Ganoderma sinensis new strain Guizhi No.3 and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a new Ganoderma sinensis strain Guizhi No.3 and application thereof. The preservation number of the Ganoderma sinensis strain Guizhi No.3 is CGMCC NO.22452, the Ganoderma sinensis strain is screened and domesticated from a wild environment by a research group, various test results show that the strain is greatly different from the existing similar variety in mycelial growth and sporocarp appearance, and the determination of inclusions also shows that the content of flavones and total phenols in sporocarp is higher than that of the existing variety, and the content of flavones and total phenols in sporocarp is higher than that of sporocarp in sporocarp appearance. From the aspect of enzyme activity, laccase, hemicellulase, filter paper enzyme and cellulase activities of the strain are all higher than those of an existing variety, lignin and cellulose in a nutrition bag can be effectively decomposed, the utilization capacity of a cellulose culture medium is effectively improved, disease resistance experiments show that the Guizhi No.3 has wide disease resistance, and the disease resistance of the Guizhi No.3 is greatly improved. The Ganoderma sinensis variety has the advantages that the disease resistance is obviously better than that of the variety in the market, the commercial value of Ganoderma sinensis can be greatly improved, and meanwhile, richer germplasm resources can be provided for Ganoderma sinensis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a new Ganoderma lucidum strain, Guizhi No. 3, and applications thereof. Background Art

[0002] Ganoderma lucidum is a higher fungus that can be used as both medicine and food. Its main active ingredients are polysaccharides, triterpenes, flavonoids, selenium, and sterols. It has benefits such as nourishing qi and calming the mind, relieving cough and asthma, alleviating fatigue, beautifying the skin, and delaying aging. It has varying degrees of efficacy in treating conditions such as chronic bronchitis, coronary heart disease, hepatitis, hyperlipidemia, neurasthenia, tumors, leukopenia, and dermatomyositis. Wild resources of Ganoderma lucidum are scarce. To improve its cultivation, it is necessary to identify new strains of Ganoderma lucidum from provenance sources.

[0003] In the existing technology, Ganoderma lucidum is a wood-dwelling edible fungus with a strong ability to secrete laccase and cellulase, which helps to improve the decomposition of lignin and cellulose. If these enzymes can be extracted and applied, the application of wood-based culture media in the existing technology will be greatly expanded.

[0004] With the continuous expansion of cultivation and production scale, the incidence of Ganoderma diseases has also shown an increasing trend year by year. Among them, Penicillium pathogens ( Penicillium spp. )、Trichoderma viride pathogen ( Trichoderma spp .)、Yellow rot pathogens ( Xylogone ganodermophthora ), Neurospora causative agent ( Neurospora spp .)、Brown rot pathogens ( Hypomyces perniciosus ) and spiderweb disease pathogens ( Cladobotryum spp .). etc. often occur in Ganoderma lucidum production. In the existing technology, in order to effectively improve the disease resistance of Ganoderma lucidum, it is essential to screen high-quality germplasm resources. Therefore, in order to strengthen the utilization of Ganoderma lucidum germplasm resources and develop more and better germplasm resources, it is necessary to continuously optimize Ganoderma lucidum germplasm resources and cultivate more commercial Ganoderma lucidum varieties. Summary of the Invention

[0005] In view of the above, the purpose of the present invention is to provide a new strain of Ganoderma lucidum: Guizhi No. 3. This variety was selected and domesticated by the applicant from the wild. Compared with other existing Ganoderma lucidum varieties, this variety has a better appearance, better quality, and higher ability to produce laccase, filter paper enzyme, cellulase and hemicellulase. In addition, it is resistant to most pathogens of Ganoderma lucidum, and its ability to utilize wood culture medium is better than other varieties, which can provide more excellent germplasm resources for Ganoderma lucidum.

[0006] In order to achieve the above object, the present invention screened out a Ganoderma lucidum ( Ganoderma lucidum) strain Gui Zhi No. 3, the preservation number of which is CGMCC NO. 22452. It belongs to edible fungi and belongs to Ganoderma lucidum Ganoderma lucidum ) The Ganoderma lucidum is preserved in the China General Microbiological Culture Collection Center, the address of which is No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, the preservation number of which is CGMCC NO. 22452, and the preservation date of which is June 28, 2021.

[0007] The application further includes the Ganoderma lucidum Ganoderma lucidum ) strain Gui Zhi No. 3 as a parent in hybrid breeding.

[0008] The application further includes the Ganoderma lucidum Ganoderma lucidum ) strain Gui Zhi No. 3.

[0009] The application further includes the Ganoderma lucidum Ganoderma lucidum ) strain Gui Zhi No. 3.

[0010] The application further includes the Ganoderma lucidum Ganoderma lucidum ) strain Gui Zhi No. 3.

[0011] The application further includes a breeding method for improving the disease resistance of the Ganoderma lucidum Ganoderma lucidum ) strain Gui Zhi No. 3 as a parent for hybrid breeding. Ganoderma lucidum

[0012] Further, the disease-resistant pathogenic bacteria are Penicillium pathogenic bacteria Penicillium spp. , Trichoderma viride pathogenic bacteria Trichoderma spp , Neurospora pathogenic bacteria Neurospora spp , brown rot pathogenic bacteria Hypomyces perniciosus and / or orbicular pathogenic bacteria Cladobotryum spp .

[0013] The application further includes an application of the mycelium in extracting laccase, hemicellulase, filter paper enzyme and cellulase.

[0014] The application has the following beneficial effects: 1. The Ganoderma lucidum Ganoderma lucidum ​) strain Guizhi No. 3 was screened and domesticated by the research team from the wild environment. The test results show that: the mycelium growth and fruiting body appearance of this strain are quite different from those of existing similar varieties. The determination of the contents also found that the flavonoids and total phenol content of the fruiting body are higher than those of the existing Ganoderma lucidum varieties. From the enzyme activity point of view: the laccase, hemicellulase, filter paper enzyme and cellulase activities of the above strain are greater than those of the existing Ganoderma lucidum varieties, which can effectively decompose the lignin and cellulose in the nutrient bags and effectively improve the utilization capacity of cellulose culture medium. After disease resistance experiments, Guizhi No. 3 has a wide range of disease resistance and is very effective against Penicillium pathogens ( Penicillium spp. )、Trichoderma viride pathogen ( Trichoderma spp. ), Neurospora spp. pathogens ( Neurospora spp .)、Brown rot pathogens( Hypomyces perniciosus ) and spiderweb disease pathogens ( Cladobotryum spp. ), and its disease resistance is significantly better than that of the varieties on the market. The discovery of this variety can greatly enhance the commercial value of Ganoderma lucidum, and at the same time, it can also provide more abundant germplasm resources for Ganoderma lucidum. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the results of the antagonistic experiment between Guizhi No. 3 and similar varieties; in the figure: the upper one is Guizhi No. 3, and the lower one is Wuzhi No. 2.

[0016] Figure 2 This is a comparison of the thickness of the mycelial bundles of Guizhi No. 3 strain and Wuzhi No. 2 strain.

[0017] Figure 3 This is a comparison of the colony density morphology of Guizhi No. 3 strain and Wuzhi No. 2 strain.

[0018] Figure 4 This is a comparison of the colony pigments of Guizhi No. 3 and Wuzhi No. 2 strains.

[0019] Figure 5 This is a picture of the upper surface of the cap of the fruiting body of Guizhi No. 3 strain.

[0020] Figure 6 This is the lower surface of the cap of the fruiting body of the control variety Wuzhi No. 2 strain.

[0021] Figure 7 Microscopic images of basidiospores of Guizhi No. 3 and Wuzhi No. 2.

[0022] Figure 8 These are pictures of the mushroom bags of Guizhi No. 3 and Wuzhi No. 2.

[0023] Figure 9 This is a cross-sectional view of the fruiting bodies of Guizhi No. 3 and Wuzhi No. 2. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the accompanying drawings, examples and tests. Example 1

[0025] This example screened out a Ganoderma lucidum ( Ganoderma lucidum ) strain Guizhi No. 3, its preservation number is CGMCC NO.22452. It is an edible fungus and belongs to Ganoderma lucidum ( Ganoderma lucidum ) species. The Ganoderma lucidum is deposited at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO. 22452, and the deposit date is June 28, 2021.

[0026] The above-mentioned Ganoderma lucidum ( Ganoderma lucidum ) strain Guizhi No. 3 was isolated, domesticated and cultivated by the applicant from wild Ganoderma lucidum in Danzhou Town, Sanjiang County, Liuzhou City. The antagonism diagram of this strain with other varieties of Ganoderma lucidum is shown in Figure 1 Among them, the one on the left is Guizhi No. 3, and the one on the right is Wuzhi No. 2; from the antagonism diagram, there is an obvious antagonism line between Guizhi No. 3 and similar varieties, and they can be determined to be different varieties.

[0027] Comparison of fruiting body morphology between Guizhi No. 3 and Wuzhi No. 2 can be found in Figure 5 、 Figure 6 、 Figure 8 and Figure 9 :in Figure 5 This is the upper surface of the cap of the fruiting body of Guizhi No. 3 strain. Figure 6 The lower surface of the fruiting body cap of the control strain Wuzhi No. 2 is shown. Figure 8 The mushroom bags of Guizhi No. 3 and Wuzhi No. 2 are shown. Figure 9 This is a cross-sectional view of the fruiting bodies of Guizhi No. 3 and Wuzhi No. 2; it can be seen from the figure that the fruiting body cap of Guizhi No. 3 is formed early, the stipe is short, the stipe is flat cylindrical, the cap is thick, the cap is small to medium in size, the cap transverse diameter / stipe length is large, the cap thickness / cap transverse diameter is large, the cap is fan-shaped, the upper surface of the fruiting body is purple-black, the lower surface is gray, there are radial longitudinal ridges on the upper surface of the cap, there are many ring marks on the upper surface of the cap, the edge of the cap is round and blunt, the flesh is obviously layered, the flesh is brown, the stipe is hard, and the cap is hard.

[0028] from Figure 5 and Figure 6 It can be seen that although the upper surfaces of the caps of Guizhi No. 3 and Wuzhi No. 2 are both purple-black, the color of Guizhi No. 3 is lighter and tends to be purple, while the color of the upper surface of Wuzhi No. 2 is darker and tends to be black; although the lower surfaces are both gray, the color of Guizhi No. 3 is darker and tends to be brown, while the color of the lower surface of Wuzhi No. 2 is lighter and tends to be white. Although there are annular lines on the upper surfaces of Guizhi No. 3 and Wuzhi No. 2, from a morphological point of view, the annular lines of Guizhi No. 3 are more obvious and regular, and the appearance is more beautiful. Example 2

[0029] This example is a method for screening the Ganoderma lucidum ( Ganoderma lucidum ) Morphological identification of strain Guizhi No. 3. Comparative identification test of fruiting body traits: Cultivation and production of mushrooms: (1) Preparation of original seed: Transfer the PDA slant culture to sawdust culture medium and culture at 25℃ for use. (2) Preparation of mushroom bags: Fill each bag with 1 / 2 to 2 / 3 of the volume of wood culture medium, sterilize at 0.14 MPa to 0.15 MPa (temperature 126℃ to 127℃) for 2.5 hours, and cool for use. (3) Inoculation: Transfer the original seed to the mushroom bag, and fill 10 bags for each number. After the mycelium fills the bag for 3 to 7 days, transfer it to the culture rack for ventilation management. The temperature during the spawning period is set at 20℃ to 30℃, and the temperature during the mushroom production period is set at 24℃ to 26℃. The entire production process is routinely managed, and the entire cultivation experiment lasts for 5 months.

[0030] Comparison of fruiting body agronomic traits: This was performed in accordance with the industry standard "Guidelines for Specificity, Consistency, and Stability Testing of New Plant Varieties (Ganoderma lucidum)". All data were calculated and averaged. Specific traits are shown in Table 1: .

[0031] As shown in Table 1, the mycelium growth rate of Guizhi No. 3 at 20-30℃ is significantly higher than that of the similar variety Wuzhi No. 2.

[0032] Comparison of fruiting body morphology between Guizhi No. 3 and Wuzhi No. 2 can be found in Figure 5 、 Figure 6 、 Figure 8 and Figure 9 :in Figure 5 This is the upper surface of the cap of the fruiting body of Guizhi No. 3 strain. Figure 6 The lower surface of the fruiting body cap of the control strain Wuzhi No. 2 is shown. Figure 8 The mushroom bags of Guizhi No. 3 and Wuzhi No. 2 are shown. Figure 9 This is a cross-sectional view of the fruiting bodies of Guizhi No. 3 and Wuzhi No. 2; it can be seen from the figure that the fruiting body cap of Guizhi No. 3 is formed early, the stipe is short, the stipe is flat cylindrical, the cap is thick, the cap is small to medium in size, the cap transverse diameter / stipe length is large, the cap thickness / cap transverse diameter is large, the cap is fan-shaped, the upper surface of the fruiting body is purple-black, the lower surface is gray, there are radial longitudinal ridges on the upper surface of the cap, there are many ring marks on the upper surface of the cap, the edge of the cap is rounded and blunt, the flesh is obviously layered, the flesh is brown, the stipe is hard, the cap is hard, the spores are oval, and the spore length / width is large.

[0033] from Figure 5 and Figure 6It can be seen that although the upper surfaces of both Gui Zhi No. 3 and Wu Zhi No. 2 are purple black, the color of Gui Zhi No. 3 is lighter and more purple, while the upper surface color of Wu Zhi No. 2 is darker and more black. Although the lower surfaces of both are gray, the color of Gui Zhi No. 3 is darker and more brown, while the lower surface color of Wu Zhi No. 2 is lighter and more white. Although both the upper surfaces of Gui Zhi No. 3 and Wu Zhi No. 2 have annular lines, the annular lines of Gui Zhi No. 3 are more obvious and regular in morphology, and the appearance is more beautiful. Example 3

[0034] Analysis of the differences in the contents of new strain Gui Zhi No. 3 and similar variety Wu Zhi No. 2: 1. Nutritional ingredients:

[0035] Under the same conditions, the cultivation of Gui Zhi No. 3 and similar variety Wu Zhi No. 2 was carried out. After the fruiting, the same batch of mature fruiting bodies was collected. According to the relevant detection standards of nutritional ingredients, the contents of protein, polysaccharide and total amino acids of Ganoderma lucidum fruiting bodies were detected. The specific analysis results are shown in Table 2: .

[0036] Note: The same row data with the same lowercase letter or no letter indicates that the difference is not significant ( P>0.05 ), and the lowercase letters are different, indicating that the difference is significant ( P<0.05 ).

[0037] As can be seen from Table 2, through detection and analysis, the total flavone and total phenol contents of new strain Gui Zhi No. 3 were significantly higher than those of similar variety Wu Zhi No. 2 ( P<0.05 ), and the polysaccharide, triterpenes and similar variety Wu Zhi No. 2 had no significant difference ( P<0.05 ), and both varieties did not contain selenium.

[0038] 2. Extracellular enzyme activity: The applicant also determined the mycelial extracellular enzyme activity of new strain Gui Zhi No. 3 and similar variety Wu Zhi No. 2 Ganoderma. The specific contents are as follows: (1) Preparation of crude enzyme solution: after the mycelium of Ganoderma lucidum was fully grown in the bag, 20 bags of each strain were taken. The upper 5 cm substrate was frozen, dried, and then crushed at low temperature and passed through a 20 mesh sieve. After mixing evenly, an appropriate amount of substrate powder was taken, and distilled water or extract was added at a mass-volume ratio of 1:10. After immersion in a constant temperature water bath and centrifugation, the supernatant obtained was the crude enzyme solution.

[0039] (2) Enzyme activity determination: ABTS method was used to determine the laccase activity: the amount of enzyme required to oxidize 1 μmol of substrate ABTS per milligram of protein per minute was defined as one enzyme activity unit; 3, 5-dinitrosalicylic acid (DNS) colorimetric method was used to determine the cellulase, hemicellulase and filter paper enzyme activity: the amount of enzyme required to catalyze 1 mg of glucose per milligram of protein per minute was defined as one enzyme activity unit, respectively.

[0040]

[0041] Note: The same lowercase letters or no letters in the superscript indicate no significant difference P>0.05 , different lowercase letters in the superscript indicate significant difference P<0.05 .

[0042] From Table 3, the laccase, hemicellulase, filter paper enzyme and cellulase activities of Gui Zhi No. 3 are significantly greater than those of the similar varieties, indicating that the new strain Gui Zhi No. 3 of the application has better decomposition ability for lignin and cellulose. Example 4

[0043] Disease resistance study of the new strain Gui Zhi No. 3 and the similar varieties: 1. Plate test: the test Ganoderma lucidum strains were respectively subjected to plate confrontation test with Penicillium pathogenic bacteria (P. Penicillium spp. ), Trichoderma viride pathogenic bacteria (T. Trichoderma spp .), yellow rot pathogenic bacteria (P. Xylogone ganodermophthora ), Neurospora pathogenic bacteria (N. Neurospora spp .), brown rot pathogenic bacteria (P. Hypomyces perniciosus ) and cobweb pathogenic bacteria (P. Cladobotryum spp ).

[0044] 2. Resistance determination: the resistance of the Ganoderma lucidum strains to the pathogenic bacteria was determined by "+" and "-". "-" indicates no resistance, and the mycelium of the pathogenic bacteria can cover the mycelium of the Ganoderma lucidum strain; "+" indicates resistance, and the mycelium of the Ganoderma lucidum strain and the mycelium of the pathogenic bacteria grow in confrontation; "+-" indicates weak resistance, and the mycelium of the Ganoderma lucidum strain and the mycelium of the pathogenic bacteria grow in confrontation, and part of the mycelium of the pathogenic bacteria can invade the Ganoderma lucidum strain.

[0045] The results of the confrontation test are shown in Table 4: .

[0046] From Table 4, Gui Zhi No. 3 has resistance to Penicillium pathogenic bacteria (P. Penicillium spp. ), Trichoderma viride pathogenic bacteria (T. Trichoderma spp .), Neurospora pathogenic bacteria (N. Neurospora spp .), brown rot pathogenic bacteria (P. Hypomyces perniciosus ) and cobweb pathogenic bacteria (P. Cladobotryum spp .), but has no resistance to yellow rot pathogenic bacteria (P. Xylogone ganodermophthora ), and Wu Zhi No. 2 has resistance only to Trichoderma viride pathogenic bacteria (T. Trichoderma spp.) and yellow rot pathogens ( Xylogone ganodermophthora ) has disease resistance, indicating that Guizhi No. 3 has better disease resistance than the existing strains of Ganoderma lucidum. Example 5

[0047] The above-mentioned Ganoderma lucidum strain Guizhi No. 3 can be used as a parent to hybridize with Ganoderma lucidum strains of other parents to obtain Ganoderma lucidum germplasm resources with richer colors and more special shapes.

[0048] In summary, the Ganoderma lucidum strain Guizhi No. 3 of the present application was screened and domesticated by the applicant from the wild environment. It was verified that: the mycelial bundles of the strain are finer, the mycelium is dense, the colony morphology is fluffy, the colony has pigment, the mycelium grows faster at 20-30°C, the primordium is formed early, the fruiting body cap is formed early, the stipe is short, the stipe is flat cylindrical, the cap is thick, the cap size is small to medium, the cap transverse diameter / stipe length is large, the cap thickness / cap transverse diameter is large, the cap is fan-shaped, the upper surface of the fruiting body is purple-black, purple-ish, the lower surface is gray-brown, and the upper surface of the cap has radial The longitudinal ridges, the upper surface of the cap has many regular ring patterns, the edge of the cap is round and blunt, the mushroom flesh is obviously layered, the color of the mushroom flesh is brown, the texture of the stipe is hard, the texture of the cap is hard, the spores are oval, and the spore length / width is large. Compared with the internal substances: the content of flavonoids and total phenols in the fruiting body is higher than that of the Ganoderma lucidum varieties in the prior art. From the perspective of enzyme activity: the laccase, hemicellulase, filter paper enzyme and cellulase activities of the above strains are all greater than those of the existing Ganoderma lucidum varieties, indicating that the new strain Guizhi No. 3 of the present application has better decomposition ability for lignin and cellulose. After disease resistance experiments, Guizhi No. 3 has a wide range of disease resistance and is resistant to Penicillium pathogens ( Penicillium spp. )、Trichoderma viride pathogen ( Trichoderma spp. ), Neurospora causative agent ( Neurospora spp .)、Brown rot pathogens( Hypomyces perniciosus ) and spiderweb disease pathogens ( Cladobotryum spp. ) are resistant to all pathogens and are a Ganoderma lucidum strain with excellent performance.

[0049] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. Ganoderma lucidum Ganoderma lucidum ) strain Guizhi No. 3, the preservation number is CGMCC NO.22452.

2. The Ganoderma lucidum as claimed in claim 1 Ganoderma lucidum ) Application of strain Guizhi No. 3 as a parent in hybrid breeding.

3. by culturing the Ganoderma lucidum as claimed in claim 1 ( Ganoderma lucidum ) spores obtained from strain Guizhi No.

3.

4. by culturing the Ganoderma lucidum as claimed in claim 1 ( Ganoderma lucidum ) Mycelia obtained from strain Guizhi No.

3.

5. by culturing the Ganoderma lucidum as claimed in claim 1 ( Ganoderma lucidum ) Fruiting bodies obtained from strain Guizhi No.

3.

6. Use of the fruiting body according to claim 5 in food processing.

7. A method for improving the Ganoderma lucidum ) A method for breeding for disease resistance, characterized in that The method comprises the following steps: utilizing Ganoderma lucidum ( Ganoderma lucidum ) strain Guizhi No. 3 was used as the parent for hybrid breeding.

8. The breeding method according to claim 7, characterized in that The disease-resistant pathogen is Penicillium pathogen ( Penicillium spp. )、Trichoderma viride pathogen ( Trichoderma spp .), Neurospora causative agent ( Neurospora spp .)、Brown rot pathogens( Hypomyces perniciosus ) and / or spider web disease pathogens ( Cladobotryum spp .).

9. Use of the mycelium according to claim 4 in extracting laccase, hemicellulase, filter paper enzyme and cellulase.

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