plus Taq HiFi PCR mix 10 μL, and ddH2O to 25 μL.

[0020] PCR program: 94°C for 5 min; 94°C for 30 s; 54°C for 30 s; 72°C for 30 s; 30 cycles of the above temperature and time; and finally 72°C for 5 min.

[0021] The sequence of the ITS primer ITS1 is 5'-TCCGTAGGTGAACCTGCGG-3';

[0022] The sequence of the ITS4 is 5'-TCCTCCGCTTATTGATATGC-3';

[0023] The identification sequence results of the strain 41GL-4 of the present invention are as follows:

[0024] .

[0025] The PCR amplification system was as follows: total volume 25 μL, including: genomic template 7 μL, tef1-α primer 983F 0.5 μL, 1567R 0.5 μL, 2×M5 Hiper plus Taq HiFi PCR mix 10 μL, and ddH2O to 25 μL.

[0026] The PCR program was: 94°C for 3 min; 94°C for 30 s; 49°C for 35 s; 72°C for 30 s; 30 cycles of the aforementioned temperature and time; and finally 72°C for 4 min.

[0027] The sequence of the tef1-α primer 983F is: 5'-GCYCCYGGHCAYCGTGAYTTYAT-3';

[0028] The sequence of 1567R is: 5'-ACHGTRCCRATACCACCSATCTT-3';

[0029] The identification sequence results of the strain 41GL-4 of the present invention are as follows:

[0030] .

[0031] The PCR amplification system was as follows: total volume 25 μL, including: genomic template 7 μL, RPB2 primer 6F 0.5 μL, 7CR 0.5 μL, 2×M5 Hiper plus Taq HiFi PCR mix 10 μL, and ddH2O to 25 μL.

[0032] PCR program: 93°C for 2 min; 93°C for 1 min; 53°C for 1 min; 72°C for 1 min; 33 cycles of the above temperature and time; and finally 72°C for 10 min.

[0033] The sequence of the RPB2 primer 6F is 5'-TGGGGYATGGTNTGYCCYGC-3';

[0034] The sequence of the 7CR is 5'-CCCATRGCTTGYTTRCCCAT-3';

[0035] The identification sequence results of the strain 41GL-4 of the present invention are as follows:

[0036] .

[0037] The PCR amplification system was as follows: total volume 25 μL, including: genomic template 7 μL, LSU primer LROR 0.5 μL, LR5 0.5 μL, 2×M5 Hiper plus Taq HiFi PCR mix 10 μL, and ddH2O to 25 μL.

[0038] PCR program: 94°C for 3 min; 94°C for 30 s; 54°C for 30 s; 72°C for 90 s; 32 cycles of the above temperature and time; and finally 72°C for 8 min.

[0039] The sequence of the LSU primer LROR is 5'-ACCCGCTGAACTTAAGC-3';

[0040] The sequence of the LR5 is 5'-ATCCTGAGGGAAACTTC-3';

[0041] The identification sequence results of the strain 41GL-4 of the present invention are as follows:

[0042] .

[0043] In summary, the results of DNA sequencing using ITS primer, tef1 primer, RPB2 primer and LSU primer respectively indicate that the strain 41GL-4 of the present invention is a Ganoderma lucidum strain.

[0044] Example 2 Biological characteristics of strain 41GL-4 of the present invention

[0045] The present invention conducted a plate culture antagonism test on strain 41GL-4 and a control group (the control group selected five main cultivated Ganoderma lucidum strains in Northeast China, numbered as 32GL-26, 22GL-23, 11GL-16, 22GL-9, and 41GL-14, respectively) (the antagonism test process is described in the subsequent content). Through confrontation culture with the five control strains, it was found that the strain 41GL-4 of the present invention can produce different degrees of antagonism, among which it produced an obvious growth mutual inhibition zone with strain 22GL-23, and produced obvious raised antagonism lines with strains 32GL-26, 11GL-16, 22GL-9, and 41GL-14, respectively. Therefore, through the plate antagonism test with the typical strains of the control group, it was found that the strain 41GL-4 of the present invention can produce different degrees of antagonism. Through the genetic characteristics of the hyphae, it can be preliminarily judged that the 41GL-4 strain has an obvious different genetic relationship with other strains. See the plate confrontation diagram. Figure 1 , 2 、3.

[0046] Compared with the existing Ganoderma lucidum strains, the fruiting body phenotypic characteristics of the strain 41GL-4 of the present invention are significantly different, as follows (the following data are all based on Mongolian oak bundles with a diameter of 20 cm and a height of 11-13 cm):

[0047] 1. Morphological characteristics

[0048] Mycelium: White, dense, with well-developed aerial mycelium, slightly yellowish when exposed to light;

[0049] Cap: 19.88±2.37 cm in diameter, smooth upper surface, orange-red to reddish brown, with distinct stratification, no radial longitudinal ridges, and few annular patterns;

[0050] Pore: pale yellow to white;

[0051] The spores are dark brown in color;

[0052] Stipe: 8.36±0.94 cm in length, purple-brown in color with lacquer-like luster, mesophotic;

[0053] The effective stem weight ratio is over 95%.

[0054] 2. Biological characteristics

[0055] Sources of nutrients: sawdust, bran, glucose, etc., of which the water content is 60%.

[0056] Cultivation and cultivation conditions: The mycelium culture temperature is 25-30℃, the humidity is 60%, and the CO2 concentration is 300-600ppm. The culture period is 50-65 days. The suitable temperature for growing mushrooms is 26-28℃, the humidity is 85-90%, the CO2 concentration is 300-600ppm, the light intensity is 300-1000 Lx, and the scattered light stimulates the growth of mushrooms. The growth period is 21-26 days.

[0057] Bacterial age test:

[0058] This section summarizes the advantages and characteristics of the Ganoderma lucidum strain 41GL-4 of the present invention by comparing the cultivation cycle and yield per unit area of ​​the Ganoderma lucidum strain 41GL-4 of the present invention with the existing Ganoderma lucidum strain 11GL-16.

[0059] The culture medium is:

[0060] 78% sawdust, 20% bran, 1% lime, 1% glucose, pH adjusted to neutral (all percentages are by mass).

[0061] The planting method is:

[0062] A high-pressure folding bag was used, with a folding diameter of 36 cm and a bag length of 46 cm. Short wood segments (Mongolian oak wood segments, length 11-13 cm) were bundled into wood segments with a diameter of 20 cm and placed in them. Sterilize with a normal pressure steam autoclave at 100℃ for 12 h, wait for the fungus segments to cool to 18℃~23℃, and then inoculate. After inoculation, move to the fungus culture room. The fungus culture environment is 20~25℃, 60% relative humidity, 3000 mg / L~3500 mg / L, and culture in the dark for 40~60 days. After the fungus culture is completed, move to the Zhizhi shed for Zhizhi management. Zhizhi covering management is carried out in Jilin Province around May 15 (average daily temperature of about 10℃). The fruiting body disappears during the white growth period at the edge of the cap and is harvested when the powder production is basically over. See Table 1 for the test results.

[0063] Table 1 41GL-4 and 11GL-16 bacterial age test

[0064]

[0065] By comparing with strain 11GL-16, it can be seen that the average bag yield of strain 41GL-4 is higher than that of strain 11GL-16, and the cultivation cycle is not much different, which shows that the strain 41GL-4 of the present invention has an advantage in yield.

[0066] Morphological characteristics and biological characteristics:

[0067] Morphological characteristics of strain 41GL-4:

[0068] Mycelium: white, dense, with well-developed aerial mycelium; Cap: diameter 19.88±2.37 cm, smooth upper surface, no radial longitudinal ridges, few annular patterns, slightly blunt edges. Cap shape irregular (often multiple kidney-shaped combined together), thick cap. Mushroom flesh: obvious stratification, white with dark brown; spore color is dark brown; Stem: length 8.36±0.94 cm, color purple brown with lacquer-like luster, mesogenous; effective stem weight ratio is more than 95%. Please refer to Table 2 for the specific morphological differences between strains 41GL-4 and 11GL-16.

[0069] Table 2 Comparison of morphological characteristics between 41GL-4 and 11GL-16

[0070]

[0071] Biological characteristics of strain 41GL-4:

[0072] Sources of nutrients: sawdust, bran, etc., of which the water content is 60%.

[0073] Cultivation and cultivation conditions: The mycelium culture temperature is 20-25℃, the humidity is 60%, and the CO2 concentration is 3000 mg / L-3500 mg / L. The culture period is 50-65 days. The suitable temperature for mycelium growth is 26-28℃, the humidity is 85-90%, and the light intensity is 300-1000 Lx. Light stimulates mycelium growth, and the growth time is about 15-20 days after the mycelium is covered with soil (depending on the physiological maturity of the mycelium).

[0074] Antagonism test:

[0075] In this test example, the Ganoderma lucidum strain 41GL-4 of the present invention and five control strains 32GL-26, 22GL-23, 11GL-16, 22GL-9, and 41GL-14 were used as test materials, and the culture medium used was PDA culture medium. 41GL-4 and 32GL-26, 22GL-23, 11GL-16, 22GL-9, and 41GL-14 were inoculated and cultured on a flat plate culture medium, respectively, with the inoculation points 3 cm apart, and cultured at a constant temperature of 25°C for 7 days.

[0076] The results of this experiment refer to Table 3. On the front and back sides of the junction of the confrontation cultures, 41GL-4 and 32GL-26, 41GL-4 and 22GL-23, 41GL-4 and 11GL-16, 41GL-4 and 22GL-9, 41GL-4 and 41GL-14 have antagonistic lines. It is preliminarily identified that 41GL-4 and 32GL-26, 22GL-23, 11GL-16, 22GL-9 and 41GL-14 are different new strains of Ganoderma lucidum.

[0077] Table 3 Antagonism experiment between different Ganoderma lucidum strains

[0078]

[0079] Note: + indicates antagonism

[0080] Cultivation comparison test:

[0081] This section statistically analyzed the uniformity of single-segment yield, cap diameter and stipe length of the two strains 41GL-4 and 11GL-16.

[0082] The culture medium used was: oak wood segments with a diameter of 20 cm and a water content of 40-45% (all percentages are mass percentages). 41GL-4 and 11GL-16 with a culture period of 126 days were used as statistical objects, and 10 cultivation segments of different strains were randomly selected as samples. The statistical results are shown in Table 4-6. Among them, the coefficient of variation (%) = standard deviation / mean value × 100% (the same below).

[0083] Table 4 Yield uniformity of two Ganoderma lucidum strains

[0084]

[0085] According to Table 4, the yield variation coefficient of 41GL-4 is 20.56%, and the yield variation coefficient of 11GL-16 is 23.42%, indicating that the relative variation of the yield of 41GL-4 is smaller, indicating that the yield of 41GL-4 is more uniform.

[0086] Table 5 Uniformity of cap size of different Ganoderma lucidum fruiting bodies

[0087]

[0088] According to Table 5, the average cap diameter of 41GL-4 is 19.88 cm, the average cap diameter of 11GL-16 is 19.72 cm, the coefficient of variation of the cap diameter of 41GL-4 is 11.92%, and the coefficient of variation of the cap diameter of 11GL-16 is 12.68%. It can be seen that the relative coefficient of variation of the cap diameter of 41GL-4 is smaller, indicating that the cap diameter of 41GL-4 is more uniform.

[0089] Table 6 Uniformity of stipe length of different Ganoderma lucidum fruiting bodies

[0090]

[0091] According to Table 6, the average stipe length of 41GL-4 is 8.36 cm, the average stipe length of 11GL-16 is 6.77 cm, the coefficient of variation of stipe length of 41GL-4 is 11.24%, and the coefficient of variation of stipe length of 11GL-16 is 15.21%. It can be seen that the relative coefficient of variation of stipe length of 41GL-4 is smaller, indicating that the stipe length of 41GL-4 is more uniform.

[0092] Example 3 Genetic characteristics of strain 41GL-4 of the present invention

[0093] In order to determine the relationship between the strain 41GL-4 of the present invention and the mainstream Ganoderma lucidum strains, the genetic characteristics of the Ganoderma lucidum strain 41GL-4 of the present invention were further identified by SRAP analysis technology. The PCR amplification system is: a total volume of 20 μL, including: 1 μL of genomic DNA, 1 μL of upstream primers and downstream primers, 8.5 μL of 2×Es Taq MasterMix, and ddH2O is supplemented to 20 μL. The PCR amplification program is: 95℃ 5 min; 95℃ 30 s; 35℃ 1 min; 72℃ 1 min; 5 cycles according to the above temperature and time; then 95℃ 30 s; 50℃ 1 min; 72℃ 1 min; 35 cycles according to the above temperature and time; and finally 72℃ 10 min. After the PCR is completed, the PCR product is detected on a 1.0% agarose gel (voltage 120 V, electrophoresis 40 min).

[0094] The sequences of the 26 random primers used in the present invention are shown in Table 7. In the SRAP analysis, three pairs of random primers were selected, which were numbered me2 and em2; me2 and em6; me4 and em1, respectively. These three primer pairs can distinguish the strain 41GL-4 of the present invention from the control strain used.

[0095] Table 7 Random primers

[0096]

[0097] The UPGMA cluster analysis based on SRAP of the strain 41GL-4 of the present invention and the existing typical strains of Ganoderma lucidum 22GL-9, 41GL-14, 11GL-16, 32GL-26, and 22GL-23, the cluster analysis results corresponding to the upstream primers me1, me2, me3, and me4, and the combined downstream primers me1-me17 are shown in FIG. Figure 4 As can be seen from the figure, the strain 41GL-4 of the present invention is a separate cluster, so the above primers can distinguish the strain 41GL-4 of the present invention from the existing 5 control strains.

[0098] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A kind of Ganoderma lucidum ( Ganoderma lucidum ) 41GL-4 strain, characterized in that The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on October 15, 2024, with the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the deposit number is: CGMCC No. 41573.

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