A strain of Armillaria mellea ZTMH-21 and its application in the cultivation of Gastrodia elata.
By selecting and breeding Armillaria mellea ZTMH-21 and combining it with specialized cultivation methods and substrates, the problems of slowed growth rate and insufficient nutrient supply of Armillaria mellea were solved, resulting in improved yield and quality of Gastrodia elata and adaptation to different cultivation environments.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHAOTONG GASTRODIA RES INST
- Filing Date
- 2026-03-27
- Publication Date
- 2026-06-26
AI Technical Summary
The current cultivation of Gastrodia elata uses a single strain of Armillaria mellea. Long-term artificial propagation has led to a slowdown in growth rate, a decrease in lignin degradation capacity, insufficient infection and nutrient supply capacity, and weak resistance to stress and contamination, resulting in a reduction in Gastrodia elata yield.
A strain of Armillaria mellea ZTMH-21 was selected and applied. This strain was isolated from the wild and exhibits rapid mycelial growth, strong mycelial cords, and no browning. It was combined with a specific nucleotide sequence for the cultivation of Gastrodia elata. Specialized cultivation substrates and methods were used, including the preparation of primary, secondary, and cultivars, combined with the cultivation method of Gastrodia elata.
It improved the yield and quality of Gastrodia elata. The combination of Armillaria mellea ZTMH-21 and Gastrodia elata significantly increased the yield, showed good stability, and was adapted to different regional cultivation environments, thus promoting the development of the Gastrodia elata industry.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a strain of Armillaria mellea ZTMH-21 and its application in the cultivation of Gastrodia elata. Background Technology
[0002] Armillaria mellea, belonging to the order Agaricales, family Penaeaceae, and genus Armillaria, is also known as hazel mushroom or small honey fungus. The fruiting body is medium to large, with a cap diameter of 4-14 cm. It is pale yellowish-brown, honey-colored to light yellowish-brown, turning brownish-brown with age. The center has small, appressed or erect scales, and the margin is striated. The flesh is white, and the gills are white or slightly pinkish-white, developing dark brown spots with age. The fruiting bodies are adnate to derivatized. Armillaria mellea fruiting bodies are not only delicious and crisp, but also rich in various bioactive components, making them highly sought after. Currently, 16 species of Armillaria mellea have been identified in China, of which only 7 phylogenetic species are suitable for cultivation of Gastrodia elata.
[0003] Armillaria mellea is an essential symbiotic fungus for the cultivation of the precious Chinese medicinal herbs Gastrodia elata and Polyporus umbellatus. The two have a special symbiotic relationship of exchanging nutrients. After the mycelial cords of Armillaria mellea invade the tuber of Gastrodia elata or the sclerotium of Polyporus umbellatus, they are digested and utilized by the latter, which obtain nutrients by digesting Armillaria mellea.
[0004] Currently, the source of Armillaria mellea strains used in the cultivation of Gastrodia elata in my country's main producing areas is relatively limited, and long-term artificial subculturing has led to a slowdown in mycelial growth rate and a decline in lignin degradation capacity. This results in a significant decrease in Armillaria mellea's growth vigor, insufficient infection and nutrient supply capacity, weak resistance to stress and contamination, and a decline in genetic and physiological indicators, leading to severe yield reductions in Gastrodia elata.
[0005] Therefore, based on the cultivation environment in different regions, robust Armillaria mellea germplasm resources are selected and bred through systematic collection and integration. Armillaria Superior strains of *Gastrodia elata* (spp.) were continuously improved to enhance its stress resistance and genetic stability. The domestication and widespread application of high-quality *Armillaria mellea* strains can increase the income of Gastrodia elata farmers and support the development of regional specialty economies, thus promoting the development of *Gastrodia elata* (spp.). Gastrodia elata This is of great significance to the high-quality development of the industry. Summary of the Invention
[0006] The purpose of this invention is to provide a strain of Armillaria mellea ZTMH-21 and its application in the cultivation of Gastrodia elata. This strain was isolated from the wild, has strong viability, white mycelium, fast mycelial growth rate, does not produce melanin, does not produce brown shell-like hard lumps during the transfer of the strain, and has a strong affinity with Gastrodia elata.
[0007] The above-mentioned technical objective of the present invention is achieved through the following technical solution: A strain of Armillaria pungentisquamosa, ZTMH-21, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 25, 2024, with accession number CGMCC No. 41670. It is classified and named Armillaria pungentisquamosa.
[0008] The strain exhibits rapid mycelial growth, with robust, vigorous, and highly branched mycelial cords that show no browning.
[0009] The strain has the specific nucleotide sequence shown in SEQ ID NO: 1.
[0010] The present invention also provides a fungal agent made from Armillaria mellea ZTMH-21, which is obtained by culturing and processing Armillaria mellea ZTMH-21.
[0011] This invention also provides the application of Armillaria mellea ZTMH-21 in the cultivation of Gastrodia elata.
[0012] The present invention also provides a cultivation substrate for cultivating the *Armillaria mellea* strain ZTMH-21, wherein the cultivation substrate comprises, by weight percentage: 40%–60% hardwood sawdust, 20%–30% corn cob particles, 20%–30% wheat bran, and additionally 0.5%–2.0% glucose, 0.2%–1.0% sucrose, 0.5%–2.0% gypsum, 0.1%–0.5% potassium dihydrogen phosphate, 0.05%–0.2% magnesium sulfate, and 0.005%–0.02% vitamin B1, and the moisture content of the cultivation substrate is 65%–75%.
[0013] This invention also provides a method for culturing Armillaria mellea ZTMH-21, comprising the following steps: S1. Preparation of primary strain: Under aseptic conditions, the purified Armillaria mellea strain ZTMH-21 was inoculated onto a solid culture medium and cultured at 25°C in the dark. Once the mycelium had fully grown on the culture medium and was free from contamination, the primary strain was obtained. S2, Preparation of secondary seed: Under aseptic conditions, the primary seed obtained in step (1) is inoculated into a sterilized secondary solid or semi-solid culture medium and cultured at 25°C in the dark until the mycelial cords are distributed throughout the culture medium to obtain the secondary seed; S3. Preparation of spawn: Select small wood segments with a length of about 1.5 cm and a diameter of about 2-3 cm, soak them in water for 6-24 hours, put them into a spawn bottle, add an appropriate amount of water, sterilize and cool to room temperature, inoculate with the secondary spawn obtained in step (2), and culture at a constant temperature of 25-28℃ until the mycelium fills the entire bottle, thus obtaining the spawn.
[0014] The present invention also provides a method for cultivating the Armillaria mellea ZTMH-21 cultivar, wherein in step S3, the ratio of wood segments to water is 50-60 wood segments per 500 ml cultivar bottle, filling the bottle to 4 / 5 of its capacity, and then adding an appropriate amount of water.
[0015] This invention also provides a method for cultivating Gastrodia elata using Armillaria mellea ZTMH-21, comprising the following steps: S1. Prepare mushroom bags: containing the cultivation strains as described in claim 7; S2. Bagging and planting: Lay a layer of sand and sawdust mixture at the bottom of the planting container, sow the sexual reproduction powder of Gastrodia elata, place the fungal substrate, break the fungal sticks in the cultivation spawn and place them at the interface of the fungal substrate, cover with the sand and sawdust mixture, and repeat the above operation to complete multi-layer bagging. S3. Cultivation and Management: After cultivating the filled planting bags under suitable conditions for 10-18 months, replant and harvest.
[0016] In summary, the present invention has the following beneficial effects. Firstly, this invention isolates a strain of Armillaria mellea ZTMH-21 from wild Armillaria mellea fruiting bodies. This strain has a fast growth rate, strong mycelial cords, and many branches, and can be used as a high-quality Armillaria mellea resource for Gastrodia elata cultivation.
[0017] Secondly, the white-headed hemp cultivated by Armillaria mellea ZTMH-21 provided by this invention has high yield and good quality.
[0018] Thirdly, the Armillaria mellea ZTMH-21 provided by this invention, when used in combination with Gastrodia elata, can effectively increase the yield of Gastrodia elata and is a high-quality and stable Armillaria mellea. Attached Figure Description
[0019] Figure 1 This is a diagram showing the growth of Armillaria mellea ZTMH-21 mycelial cords on tree stumps in the wild; Figure 2 This is a diagram showing the mycelial germination of Armillaria mellea ZTMH-21 on a plate 3 days after inoculation; Left: mycelium 10x magnification, Right: mycelium 40x magnification. Figure 3 This is a graph showing the growth of mycelial cords on a plate 5 days after inoculation with Armillaria mellea ZTMH-21; Figure 4 This is a phylogenetic tree of Armillaria mellea ZTMH-21; Figure 5 This is a graph showing the growth of Armillaria mellea ZTMH-21 on a plate 15 days after inoculation. Figure 6 This is a graph showing the growth of Armillaria mellea ZTMH-21 strain 17 days after inoculation; Figure 7 This is a diagram showing the growth of Gastrodia elata and Gastrodia elata planted with Armillaria mellea ZTMH-21. Detailed Implementation
[0020] The present invention will be further described in detail below with reference to the accompanying drawings.
[0021] Example 1: A strain of Armillaria mellea ZTMH-21, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 25, 2024, with accession number CGMCC No. 41670. It is classified and named *Armillaria pungentisquamosa*. The mycelial cords of this strain exhibit vigorous growth, appearing white, robust, highly branched, and without browning.
[0022] Example 2: Isolation and purification of Armillaria mellea ZTMH-21 1. Materials and Methods The ZTMH-21 strain originated from Xiaocaoba State-owned Forest Farm, Yiliang County, Zhaotong City, Yunnan Province. The mycelial cords were found on dead tree stumps. The mycelial cords, along with the stumps and soil, were wrapped in sterile kraft paper bags and brought back to the laboratory. The tree stumps containing the mycelial cords were wrapped in a plastic bag and placed in a foam box, with relative humidity controlled at approximately 85%, and incubated at 25°C in the dark for 5 days. Figure 1 As shown.
[0023] 2. Separation and purification Once fresh mycelial cords have grown, select robust, fresh mycelial cords without visible mold. Collect the tip of the mycelial cord and rinse the sample with running water (tap water or sterile water) to remove most of the attached material. Then, blot the moisture with sterile filter paper for later use.
[0024] Place the cleaned mycelial cords in a sterile operating table and immerse them in 70%-75% medical alcohol for 30 seconds. This step quickly removes surface grease and some microorganisms. Soak in 1%-3% sodium hypochlorite solution for 5 minutes. Quickly transfer the disinfected mycelial cords to sterile water and rinse repeatedly 3-5 times, approximately 30 seconds to 1 minute each time, ensuring that any residual disinfectant is thoroughly removed.
[0025] Gently blot off excess moisture from the surface of the mycelial cords with sterile filter paper, or place them in a sterile petri dish to dry slightly. In a laminar flow hood, use a sterile scalpel or forceps to cut the mycelial cords into 0.5-1 cm segments. After cutting, expose the relatively clean internal tissue and immediately transfer the segments to PDA medium (200g potato starch, 20g glucose, 20g agar, 1000ml water, natural pH). Incubate the inoculated plates at 25°C in the dark. Regularly observe the recovery and growth of the mycelial cords and check for bacterial or fungal contamination. Remove any contaminated sections immediately. When new hyphae or mycelial cords grow, transfer them to other plates or test tubes for further culture. Successfully subculture the culture twice on the medium; if no contamination and normal growth are observed, a pure culture has been obtained.
[0026] Example 3: Species identification of Armillaria mellea ZTMH-21 (a) Morphological identification The pure strain ZTMH-21, isolated from tree stumps, showed sparse, white, fluffy mycelia at the edge of the inoculation site (mycelial cord / herb block) radiating outwards 72 hours after inoculation. The mycelia were fine and transparent, with few aerial hyphae, adhering closely to the surface of the culture medium. Figure 2 As shown.
[0027] Five days after inoculation, white, thread-like structures appear, with reddish-brown roots; these are the juvenile mycelial cords. The tips of the cords retain white growth points and branch dichotomously. The colony diameter expands to 3-5 cm, with the cords extending at a significantly faster rate (1-2 mm / day) than the mycelia. Figure 3 As shown. Fifteen days after inoculation, the mycelial cords thickened (0.2-1 mm), the PDA color darkened, partial browning appeared in the mycelial cord area, and the base of the mycelial cords turned dark brown (mature part). The mycelial cords became robust, with increased branching, forming a reticulate structure, and the tips remained white. Figure 5 As shown.
[0028] (II) Molecular biological identification 1. Extraction of genomic DNA from Armillaria mellea ZTMH-21 Genomic DNA was extracted from fresh mycelia using a modified CTAB method. Using the extracted genomic DNA as a template, PCR amplification was performed with primer sequences HPf1655 (5′-CGAGGAAAGGCGATGTGTT-3′) as shown in SEQ ID NO: 2 and HPr3131 (5′-CCCTTGAGAACGGCCGTTATG-3′) as shown in SEQ ID NO: 3. The PCR reaction system (25 μL) contained: 1× PCR buffer, 0.2 mM dNTPs, 0.5 μM of each primer, 1 U Taq DNA polymerase, and 50 ng template DNA. The amplification program was as follows: 94 °C pre-denaturation for 5 min; 35 cycles (94 °C 40 s, 50 °C 40 s, 72 °C 50 s); 72 °C final extension for 8 min. After the amplified products were detected by 1% agarose gel electrophoresis, Sanger sequencing was performed using the same primers on an ABI-3730 XL sequencer (Applied Biosystems, USA) manufactured by Beijing Qingke Biotechnology Co., Ltd. (China). The obtained sequences were aligned to the NCBI BLAST database. The results showed that the strain was a *Armillaria pungentis* quamosa, and its Hispidin synthase gene sequence is shown in SEQ ID NO: 1.
[0029] A BLAST comparison of this strain on the NCBI website revealed a 100% similarity to a known Armillaria mellea (sequence ID: MT673981.1). To further understand the phylogenetic position of this strain, a phylogenetic tree was constructed using the neighbor-joining (NJ) method combined with existing Armillaria mellea sequences. The phylogenetic relationships are as follows: Figure 4 The phylogenetic tree shows that this strain belongs to the genus Armillaria pungentisquamosa.
[0030] Example 4: Preparation of Armillaria mellea ZTMH-21 cultivar 1. Preparation of sawdust culture medium: 50% hardwood sawdust, 25% corn cob (crushed into small particles), 25% wheat bran, plus 1% glucose, 0.5% sucrose, 1% gypsum, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, and 0.01% vitamin B1. Mix the raw materials with the gypsum, lime, and other dry materials thoroughly. Then dissolve all the above-mentioned soluble additives in water and mix them into the raw materials to achieve a moisture content of 70%. Mix thoroughly and fill the test tubes (18mm*180mm), compacting them appropriately. Fill the test tubes to 2 / 3 of their volume, then seal them with silicone sealant. Autoclave at 121℃, cool, and then inoculate.
[0031] 2. Preparation method of primary seed Under aseptic conditions, a small piece (5mm*5mm) of the purified strain ZTMH-21 was inoculated onto sawdust culture medium and cultured at 25℃ in the dark with a relative humidity of 60%–70%. The growth of Armillaria mellea was monitored periodically. Results showed that strain ZTMH-21 reached full colony status in approximately 25 days. Figure 6 As shown, once the tube is full and free from contamination by other microorganisms, the primary seed can be confirmed and used for the transfer of secondary seed.
[0032] 3. Preparation method of secondary seeds In a sterile environment, inoculate a piece of primary inoculum into a sterilized and cooled secondary solid or semi-solid culture medium, then seal the bottle and place it in a dark, temperature-controlled incubation room at 25°C. After two months of cultivation, when the Armillaria mellea mycelia have spread throughout the entire culture medium, the secondary inoculum is obtained.
[0033] 4. Methods for preparing cultivars For inoculation and cultivation of Armillaria mellea, the formula consists of small tree segments and water. First, cut finger-thick branches into 1.5cm segments and soak them in water for about 10 hours to ensure they are fully hydrated. Then, pack the tree segments into 500ml inoculum bottles, filling each bottle to about 4 / 5 capacity with 50-60 segments. Add a small amount of water, cap the bottle, and autoclave (0.1-0.15 MPa pressure, 121-126℃) for 5 hours. Cool to 25-30℃, inoculate with secondary inoculum, and incubate at a constant temperature of 25℃. After 60 days, once the entire bottle has grown, it is ready for use in Gastrodia elata cultivation.
[0034] Example 5: Experiment on treating Gastrodia elata seed with Armillaria mellea ZTMH-21 1. Materials and Methods The cultivation experiment was conducted at the Xiaocaoba Gastrodia elata planting base in Yiliang County, Yunnan Province. Four Armillaria mellea strains were selected for the co-cultivation experiment with Gastrodia elata. Comparative Example 1 was provided by Yunnan Senhao Fungi Industry Co., Ltd., Comparative Example 2 was provided by the Kunming Institute of Botany, Chinese Academy of Sciences, and Comparative Example 3 was collected and isolated from the wild by our laboratory. The method for preparing and cultivating the fungal bags was the same as in Example 3. The field experiment used bag cultivation, with each bag having an area of approximately 0.09 m². 2 Set up 6 replicates, with a total of 3 layers. Place a layer of sand and sawdust mixture at the bottom of the bag, sprinkle in the seed powder, arrange the substrate, break the mushroom stick in the bag and place it at the interface of the substrate, cover with sand and sawdust mixture, repeat the operation more than twice to complete the bagging, and replant and measure the yield after 10 months.
[0035] 2. Experimental Results As can be seen from the data in Table 1, based on the yield measurement results of Gastrodia elata and Gastrodia elata with white tips, the average yield of ZTMH-21 (0.91) is the highest compared with other Armillaria mellea strains, exceeding that of control group 1 by approximately 4.6%, demonstrating a significant yield advantage. Furthermore, the yield of Armillaria mellea strain ZTMH-21 is stable and reliable, and the growth of Gastrodia elata with white tips is as follows... Figure 7 As shown.
[0036] Table 1 Comparison of Production Results from Production Trial Sites This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.
Claims
1. A strain of Armillaria mellea ZTMH-21, characterized in that: The strain Armillaria pungentisquamosa has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 25, 2024, with accession number CGMCC No. 41670. It is classified as Armillaria pungentisquamosa.
2. The Armillaria mellea ZTMH-21 according to claim 1, characterized in that: The strain exhibits rapid mycelial growth, with robust, vigorous, and highly branched mycelial cords that show no browning.
3. The Armillaria mellea ZTMH-21 according to claim 1, characterized in that: The strain has the specific nucleotide sequence shown in SEQ ID NO:
1.
4. The fungal agent prepared from Armillaria mellea ZTMH-21 according to any one of claims 1-3, characterized in that: The bacterial agent is obtained by culturing and processing Armillaria mellea ZTMH-21.
5. The application of Armillaria mellea ZTMH-21 according to claim 1 in the cultivation of Gastrodia elata.
6. A cultivation substrate for cultivating the honey fungus strain ZTMH-21 according to claim 1, characterized in that, The cultivation substrate is composed of the following components by weight percentage: 40%–60% hardwood sawdust, 20%–30% corn cob pellets, 20%–30% wheat bran, plus 0.5%–2.0% glucose, 0.2%–1.0% sucrose, 0.5%–2.0% gypsum, 0.1%–0.5% potassium dihydrogen phosphate, 0.05%–0.2% magnesium sulfate, and 0.005%–0.02% vitamin B1. The moisture content of the cultivation substrate is 65%–75%.
7. The method for cultivating Armillaria mellea ZTMH-21 cultivar according to claim 6, characterized in that, Includes the following steps: S1. Preparation of primary strain: Under aseptic conditions, the purified Armillaria mellea strain ZTMH-21 was inoculated onto a solid culture medium and cultured at 25°C in the dark. Once the mycelium had fully grown on the culture medium and was free from contamination, the primary strain was obtained. S2, Preparation of secondary seed: Under aseptic conditions, the primary seed obtained in step (1) is inoculated into a sterilized secondary solid or semi-solid culture medium and cultured at 25°C in the dark until the mycelial cords are distributed throughout the culture medium to obtain the secondary seed; S3. Preparation of spawn: Select small wood segments with a length of about 1.5 cm and a diameter of about 2-3 cm, soak them in water for 6-24 hours, put them into a spawn bottle, add an appropriate amount of water, sterilize and cool to room temperature, inoculate with the secondary spawn obtained in step (2), and culture at a constant temperature of 25-28℃ until the mycelium fills the entire bottle, thus obtaining the spawn.
8. The method for cultivating Armillaria mellea ZTMH-21 cultivar according to claim 7, characterized in that: In step S3, the ratio of small wood segments to water is 50-60 small wood segments per 500 ml culture bottle, filling the bottle to 4 / 5 of its capacity, and then adding an appropriate amount of water.
9. The method for cultivating Gastrodia elata using Armillaria mellea ZTMH-21 according to claim 7, characterized in that, Includes the following steps: S1. Prepare mushroom bags: containing the cultivation strains as described in claim 7; S2. Bagging and planting: Lay a layer of sand and sawdust mixture at the bottom of the planting container, sow the sexual reproduction powder of Gastrodia elata, place the fungal substrate, break the fungal sticks in the cultivation spawn and place them at the interface of the fungal substrate, cover with the sand and sawdust mixture, and repeat the above operation to complete multi-layer bagging. S3. Cultivation and Management: After cultivating the filled planting bags under suitable conditions for 10-18 months, replant and harvest.