A method for cultivating microdermabranch fruiting bodies using agarwood forestry waste

Abstract

The present application relates to a method for culturing the fruiting bodies of Microdermabranch mushroom using agarwood forestry waste, comprising the following steps: (1) culturing the Microdermabranch mushroom species, an endophytic fungus of agarwood, in a PDA culture medium to obtain mycelium; (2) culturing the mycelium using agarwood forestry waste as the main component of a secondary culture medium to obtain a secondary culture medium; (3) inoculating the secondary culture medium into an artificial fruiting body culture medium containing agarwood forestry waste as the main component of the culture medium to obtain an artificial fruiting body of Microdermabranch mushroom.

Description

Technical Field

[0001] The present application relates to the technical field of agarwood fungus cultivation, and in particular to a method for cultivating the fruiting bodies of Microdermabrax agaricus using agarwood forestry waste. Background Art

[0002] Agarwood, a species of plant in the genus Aquilaria (Thymelaeaceae), is produced when the tree is injured and infected by fungi, and the resin deposited therein becomes agarwood. The fungus Marasmiellus Murrill, a member of the genus Basidiomycota, order Agaricales, and family Omphalotaceae, is a common macrofungus in the subtropical zone and an important pathogen and medicinal fungus for subtropical plants. Research on the genus Marasmiellus began relatively late and has received insufficient attention, resulting in confusion about its scientific name and significant misidentification. As an endophytic fungus of agarwood, Marasmiellus Murrill's fruiting bodies are essential for studying its morphology, medicinal properties, and potential applications.

[0003] However, since naturally collected Microdermabranchs often grow on rotten wood and plant humus, it is very difficult to identify their specific hosts, making it difficult to find suitable culture substrates for Microdermabranchs to form artificial fruiting bodies; or they appear in the form of plant endophytes, and endophytic Microdermabranchs do not naturally form fruiting bodies.

[0004] There has been no successful report on the cultivation of artificial fruiting bodies of the endophytic Microdermabranch of Agaricus sinensis, let alone examples of factory production.

[0005] Given the extreme scarcity of natural sources of endophytic microdermabranchs in agarwood, it would be very meaningful to find a way to successfully artificially cultivate endophytic microdermabranchs in agarwood to obtain fruiting bodies. Summary of the Invention

[0006] In order to solve or partially solve the problems existing in the relevant technology, the present application provides a method for culturing artificial fruiting bodies of endophytic Microdermabranchs of Agaricus sinensis, which is easy to operate and can obtain sufficient Microdermabranchs fruiting bodies for related scientific research and development.

[0007] A method for culturing the fruiting bodies of Agaricus spp. using agarwood forestry waste in the present application comprises the following steps:

[0008] (1) The endophytic fungus Microdermabrax species of agarwood was cultured in PDA culture medium to obtain mycelium;

[0009] (2) Using agarwood forestry waste as the main component of the secondary culture medium to cultivate mycelium and obtain secondary strains;

[0010] (3) The secondary strain was transferred and inoculated into an artificial fruiting body culture medium with agarwood forestry waste as the main component of the culture medium to obtain artificial fruiting bodies of Microdermabrax.

[0011] In this application, the species of Microdermabrax endophyte of Agarwood is GX-6-1, which is a strain of the literature "Taxonomy, phylogeny, and successful cultivation of Marasmiellus scandens (Basidiomycota)associated with Aquilaria sinensis (agarwood tree) in China》(Zhang. et al The strain disclosed in [1] is deposited in the Kunming Institute of Botany's Microbial Culture Collection, with the accession number KUNCC 22-12451. It should be noted that the general public can apply for GX-6-1 through the above-mentioned collection, provided that the holder of the above-mentioned biological material provides a certificate guaranteeing that the biological material will be released to the public within 20 years from the date of application.

[0012] In this application, the mass ratio of the secondary bacterial culture medium is: 50% agarwood sawdust, 30% buckwheat, 5% agarwood dead branches, 5% agarwood fallen leaves, 10% glucose, and a moisture content of 75%-80%.

[0013] In the present application, the mass ratio of the artificial fruiting body culture medium is: 70% of soil agarwood sawdust, 15% of soil agarwood dead branches, 13% of soil agarwood fallen leaves, 1% of lime, and 1% of gypsum.

[0014] In the present application, the culture conditions after the secondary strain is inoculated in step (3) are a temperature of 30°C and culture in the dark for 25 days.

[0015] In the present application, when preparing the secondary bacterial strain in step (2), the inoculation amount is 100 mg of PDA culture mycelium / 500 g of secondary bacterial strain culture bag.

[0016] In the present application, during the transfer inoculation in step (3), the inoculation amount of the secondary strain is 2cm x 2cm or 1cm x 1cm size bacterial blocks / 500g culture bag.

[0017] In the present application, in step (3), when the secondary strain is inoculated and cultured, the temperature for the formation of the artificial fruiting body primordium is controlled to be 28-30°C, the temperature for the artificial fruiting body fruiting is controlled to be 30-35°C, the humidity for fruiting is controlled to be 80%-85%, and the light intensity for the artificial fruiting body fruiting is controlled to be 100 lux.

[0018] In this application, the specification of the culture bags is 500 grams and they are sterilized at 121° C. for 4 hours.

[0019] The technical solution provided by this application may have the following beneficial effects:

[0020] 1. In this application, the endophytic fungus Microdermabrax species of agarwood was pre-cultured in PDA medium to form mycelium. It was then inoculated into a secondary culture medium composed primarily of sterilized agarwood forestry waste. The culture was protected from light to produce a secondary culture. The cultured secondary culture was then inoculated into a culture medium composed primarily of sterilized agarwood forestry waste. After 30 days of culture, the agarwood forestry waste substrate fermented and grew artificial fruiting bodies of Microdermabrax.

[0021] This method is easy to operate and has wide applicability. It can effectively obtain sufficient artificial fruiting bodies of Microdermabrax in a short period of time for scientific and applied research and development. This culture method is currently the only way to obtain artificial fruiting bodies of Microdermabrax endophyticus from Aquilaria sinensis.

[0022] 2. This method also achieves the effective decomposition and reuse of Aquilaria sinensis forestry waste, does not pollute the environment, and does not affect the normal growth and metabolism of Aquilaria sinensis trees, and is suitable for promotion and application in actual production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Preparation of fungus bag culture medium using Aquilaria sinensis forestry waste;

[0024] Figure 2 Mycelium of Microdermabrax spp. growing in fruiting body culture medium;

[0025] Figure 3 Mycelium of Microdermabranchus (on PDA medium);

[0026] Figure 4: Artificial fruiting bodies of Microdermabrax microcarpa grown on agarwood forestry waste substrate;

[0027] Figure 5 Secondary fungus species of Microdermabrasion. DETAILED DESCRIPTION

[0028] The following describes embodiments of the present application in more detail with reference to the accompanying drawings. Although the accompanying drawings illustrate embodiments of the present application, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0029] It should be understood that although the terms "first", "second", "third", etc. may be used in this application to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from each other. For example, without departing from the scope of this application, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0030] In the description of this application, it should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on this application.

[0031] Example 1

[0032] Microdermabranch GX-6-1 was isolated from the leaf tissue of Aquilaria sinensis grown in Pubei County, Qinzhou City, Guangxi Zhuang Autonomous Region. Microdermabranch GX-6-1 was cultured in PDA medium at 28°C in a constant temperature incubator for 14 days. Freshly cultured fungi (mycelium such as Figure 3 Divide into 100 mg pieces for later use;

[0033] Aquilaria sinensis forestry waste was collected from the Aquilaria sinensis planting base in Lushui County, Jiang Lisu Autonomous Prefecture, Yunnan Province. The remaining Aquilaria sinensis forestry waste after logging and construction was collected at the Aquilaria sinensis planting base in Lushui County, Nujiang Lisu Autonomous Prefecture, Yunnan Province: unfruitful sawdust, Aquilaria sinensis dead branches and Aquilaria sinensis fallen leaves.

[0034] At the Kunming Institute of Botany, crushed Aquilaria sinensis forestry waste was prepared in different proportions to obtain secondary bacterial culture medium and fruiting body culture medium, wherein the mass ratio of the secondary bacterial culture medium is: 50% Aquilaria sinensis sawdust, 30% Tartary buckwheat, 5% Aquilaria sinensis dead branches, 5% Aquilaria sinensis fallen leaves, 10% glucose, and a moisture content of 78%; the mass ratio of the artificial fruiting body culture medium is: 70% Aquilaria sinensis sawdust, 15% Aquilaria sinensis dead branches, 13% Aquilaria sinensis fallen leaves, 1% lime, and 1% gypsum; and then packed into 35cm x 15cm (500g) fungus bags, as shown in Figure 2. Figure 1As shown, after sterilization at 121°C for 4 hours, 100 mg of fresh bacteria were inoculated into the secondary culture bags. The bags were sealed with airtight sealing rings to prevent contamination. The inoculated bags were transferred to a greenhouse and cultured in the dark at 30°C for 25 days to obtain the secondary bacteria. Figure 5 shown.

[0035] Use a sterile blade to cut the cultured secondary strains into 2cm x 2cm blocks in a clean bench. Then inoculate the 2cm x 2cm blocks into the fruiting body culture bags. Seal the bags with a breathable sealing ring to prevent contamination. Figure 2 The figure shows the mycelium of Microdermabranchium growing in a fruiting body culture medium. The inoculated fruiting body culture bag was placed at 30-35°C, 80%-85% humidity, and 100 lux light conditions.

[0036] Record the fruiting of the bags after 30-50 days of cultivation. On the 30th day of cultivation, if Figure 4 As shown, the average artificial fruiting body production was 5 per bag; on the 45th day, the average artificial fruiting body production was 11.5 per bag; on the 50th day, the average artificial fruiting body production was 16 per bag.

[0037] Example 2

[0038] Microdermabranch GX-6-1 was isolated from leaf tissue of Aquilaria sinensis grown in Pubei County, Qinzhou City, Guangxi Zhuang Autonomous Region. Microdermabranch GX-6-1 was cultured in PDA medium at 28°C in a constant-temperature incubator for 14 days. Fresh cultured fungi (fruiting bodies) were divided into 100 mg pieces using sterile razor blades in a clean bench for later use. Aquilaria sinensis forestry waste was collected from the Aquilaria sinensis plantation in Lushui County, Jiangjiang Lisu Autonomous Prefecture, Yunnan Province. Aquilaria sinensis forestry waste remaining after logging and construction, including unfruitful sawdust, dead branches, and fallen leaves, was collected from the Aquilaria sinensis plantation in Lushui County, Nujiang Lisu Autonomous Prefecture, Yunnan Province.

[0039] At the Honghe Mountain Future Germplasm Resource Innovation Center, crushed agarwood forestry waste was placed in varying proportions (secondary culture medium and fruiting body culture medium) into 35cm x 15cm (500g) bags and sterilized at 121°C for 4 hours. Then, 100mg of fresh, pre-split spawn was inoculated into the secondary culture bags, which were sealed with air-permeable rings to prevent contamination. The inoculated bags were transferred to a greenhouse and incubated in the dark at 30°C for 25 days to obtain secondary culture.

[0040] In a clean bench, use a sterile razor blade to cut the fresh culture (secondary culture) into 1cm x 1cm pieces for later use. Then, inoculate the fruiting body culture bags with these 1cm x 1cm pieces. Seal the bags with a breathable sealing ring to prevent contamination. Incubate the fruiting body culture bags at 30°C, 85% humidity, and 100 lux of diffuse light.

[0041] The fruiting of the bags was recorded after 30-50 days of cultivation. On the 30th day, the average number of artificial fruiting bodies produced was 3 per bag; on the 45th day, the average number of artificial fruiting bodies produced was 12 per bag; and on the 50th day, the average number of artificial fruiting bodies produced was 18.5 per bag.

[0042] Comparative Example 1

[0043] Mycelium was prepared according to the method described in Example 1. The secondary culture medium in this comparative example contained the following mass ratios: 50% agarwood sawdust, 30% tartary buckwheat, 5% agarwood dead branches, 5% agarwood fallen leaves, 10% glucose, and 78% moisture content. The fruiting body culture medium contained the following mass ratios: 70% elm sawdust, 15% wheat bran, 13% cottonseed hulls, 1% lime, and 1% gypsum. After inoculation with the secondary culture, the fruiting body culture bags were incubated at 30°C, 85% humidity, and 100 lux of diffuse light at the Honghe Mountain Future Germplasm Resource Innovation Center.

[0044] After culturing for 30 days, the mycelium of Microdermabrasion could grow in the secondary fungus culture medium, and the secondary fungus could also grow in the fruiting body culture medium, but no fruiting body was observed.

[0045] Comparative Example 2

[0046] Mycelium was prepared according to the method described in Example 1. The secondary culture medium in this comparative example contained the following mass ratios: 50% elm sawdust, 30% tartary buckwheat, 5% wheat bran, 5% cottonseed hulls, and 10% glucose; the fruiting body culture medium contained the following mass ratios: 70% elm sawdust, 15% wheat bran, 13% cottonseed hulls, 1% lime, and 1% gypsum. After inoculation with the secondary culture, the inoculated fruiting body culture bags were incubated in a greenhouse at the Honghe Mountain Future Germplasm Resource Innovation Center at 30-35°C, 80%-85% humidity, and 100 lux of light.

[0047] After culturing for 30 days, the mycelium of Microdermabrasion could grow in the secondary fungus culture medium, and the secondary fungus could also grow in the fruiting body culture medium, but no fruiting body was observed.

[0048] Comparative Example 3

[0049] Mycelium was prepared according to the method described in Example 1. The secondary culture medium in this comparative example contained the following weight ratios: 50% elm sawdust, 30% tartary buckwheat, 5% wheat bran, 5% cottonseed hulls, and 10% glucose; the artificial fruiting body culture medium contained the following weight ratios: 70% agarwood sawdust, 15% agarwood dead branches, 13% agarwood fallen leaves, 1% lime, and 1% gypsum. After inoculation with the secondary culture, the inoculated fruiting body culture bags were incubated in a greenhouse at the Honghe Mountain Future Germplasm Resource Innovation Center at 30-35°C, 80%-85% humidity, and 100 lux of light.

[0050] After culturing for 30 days, the mycelium of Microdermabrax can grow in the secondary strain culture medium, and the secondary strain can also grow, but ultimately no fruiting body is observed.

[0051] According to the results of Examples 1 and 2, the method provided by the present invention can effectively utilize Aquilaria sinensis forestry waste to cultivate and obtain artificial fruiting bodies of Microdermabranch, providing valuable experimental materials for subsequent scientific research and application development. The present invention can obtain artificial fruiting bodies of Microdermabranch after 30 days of fermentation and cultivation, with a short production cycle, simple operation, low cost, no environmental pollution, and the realization of forestry waste recycling. It can be seen that the present invention effectively produces artificial fruiting bodies of Microdermabranch in a short period of time, which has great scientific research value and application potential.

[0052] Furthermore, it can be seen from Comparative Example 1 that when the components of the secondary bacterial culture medium are not changed and the agarwood waste as the main ingredient in the fruiting body culture medium is changed, no fruiting body can be obtained;

[0053] Furthermore, it can be seen from Comparative Example 2 that even after the agarwood waste as the main material in the secondary bacterial culture medium and the fruiting body culture medium are changed, fruiting bodies cannot be obtained.

[0054] Furthermore, it can be seen from Comparative Example 3 that when the composition of the agarwood waste as the main ingredient in the secondary strain culture medium is changed, the fruiting body culture medium composition is not changed and the fruiting body cannot be obtained.

[0055] That is to say, the presence of agarwood waste as the main material in the secondary bacterial culture medium and fruiting body culture medium in this application is crucial to the production of fruiting bodies and neither is indispensable.

[0056] The embodiments of the present application have been described above. The above description is illustrative and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to the technology in the market, or to enable other persons skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for cultivating the fruiting body of Agaricus spp. using agarwood forestry waste, characterized by: The following steps are involved: (1) The endophytic fungus Microdermabrax species of agarwood was cultured in PDA culture medium to obtain mycelium; (2) using agarwood forestry waste as the main component of a secondary culture medium to cultivate mycelium and obtain a secondary culture medium, wherein the mass ratio of the secondary culture medium is: 50% agarwood sawdust, 30% tartary buckwheat, 5% agarwood dead branches, 5% agarwood fallen leaves, 10% glucose, and a moisture content of 75%-80%; (3) The secondary strain was transferred to an artificial fruiting body culture medium with agarwood forestry waste as the main component of the culture medium for cultivation to obtain artificial fruiting bodies of Microdermabranch, wherein the mass ratio of the artificial fruiting body culture medium is: 70% of agarwood sawdust, 15% of agarwood dead branches, 13% of agarwood fallen leaves, 1% of lime, and 1% of gypsum; the agarwood endophyte Microdermabranch species is Microdermabranch GX-6-1, which is preserved in the Microbial Culture Collection Center of Kunming Institute of Botany with the preservation number KUNCC 22-12451.

2. The method for culturing the fruiting body of Agaricus spp. using agarwood forestry waste according to claim 1, characterized in that: In step (3), the culture conditions after the secondary strain is inoculated are a temperature of 30° C. and culture in the dark for 25 days.

3. The method for culturing the fruiting body of Agaricus spp. using agarwood forestry waste according to claim 1, characterized in that: When preparing the secondary bacterial strain in step (2), the inoculation amount is 100 mg of PDA culture mycelium / 500 g of the secondary bacterial strain culture bag.

4. The method for culturing the fruiting body of Agaricus spp. using agarwood forestry waste according to claim 1, characterized in that: During the transfer inoculation in step (3), the inoculation amount of the secondary strain is 2 cm x 2 cm or 1 cm x 1 cm size bacterial blocks per 500 g culture bag.

5. The method for culturing the fruiting body of Agaricus spp. using agarwood forestry waste according to claim 1, characterized in that: In the step (3), when the secondary strain is inoculated and cultured, the temperature for forming the artificial fruiting body primordium is controlled at 28-30° C., the temperature for fruiting of the artificial fruiting body is controlled at 30-35° C., the humidity for fruiting is controlled at 80%-85%, and the light intensity during fruiting of the artificial fruiting body is controlled at 100 lux.

6. The method for culturing the fruiting bodies of Agaricus spp. using agarwood forestry waste according to claim 4 or 5, characterized in that: The size of the culture bags was 500 g and they were sterilized at 121°C for 4 hours.

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