Three-section type seedling raising method for cremastra appendiculata

Abstract

The invention discloses a three-section type seedling raising method for cremastra appendiculata, which comprises the following steps: carrying out seed bacteria-accompanying germination culture, carrying out protocorm bacteria-accompanying germination culture, transplanting seedlings, preparing a Coprinus albiculatus bag by using Coprinus albiculatus as bacteria for bacteria-accompanying germination culture, crushing the Coprinus albiculatus bag into particles, mixing the particles with 3cm sawdust blocks to obtain a mixed matrix, adding humus soil, and carrying out seedling raising. The method solves the problems of low seed germination rate, low seedling growth speed, low transplanting survival rate and the like of the cremastra appendiculata. According to the three-section seedling raising method, an alternative scheme with commercial feasibility is provided for large-scale production of the rhododendron simsii, and the technical principle of the method can be further expanded and applied to conservation and commercial cultivation of other rare orchid plants. The future research can further optimize the symbiotic strain combination and environmental control parameters so as to continuously improve the stability and the application range of the technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the seedling raising technical field of Cremastra appendiculata, in particular to a three-stage seedling raising method of Cremastra appendiculata. BACKGROUND

[0002] Cremastra appendiculata, also known as "mountain Cigu", is a perennial rare medicinal plant of the genus Cremastra of the Orchidaceae family. According to the Chinese Pharmacopoeia, the dry pseudobulb is used as a medicine, has the effects of clearing heat and resolving toxins, reducing phlegm and resolving nodules, and is often used for treating sores, sore throat, and scrofula caused by phlegm fire stagnation.

[0003] Due to the small size of wild Cremastra appendiculata seeds without endosperm and serious lignification of seed coat, the seeds can only break through the germination barrier by establishing an interaction relationship with specific symbiotic fungi under natural conditions, resulting in difficulty in natural propagation of wild populations and extremely weak regeneration capacity. In addition, due to overexploitation and habitat destruction, wild resources of Cremastra appendiculata have been listed as a national second-class protected plant, and artificial large-scale breeding has become a core approach to alleviate resource crisis. In traditional aseptic seeding technology, the cost of tissue culture seedling cultivation process is high, and the requirements for equipment, technology and professional personnel are strict. After the seedlings are transplanted to the natural environment, the survival rate is low due to poor environmental adaptability, and it is difficult to achieve large-scale breeding. These problems seriously restrict the artificial cultivation and popularization of Cremastra appendiculata, and therefore it is urgent to develop an efficient seedling breeding technology.

[0004] The present application aims to develop a complete "three-stage seedling raising method" (seed symbiotic germination culture → protocorm symbiotic germination culture → transplanted adult seedling), which solves the core problems of low seed germination rate, slow seedling growth rate and low survival rate after transplanting, and provides a theoretical basis and practical guidance for resource protection, large-scale seedling breeding and industrialized cultivation of Cremastra appendiculata. SUMMARY

[0005] The present application aims to provide a three-stage seedling raising method of Cremastra appendiculata, which solves the problems of low seed germination rate, slow seedling growth rate and low survival rate after transplanting by seed symbiotic germination culture → protocorm symbiotic germination culture → transplanted adult seedling.

[0006] The technical scheme of the present application is as follows: a three-stage seedling raising method of Cremastra appendiculata, which is carried out according to the following steps: (1) Cultivation of white ghost mushroom mycelium block: white ghost mushroom is inoculated into PDA culture medium slope, and incubated at 23-27℃ in the dark for 6-8 days. After the mycelium grows over the plate, the edge pure mycelium is picked and transferred to a new PDA culture medium for continuous purification for 2-4 times, and the white ghost mushroom mycelium block is obtained and stored at 3-5℃ for standby use; (2) Preparation of sawdust-bran mixed substrate: Broadleaf sawdust was crushed to pass through a 1.5-2.5 mm sieve, bran, gypsum powder and sucrose were added and mixed, the mass ratio of broadleaf sawdust, bran, gypsum powder and sucrose was 7-9:0.8-0.12:0.4-0.6:0.4-0.6, water was added to adjust the moisture content to 60%-65%, to obtain a sawdust-bran mixed substrate, ready for use; (3) Preparation of Coprinus candidus fungus package: 400-600 g of sawdust-bran mixed substrate was packed, compacted and sealed with cotton, sterilized, cooled to room temperature, and then inoculated with 2-4 pieces of Coprinus candidus mycelial block in a super-clean bench, each piece of Coprinus candidus mycelial block had a diameter of 9-11 mm, cultured at 23-27°C in the dark for 18-22 days, until the mycelium covered the fungus package and formed sclerotia, to obtain a Coprinus candidus fungus package, ready for use; (4) Preparation of humus soil: humus soil with pH 5.5-6.5 was sterilized at 121°C for 2 h to obtain sterilized humus soil, ready for use; (5) Seed companion germination culture: four layers of culture medium were laid in the germination culture container, which were bottom layer, middle layer, upper layer and surface layer, the bottom layer was laid with 3.5-4.5 cm thick sterilized humus soil, the middle layer was laid on the sterilized humus soil of the bottom layer, the structure of the middle layer was designed as follows: first, a half amount of wood chip block was laid as a base, the Coprinus candidus fungus package was distributed in a grid pattern on the wood chip block with a spacing of about 4.5-5.5 cm, then the remaining wood chip block was completely covered, the thickness of the middle layer was 4-9 cm, the volume ratio of the wood chip block and the Coprinus candidus fungus package block was 0.8-1.2:0.8-1.2, the size of the wood chip block was 1-2 cm in thickness, 1 cm in width and 1-5 cm in length, the Coprinus candidus fungus package block was obtained by crushing the Coprinus candidus fungus package into 3-4 cm 3 particles, the upper layer was sterilized humus soil, the thickness of the upper layer was 0.8-1.2 cm, the Cymbidium seed was sown on the sterilized humus soil of the upper layer, and finally the surface layer was covered, which was sterilized humus soil, the thickness of the surface layer was 0.8-1.2 cm of sterilized humus soil to cover the seed, under the conditions of relative humidity of 70%-85% and temperature of 20-22°C, the culture was carried out for 90 days to obtain the germinated Cymbidium protocorm; (6) Prothallium companion bacteria germination cultivation: the white ghost fungus bag is broken into 0.5-1cm particles and mixed with sterilized humus soil as the germination cultivation substrate, with a mass ratio of 1:2.5-3.5, and is loaded into the germination cultivation container to a height of 0.8-1.0cm. The diameter of the selected Cymbidium prothallus is ≥5mm, and the germination cultivation container is planted with the prothallus and the germination substrate with a plant spacing of 2.5-3.5cm. The prothallus is completely covered with the germination substrate and is 0.8-1.2cm higher than the prothallus. The root-fixing water is poured thoroughly, and the container is placed in a greenhouse at 22-25℃ and a medium relative humidity of 70%-85%. The light time is 12h / d, and the cultivation is carried out for 110-130 days. Water is poured every 1-3 days to keep the substrate moist. When the prothallus differentiates into leaves and roots, Cymbidium seedlings with a height of ≥8cm are obtained. (7) Seedling transplanting: The soil in the greenhouse is disinfected by spraying carbendazim at a dilution of 800-1200 times, and the soil pests are killed by spraying clothianidin. After 2-4 days of disinfection, the white ghost fungus bag is broken into 0.5-1cm particles and mixed with soil, with a mass ratio of 2:0.8-1.2. The soil and white ghost fungus bag are deeply plowed to a depth of 25-35cm and leveled into a ridge. The ridge width is 1-1.5m, and the height is 18-25cm. The Cymbidium seedlings with a height of ≥8cm are selected and transplanted to the ridge surface with a plant spacing of 10cm×15cm. The transplanting depth is suitable for the top of the prothallus to be just submerged by the soil surface. The root-fixing water is poured thoroughly. The temperature in the greenhouse is controlled at 22-28℃, and the shading net is used to shade about 75%. The cultivation is carried out for 55-65 days, and the growth of the seedlings is monitored.

[0007] In the foregoing step (1), the cultivation of the white ghost fungus mycelium block: the white ghost fungus is inoculated into the PDA medium slope, and incubated at 25℃ for 7 days. After the mycelium grows over the plate, the edge of the pure mycelium is picked and transferred to a new PDA medium for continuous purification for 3 times. The white ghost fungus mycelium block is obtained and stored at 4℃ for standby use.

[0008] In the foregoing step (1), the PDA medium is prepared by adding 200g / L of potatoes, 20g / L of glucose, and 20g / L of agar to 1L of distilled water, and then sterilizing at 121℃ for 20min.

[0009] In the foregoing step (2), the mass ratio of the sawdust of broadleaf trees, bran, gypsum powder, and sucrose is 8:1:0.5:0.5.

[0010] In the foregoing step (3), 500g of the sawdust-bran mixed substrate is packed in a bag, compacted, and sealed with cotton. The bag is sterilized at 121℃ for 2h, and then cooled to room temperature. The white ghost fungus mycelium block is inoculated into the bag in a clean bench, with 3 blocks per bag. Each white ghost fungus mycelium block has a diameter of 10mm. The bag is incubated at 25℃ in the dark for 20 days. When the mycelium grows over the bag and forms a sclerotium, the white ghost fungus bag is obtained and stored for standby use.

[0011] In the aforementioned step (3), the culture bag used for the sawdust-bran mixed matrix is ​​a polypropylene bag, and the specification of the polypropylene bag is 17 cm×33 cm.

[0012] In the aforementioned step (5), seed-associated fungus germination culture: four layers of culture substrate are laid in the germination culture container, namely the bottom layer, the middle layer, the upper layer and the surface layer. The bottom layer is laid with 4 cm thick sterilized humus soil, and the middle layer is laid on the sterilized humus soil of the bottom layer. The structure of the middle layer is designed to first use half of the sawdust blocks as a base, and then distribute the white false agaricus mushroom bags in a grid shape with a spacing of about 5 cm on the sawdust blocks, and then completely cover the remaining sawdust blocks. The thickness of the middle layer is 5-8 cm, and the volume ratio of the sawdust blocks to the white false agaricus mushroom blocks is 1:1. The size of the sawdust blocks is 1-2 cm thick, 1 cm wide, and 1-5 cm long. The white false agaricus mushroom blocks are obtained by crushing the white false agaricus mushroom bags into 3-4 cm 3 The middle layer is covered with an upper layer, which is sterilized humus with a thickness of 1 cm. The azalea seeds are sown on the sterilized humus on the upper layer. Finally, the surface layer is covered with sterilized humus with a thickness of 1 cm. The sterilized humus covers the seeds. Under the conditions of relative humidity of 70%-85% and temperature of 20-22℃, the culture is carried out for 90 days to obtain the germinated azalea protocorms.

[0013] In the above step (6), the protocorms are cultured with bacteria for germination: the white pseudo-coprinus agaricus mushroom bags are crushed into 0.5-1 cm particles and mixed with sterilized humus soil at a mass ratio of 1:3 as a germination culture medium, and the mixture is placed in a germination culture container to a height of 0.8-0.9 cm. The azalea orchid protocorms with a diameter of ≥5 mm are selected and planted together with the germination culture medium at a spacing of 3 cm into the germination culture container, and the germination culture medium is covered to completely cover the protocorms and 1 cm higher than the protocorms. The roots are irrigated with water, and the seeds are placed in a greenhouse at 22-25°C and a relative humidity of 70%-85% for cultivation, with a light duration of 12h / d for 120 days. Watering is carried out every 1-3 days to keep the medium moist. When the protocorms differentiate into leaves and roots, mature azalea orchid seedlings with a plant height of ≥8 cm are obtained.

[0014] In the above step (7), seedling transplanting: prepare the soil in the greenhouse in advance, dilute carbendazim 1000 times and spray it on the greenhouse soil for sterilization and disinfection, then spray the soil pests with clothianidin, and after disinfection for 3 days, crush the white false agaricus mushroom capsules into 0.5-1 cm particles and mix them with the soil, the mass ratio of soil to white false agaricus mushroom capsules is 2:1, and deep plowing is carried out to 30 cm and then leveled to make a bed, the bed width is 1.2 m and the height is 20 cm. Select azalea orchid with a height of ≥8 cm and transplant it with soil clumps to the bed surface, the plant spacing is 10 cm × 15 cm, the transplanting depth is suitable for the top of the bulb to be just submerged by the soil surface, and water thoroughly to establish roots; the temperature in the greenhouse is controlled at 22-28 ° C, and a shade net is used to shade about 75%. Cultivate for 60 days and monitor the growth of the seedlings.

[0015] In the aforementioned step (7), after the seedlings are transplanted, field management is performed, and weeds are removed and diluted organic fertilizer is sprayed.

[0016] Compared with the prior art, the present application has the following beneficial effects: 1. By seed associated bacteria germination culture, the treatment group 3 uses 3 cm wood chip blocks and fungus package blocks as mixed substrates, so that the germination rate of the cymbidium goeringii seeds is the highest, and the number of cymbidium goeringii protocorms reaches 590 per 100 cm 2 , which is significantly higher than that of other treatments (P<0.05); 2. In the protocorm associated bacteria germination culture stage, the fresh weight of single seedling reaches 2.72 g, the total length of single seedling reaches 14.5 cm, the underground part length reaches 4.6 cm, and the aboveground part length reaches 9.9 cm. The protocorm formed in the first stage of treatment 1 is the smallest (weight 19.3 mg, length 0.58 cm), and the fresh weight and plant height after transplanting are the lowest, only 37.5% and 51.7% of those of treatment 3. The growth indexes of seedlings of treatment 2 and treatment 4 are between those of treatment 3 and treatment 1, and are significantly positively correlated with the size of the first stage protocorm.

[0017] 3. After the seedlings are transplanted, the plant height of the seedlings in the treatment group with added fungus package blocks is (19.6±1.3) cm, which is significantly higher than that of the control (13.3±0.9) cm (P<0.05), and the plant height increases by 47.4%; in terms of fresh weight, the average fresh weight of the seedlings with added fungus package blocks is (3.8±0.25) g, which is significantly higher than that of the control (2.1±0.18) g (P<0.05), and is 1.81 times that of the control; in terms of survival rate, the survival rate of the seedlings in the treatment group with added fungus package blocks reaches 91%, which is significantly higher than that of the control (58%) (P<0.05), and the survival rate increases by 56.9%.

[0018] 4. The "three-stage seedling cultivation method" developed in this study effectively overcomes the limitations of traditional cymbidium goeringii sterile tissue culture technology by introducing associated bacteria to construct a fungus-plant symbiotic system. Studies have shown that fungi can degrade wood chip substrates by secreting cellulase and ligninase, continuously releasing nutrients such as glucose and amino acids

[12] , which not only reduces the dependence on culture medium (significantly saving material costs), but also simulates the nutrient supply mode of natural habitat through biological transformation process.

[0019] 5、The "three-stage seedling cultivation method" developed in this study breaks through the limitations of traditional Cymbidium goeringii tissue culture technology, achieving the synergistic optimization of economic feasibility, technical generalizability, and ecological adaptability. In terms of industrial application, this method has significant advantages: (1) Economic feasibility - sawdust substrate can be obtained from forestry waste factories, and fungus bag blocks can be mass-produced using simple fermentation tanks (costing about 5000 yuan), reducing the cost of cultivating a single seedling to 1.2-1.5 yuan, which is only 30-40% (1 / 3-2 / 5) of the cost of tissue culture seedlings. With 10,000 m 2 of production base, annual cost savings of 425-597 million yuan; (2) Technical generalizability - the method is simple to operate, and the entire process does not require a sterile operating room, so farmers can master the key technology after simple training, making local cultivation in remote areas possible; (3) Ecological adaptability - the root system of the symbiotic seedlings is stronger, and the survival rate after transplanting is significantly improved, solving the core problem of poor environmental adaptability of tissue culture seedlings. These innovative breakthroughs not only provide a commercially viable alternative for the large-scale production of Cymbidium goeringii, but the technical principles can also be applied to the conservation and commercial cultivation of other rare orchid plants. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 : First-stage seed inoculation and germination culture treatment 3-seal box culture flowchart (a: bottom layer of humus soil; b: middle layer of white ghost fungus bag blocks distributed on sawdust blocks; c: middle layer of sawdust blocks covering fungus bag blocks; d: side view of the complete seal box in culture); Figure 2 : White ghost fungus bag inoculation and culture evolution diagram (A: 4B strain inoculated into PDA slant medium; B: white ghost fungus bag; C: phylogenetic tree of white ghost fungus); Figure 3 : Pro-cormus formation after fungus inoculation and culture (a: pro-cormus after 20d of seed fungus inoculation and culture; b: pro-cormus after 40d of seed fungus inoculation and culture; c: pro-cormus after 55d of seed fungus inoculation and culture; d: comparison of seed fungus inoculation and culture growth, 1 in d is seed without fungus inoculation; 2 is seed fungus inoculation and culture for 20d; 3 is seed fungus inoculation and culture for 30d; 4 is seed fungus inoculation and culture for 40d; 5 is seed fungus inoculation and culture for 55d; 6 is seed fungus inoculation and culture for 70d; 7 is seed fungus inoculation and culture for 90d); Figure 4: Comparison chart of different periods of protocorm seedling (1: seed mixed with bacteria germination culture for 40d; 2: seed mixed with bacteria germination culture for 60d; 3: seed mixed with bacteria germination culture for 90d; the second stage protocorm transplanting (4: protocorm with a diameter of ≥5mm 20d after transplanting; 5: protocorm with a diameter of ≥5mm 40d after transplanting; 6: protocorm with a diameter of ≥5mm 70d after transplanting; 7: protocorm with a diameter of ≥5mm 90d after transplanting; 8: protocorm with a diameter of ≥5mm 120d after transplanting); Figure 5 Growth index and survival rate of seedling after 60d of greenhouse cultivation. DETAILED DESCRIPTION

[0021] The present application will be further described in the following examples, it is necessary to point out that the following examples are only used to further illustrate the present application, and cannot be understood as limiting the protection scope of the present application.

[0022] Example 1 (1) Culture of white ghost mushroom mycelium block: white ghost mushroom was inoculated into PDA culture medium slant, and incubated at 25℃ for 7d in dark. After the mycelium grew over the plate, the edge pure mycelium was picked and transferred to new PDA culture medium for continuous purification for 3 times, to obtain white ghost mushroom mycelium block, which was stored at 4℃ for standby use. PDA culture medium was prepared by adding 200g / L of potato, 20g / L of glucose and 20g / L of agar to 1L of distilled water, and then sterilized at 121℃ for 20min.

[0023] (2) Preparation of sawdust-bran mixed substrate: broadleaf tree sawdust was ground through 2mm sieve, and then mixed with bran, gypsum powder and sucrose. The mass ratio of broadleaf tree sawdust, bran, gypsum powder and sucrose was 8:1:0.5:0.5. Water was added to adjust the water content to 60%-65%, to obtain sawdust-bran mixed substrate, which was standby used.

[0024] (3) Preparation of white ghost mushroom bag: 500g of sawdust-bran mixed substrate was packed in a bag, and then compacted and sealed with cotton. The bag was sterilized at 121℃ for 2h, and then cooled to room temperature. In a clean bench, white ghost mushroom mycelium block was inoculated into the bag, 3 blocks per bag, and each white ghost mushroom mycelium block had a diameter of 10mm. The bag was incubated at 25℃ in dark for 20 days, until the mycelium grew over the bag and formed sclerotia, to obtain white ghost mushroom bag, which was standby used. The culture bag used for sawdust-bran mixed substrate was polypropylene bag, and the specification of the polypropylene bag was 17cm×33cm.

[0025] (4) Preparation of humus soil: humus soil with pH 5.5-6.5 was sterilized at 121℃ for 2h, to obtain sterilized humus soil, which was standby used.

[0026] (5) Seed-associated fungus germination culture: Four layers of culture substrate are laid in the germination culture container, namely the bottom layer, middle layer, upper layer and surface layer. The bottom layer is covered with 4 cm thick sterilized humus soil, and the middle layer is laid on the sterilized humus soil of the bottom layer. The structure of the middle layer is designed to first use half of the sawdust blocks as a base, and then distribute the white false agaricus mushroom bags in a grid shape with a spacing of about 5 cm on the sawdust blocks, and then completely cover the remaining sawdust blocks. The thickness of the middle layer is 5-8 cm, and the volume ratio of sawdust blocks to white false agaricus mushroom blocks is 1:1. The size of the sawdust blocks is 1-2 cm thick, 1 cm wide, and 1-5 cm long. The white false agaricus mushroom blocks are obtained by crushing the white false agaricus mushroom bags into 3-4 cm 3 The middle layer is covered with an upper layer, which is sterilized humus with a thickness of 1 cm. The azalea seeds are sown on the sterilized humus on the upper layer. Finally, the surface layer is covered with sterilized humus with a thickness of 1 cm. The sterilized humus covers the seeds. Under the conditions of relative humidity of 70%-85% and temperature of 20-22℃, the culture is carried out for 90 days to obtain the germinated azalea protocorms.

[0027] (6) Germination and cultivation of protocorms with fungi: Crush the white pseudo-coprinus caps into 0.5-1 cm particles and mix them with sterilized humus soil at a mass ratio of 1:3 as a germination and cultivation medium. Place them in a germination and cultivation container to a height of 0.8-0.9 cm. Select azalea orchid protocorms with a diameter of ≥5 mm and plant them together with the germination culture medium at a spacing of 3 cm into the germination and cultivation container. Cover the protocorms with the germination culture medium to completely cover the protocorms and 1 cm higher than the protocorms. Water thoroughly to establish roots. Place them in a greenhouse at 22-25℃ and a relative humidity of 70%-85% for cultivation. The light duration is 12h / d. Cultivate for 120 days. Water every 1-3 days to keep the medium moist. Wait until the protocorms differentiate into leaves and roots to obtain mature azalea orchid seedlings with a plant height of ≥8 cm.

[0028] (7) Transplanting seedlings: Prepare the soil in the greenhouse in advance, dilute carbendazim 1000 times and spray it on the greenhouse soil for sterilization and disinfection, then spray the soil pests with clothianidin. After disinfection for 3 days, crush the white false agaricus mushroom capsules into 0.5-1cm particles and mix them with the soil. The mass ratio of soil to white false agaricus mushroom capsules is 2:1. After deep plowing 30cm, level the soil and make a bed with a width of 1.2m and a height of 20cm. Select azalea orchid with a height of ≥8cm and transplant it to the bed surface with a row spacing of 10cm×15cm. The transplanting depth should be such that the top of the bulb is just submerged in the soil surface. Water thoroughly to allow the roots to take root. The temperature in the greenhouse is controlled at 22-28℃, and a shade net is used to shade about 75%. Cultivate for 60 days and monitor the growth of the seedlings. After transplanting the seedlings, carry out field management, weeding and spraying diluted organic fertilizer.

[0029] Example 2: (1) Cultivation of white ghost mushroom mycelium block: white ghost mushroom was inoculated into PDA medium slant, and incubated at 27℃ for 6 days in dark. After the mycelium grew over the plate, the edge pure mycelium was picked and transferred to new PDA medium for continuous purification for 4 times. The white ghost mushroom mycelium block was obtained and stored at 5℃ for standby; PDA medium was prepared by adding 200g / L potato, 20g / L glucose and 20g / L agar to 1L distilled water, and then sterilized at 121℃ for 20min.

[0030] (2) Preparation of sawdust-bran mixed substrate: broadleaf sawdust was ground through 1.5mm sieve, mixed with bran, gypsum powder and sucrose, and the mass ratio of broadleaf sawdust, bran, gypsum powder and sucrose was 7:0.8:0.6:0.6. Water was added to adjust the moisture content to 60%-65%, to obtain sawdust-bran mixed substrate for standby; (3) Preparation of white ghost mushroom bag: 400g sawdust-bran mixed substrate was packed, compacted and sealed with cotton. After sterilization, the bag was cooled to room temperature, and then inoculated with white ghost mushroom mycelium block in a clean bench. Four blocks of white ghost mushroom mycelium block with a diameter of 9mm were inoculated in each bag. The bag was incubated at 27℃ in dark for 18 days, until the mycelium grew over the bag and formed sclerotia. The white ghost mushroom bag was obtained for standby; (4) Preparation of humus soil: humus soil with pH 5.5-6.5 was sterilized at 121℃ for 2h to obtain sterilized humus soil for standby; (5) Seed associated germination culture: four layers of medium were laid in the germination culture container, which were bottom layer, middle layer, upper layer and surface layer. The bottom layer was 4.5cm thick sterilized humus soil. The middle layer was laid on the sterilized humus soil of the bottom layer. The structure of the middle layer was designed as follows: first, half amount of wood chip block was laid as a base, then white ghost mushroom bag was distributed on the wood chip block in a grid pattern with a spacing of about 5.5cm, and then the remaining wood chip block was completely covered. The thickness of the middle layer was 6-9cm, and the volume ratio of wood chip block to white ghost mushroom bag block was 1.2:0.8. The size of the wood chip block was 1-2cm in thickness, 1cm in width and 1-5cm in length. The white ghost mushroom bag block was obtained by crushing the white ghost mushroom bag into 3-4cm particles. The upper layer was sterilized humus soil, and the thickness of the upper layer was 1.2cm. The Cymbidium ensifolium seeds were sown on the sterilized humus soil of the upper layer. Finally, the surface layer was covered, which was sterilized humus soil with a thickness of 0.8cm to cover the seeds. The germinated Cymbidium ensifolium protocorms were obtained after incubation for 90 days under the conditions of relative humidity of 70%-85% and temperature of 20-22℃. 3 ​(6) Germination and cultivation of protocorms with fungi: Crush the mushroom capsules of white pseudo-coprinus into 0.5-1 cm particles and mix them with sterilized humus soil at a mass ratio of 1:3.5 as a germination and cultivation medium. Place them in a germination and cultivation container to a height of 0.8-0.9 cm. Select protocorms of azalea orchid with a diameter of ≥5 mm and plant them together with the germination culture medium at a spacing of 3.5 cm into the germination and cultivation container. Cover the protocorms with the germination culture medium to completely cover the protocorms and 1.2 cm higher than the protocorms. Water thoroughly to establish roots. Place them in a greenhouse at 22-25℃ and a relative humidity of 70%-85% for cultivation. The light duration is 12h / d. Cultivate for 110 days. Water every 3 days to keep the medium moist. Wait until the protocorms differentiate into leaves and roots to obtain mature seedlings of azalea orchid with a plant height of ≥8 cm.

[0031] (7) Transplanting seedlings: Prepare the soil in the greenhouse in advance, dilute carbendazim 1200 times and spray it on the greenhouse soil for sterilization and disinfection, then spray the soil pests with clothianidin. After disinfection for 2 days, crush the white false agaricus mushroom capsules into 0.5 cm particles and mix them with the soil. The mass ratio of soil to white false agaricus mushroom capsules is 2:1.2. After deep plowing 25 cm, level the soil and make a bed with a width of 1.5 m and a height of 18 cm. Select azalea orchid with a height of ≥8 cm and transplant it to the bed surface with a row spacing of 10 cm × 15 cm. The transplanting depth should be such that the top of the bulb is just submerged in the soil surface. Water thoroughly to allow the roots to take root. The temperature in the greenhouse is controlled at 22-28 ° C. Use a shade net to shade about 75%. Cultivate for 55-65 days and monitor the growth of the seedlings. After transplanting the seedlings, carry out field management, weeding and spraying diluted organic fertilizer.

[0032] Example 3: (1) Cultivation of mycelial blocks of Coprinus albopictus: Inoculate Coprinus albopictus onto the slant of PDA culture medium and culture in the dark at 23°C for 8 days. After the mycelium has grown all over the plate, pick the pure mycelium at the edge and transfer it to a new PDA culture medium. Purify twice to obtain mycelial blocks of Coprinus albopictus and store them at 3°C ​​for later use. PDA culture medium was prepared by adding 200 g / L potato, 20 g / L glucose, and 20 g / L agar to a volume of 1 L with distilled water, and then sterilized under high pressure at 121°C for 20 min.

[0033] (2) Preparation of sawdust-bran mixed matrix: grind broadleaf sawdust through a 2.5 mm sieve, add bran, gypsum powder and sucrose and mix them. The mass ratio of broadleaf sawdust, bran, gypsum powder and sucrose is 9:0.12:0.4:0.4. Add water to adjust the moisture content to 60%-65% to obtain a sawdust-bran mixed matrix, which is set aside. (3) Preparation of the white false agaricus fungus bag: 600 g of sawdust-bran mixed matrix was packed into a bag, compacted and sealed with cotton plugs. After sterilization, the bag was cooled to room temperature and then inoculated with white false agaricus mycelium blocks in a clean bench. Two blocks were inoculated per bag. The diameter of each white false agaricus mycelium block was 11 mm. The bags were cultured in the dark at 23 ° C for 22 days until the mycelium grew all over the bag and formed sclerotia. The white false agaricus fungus bag was obtained and set aside. (4) Preparation of humus soil: Take humus soil with a pH of 5.5-6.5 and sterilize it under high pressure at 121℃ for 2 hours to obtain sterilized humus soil for later use; (5) Seed-associated fungus germination culture: Four layers of culture substrate are laid in the germination culture container, namely the bottom layer, the middle layer, the upper layer and the surface layer. The bottom layer is paved with 3.5 cm thick sterilized humus soil, and the middle layer is laid on the sterilized humus soil of the bottom layer. The structure of the middle layer is designed to first use half of the sawdust blocks as a base, and then distribute the white pseudo-coprinus agaricus mushroom bags in a grid shape with a spacing of about 4.5 cm on the sawdust blocks, and then completely cover the remaining sawdust blocks. The thickness of the middle layer is 4-7 cm, and the volume ratio of the sawdust blocks to the white pseudo-coprinus agaricus mushroom blocks is 0.8:1.2. The size of the sawdust blocks is 1-2 cm thick, 1 cm wide, and 1-5 cm long. The white pseudo-coprinus agaricus mushroom blocks are obtained by crushing the white pseudo-coprinus agaricus mushroom bags into 3-4 cm 3 The middle layer is covered with an upper layer of sterilized humus soil with a thickness of 0.8 cm, and the azalea orchid seeds are sown on the sterilized humus soil on the upper layer; finally, the surface layer is covered with sterilized humus soil with a thickness of 1.2 cm. The sterilized humus soil covers the seeds, and the seeds are cultured for 90 days under the conditions of relative humidity of 70%-85% and temperature of 20-22°C to obtain the germinated azalea orchid protocorms; (6) Germination and cultivation of protocorms with fungi: Crush the white pseudo-coprinus caps into 0.5-1 cm particles and mix them with sterilized humus soil at a mass ratio of 1:2.5 as a germination and cultivation medium. Place them in a germination and cultivation container to a height of 0.9-1.0 cm. Select azalea orchid protocorms with a diameter of ≥5 mm and plant them together with the germination culture medium at a spacing of 2.5 cm into the germination and cultivation container. Cover the protocorms with the germination culture medium to completely cover the protocorms and to be 0.8 cm higher than the protocorms. Water thoroughly to establish roots. Place them in a greenhouse at 22-25℃ and a relative humidity of 70%-85% for cultivation. The light duration is 12h / d. Cultivate for 130 days. Water every other day to keep the medium moist. Wait until the protocorms differentiate into leaves and roots to obtain mature azalea orchid seedlings with a plant height of ≥8 cm.

[0034] (7) Transplanting seedlings: Prepare the soil in the greenhouse in advance, dilute carbendazim 800 times and spray it on the greenhouse soil for sterilization and disinfection, then spray the soil pests with clothianidin. After disinfection for 4 days, crush the white false agaricus mushroom capsules into 1 cm particles and mix them with the soil. The mass ratio of soil to white false agaricus mushroom capsules is 2:0.8. After deep plowing 35 cm, level the soil to make a bed with a width of 1 m and a height of 25 cm. Select azalea orchids with a height of ≥8 cm and transplant them with soil clumps to the bed surface. The spacing between rows and plants is 10 cm × 15 cm. The transplanting depth should be such that the top of the bulb is just submerged in the soil surface. Water thoroughly to allow the roots to take root. The temperature in the greenhouse is controlled at 22-28 ° C. Use a shade net to shade about 75%. Cultivate for 55-65 days and monitor the growth of the seedlings. After transplanting the seedlings, carry out field management, weeding and spraying diluted organic fertilizer.

[0035] Example 4: (1) Cultivation of mycelial blocks of Coprinus albopictus: Inoculate Coprinus albopictus onto the slant of PDA culture medium and culture in the dark at 25°C for 7 days. After the mycelium has grown all over the plate, pick the pure mycelium at the edge and transfer it to a new PDA culture medium. Purify the mycelium blocks three times in a row to obtain the mycelial blocks of Coprinus albopictus. Store them at 4°C for later use. PDA culture medium was prepared by adding 200 g / L potato, 20 g / L glucose, and 20 g / L agar to a volume of 1 L with distilled water, and then sterilized under high pressure at 121°C for 20 min.

[0036] (2) Preparation of sawdust-bran mixed matrix: grind broadleaf sawdust through a 2 mm sieve, add bran, gypsum powder and sucrose and mix them. The mass ratio of broadleaf sawdust, bran, gypsum powder and sucrose is 9:1:0.5:0.4. Add water to adjust the moisture content to 60%-65% to obtain a sawdust-bran mixed matrix, which is set aside. (3) Preparation of the white false agaricus fungus bag: 450 g of sawdust-bran mixed matrix was packed into a bag, compacted and sealed with cotton plugs. After sterilization, the bag was cooled to room temperature and then inoculated with white false agaricus mycelium blocks in a clean bench. Three blocks were inoculated per bag. The diameter of each white false agaricus mycelium block was 10 mm. The bags were cultured in the dark at 25 ° C for 20 days until the mycelium grew all over the bag and formed sclerotia. The white false agaricus fungus bag was obtained and set aside. (4) Preparation of humus soil: Take humus soil with a pH of 5.5-6.5 and sterilize it under high pressure at 121℃ for 2 hours to obtain sterilized humus soil for later use; (5) Seed companion bacteria germination culture: four layers of culture medium are laid in the germination culture container, which are bottom layer, middle layer, upper layer and surface layer. The bottom layer is laid with 4.5 cm thick sterilized humus, the middle layer is laid on the sterilized humus of the bottom layer, the structure of the middle layer is designed as follows: first, a half amount of wood chip block is laid as a base, then white ghost mushroom fungus packs are distributed in a grid shape on the wood chip block with a spacing of about 5 cm, and then the remaining wood chip block is completely covered, the thickness of the middle layer is 4-9 cm, and the volume ratio of the wood chip block to the white ghost mushroom fungus pack is 0.9:1.1, the upper layer is further covered on the middle layer, the size of the wood chip block is 1-2 cm in thickness, 1 cm in width and 1-5 cm in length, the white ghost mushroom fungus pack is obtained by crushing the white ghost mushroom fungus pack into 3-4 cm 3 particles, the upper layer is sterilized humus, the thickness of the upper layer is 0.9 cm, and the azoresia seed is sown on the sterilized humus of the upper layer; finally, the surface layer is covered, which is sterilized humus, and the thickness of the surface layer is 1.1 cm of sterilized humus covering the seed, under the conditions of relative humidity of 70%-85% and temperature of 20-22℃, the culture is carried out for 90 days, and the germinated azoresia protocorm is obtained; (6) Protocorm companion bacteria germination culture: the white ghost mushroom fungus pack is crushed into 0.5-1 cm particles and mixed with sterilized humus at a mass ratio of 1:2.8 as the germination culture substrate, which is loaded into the germination culture container to a height of 0.9-1.0 cm, the azoresia protocorm with a diameter of ≥5 mm is selected and planted into the germination culture container together with the germination culture substrate at a plant spacing of 3 cm, the germination culture substrate is covered to completely cover the protocorm and is higher than the protocorm by 0.9 cm, and the root fixing water is poured, which is placed in a greenhouse at a temperature of 22-25℃ and a relative humidity of 70%-85%, the light time is 12 h / d, the culture is carried out for 115 days, the water is poured every 2 days to keep the substrate moist, and the azoresia mature seedling with a plant height of ≥8 cm is obtained when the protocorm differentiates into leaves and root system.

[0037] (7) Seedling transplanting: the soil in the greenhouse is prepared in advance, the soil is disinfected by spraying with diluted 1100 times carbendazim, and the soil pests are killed by spraying with clothianidin, after 3 days of disinfection, the white ghost mushroom fungus pack is crushed into 0.5-1 cm particles and mixed with the soil, the mass ratio of the soil to the white ghost mushroom fungus pack is 2:1.1, the soil is deeply ploughed to 30 cm, then the soil is leveled to form a ridge, the ridge width is 1.3 m, and the ridge height is 20 cm, the azoresia seedling with a plant height of ≥8 cm is selected and transplanted to the ridge surface with soil clumps, the plant spacing and row spacing are 10 cm×15 cm, the transplanting depth is appropriate when the top of the protocorm is just submerged by the soil surface, and the root fixing water is poured thoroughly; the temperature in the greenhouse is controlled at 22-28℃, the shading net is used to shade about 75%, and the seedling growth is monitored during the cultivation for 55-65 days; After the seedling is transplanted, the weeds are removed and the diluted organic fertilizer is sprayed for field management.

[0038] The inventor verified the effect of the present application by a large number of experiments, and the specific experiments are as follows: 1Materials and methods 1.1 Experimental materials 1.1.1 Strain source The associated bacteria were isolated from the rhizosphere of wild Cymbidium goeringii, and the strain was identified by morphological observation and molecular biology (based on ITS sequence analysis). The seeds of Cymbidium goeringii were obtained from the healthy mature capsules of Baolin Technology Co., Ltd. in Anshun, Guizhou Province, and were stored in a refrigerator at 4°C for later use. 1.1.2 Culture medium and reagents The isolation medium was PDA medium (potato 200 g / L, glucose 20 g / L, agar 20 g / L, distilled water to 1 L, 121°C high pressure sterilization for 20 min). The materials for making the fungus package included sawdust of broad-leaved trees (passed through a 2 mm sieve), bran, gypsum powder (CaSO4·2H2O), and sucrose, which were mixed in a mass ratio of 8:1:0.5:0.5, and water was added to adjust the moisture content to 60%-65%. The cultivation substrate was humus soil (pH 5.5-6.5) which was sterilized at 121°C for 2 h. The broad-leaved wood chips used in the experiment were divided into four sizes: sawdust of ash wood chips (passed through a 0.5 mm sieve), 1 cm wood chips, 3 cm wood chips, and 5 cm shavings wood chips, all of which were sterilized. The cultivation container was a transparent sealed box with a lid in the first stage, made of PET material, with an outer diameter of 45x36x14 cm and an inner diameter of 41x32x13 cm. In the second stage, it was a transparent plastic box without a lid, with an outer diameter of 45x36x14 cm and an inner diameter of 41x32x13 cm. In the third stage, it was a greenhouse cultivation bed (1x2 m) equipped with an automatic sprinkler system.

[0039] 1.2 Isolation and propagation of C. geogorum 1.2.1 Strain isolation Rhizosphere soil of wild Cymbidium goeringii was collected, and dilution plating method was used to isolate fungi on PDA plates. Single colonies were picked and identified as C. geogorum by ITS-rDNA sequencing (primers ITS1 / ITS4). The purified strain was inoculated into PDA slant and incubated at 25°C for 7 days in the dark. When the mycelium covered the plate, the edge of the pure mycelium was transferred to a new PDA medium, and the process was repeated for 3 times to obtain a pure culture strain, which was stored at 4°C for later use.

[0040] 1.2.2 Fungus package preparation Preparation of sawdust-bran mixed substrate: Broad-leaved wood chips were crushed to pass through a 2 mm sieve, and bran, gypsum powder, and sucrose were added and mixed. The mass ratio of broad-leaved sawdust, bran, gypsum powder, and sucrose was 8:1:0.5:0.5, and water was added to adjust the moisture content to 60%-65%. The sawdust-bran mixed substrate was obtained and stored for later use.

[0041] The mixed sawdust-bran mixed substrate was filled into polypropylene bacterial bags (specification 17 cm x 33 cm), about 500 g of dry material was filled into each bag, and after compaction, it was sealed with cotton plug, 121 ℃ high pressure sterilization for 2 h. After cooling to room temperature, the white ghost mushroom mycelium block (diameter 10 mm) was connected in the clean bench, 3 blocks were inoculated in each bag, and it was placed in a 25 ℃ dark incubator for 20 days, until the mycelium grew full of the fungus bag and formed sclerotia, and the white ghost mushroom fungus bag was obtained.

[0042] 1.3 Three-stage symbiotic seedling raising technology 1.3.1 First stage: seed symbiotic germination culture (sealed box culture) The germination culture container is a transparent sealed box with a cover, PET material, outer diameter size 45x36x14 cm, inner diameter size 41x32x13 cm, and 5 treatment groups are set, each group with 3 repeats.

[0043] Treatment 1: The bottom layer was laid with sterilized humus soil (thickness 4 cm); the middle layer was mixed with sawdust sawdust (passed through 0.5 mm sieve) and fungus bag block (broken into 3-4 cm small pieces) at a volume ratio of 1:1 (thickness 5 cm); the upper middle layer was covered with sterilized humus soil (thickness 1 cm), and then evenly scattered with one duoceras fruit pod seed (the weight of the fruit pod seed sown in each box was about 50 mg); finally, the top layer was covered with a layer of sterilized humus soil to cover the seeds (thickness 1 cm).

[0044] Treatment 2: The middle layer was mixed with wood chip block (thickness 0.5-1 cm, width 1 cm, length 1 cm) and fungus bag block (broken into 3-4 cm small pieces) at a volume ratio of 1:1, the middle layer used the structure design of "wood chip block-fungus-wood chip block", half of the wood chip block was used as the bottom, the white ghost mushroom fungus bag was distributed on the wood chip block in a grid shape with a spacing of about 5 cm, and the remaining wood chip block was completely covered, and the rest was the same as treatment 1. Treatment 3: The middle layer was mixed with wood chip block (thickness 0.5-1 cm, width 1 cm, length 1-5 cm) and fungus bag block (broken into 3-4 cm small pieces) at a volume ratio of 1:1, the middle layer used the structure design of "wood chip block-fungus-wood chip block", half of the wood chip block was used as the bottom, the white ghost mushroom fungus bag was distributed on the wood chip block in a grid shape with a spacing of about 5 cm, and the remaining wood chip block was completely covered, and the rest was the same as treatment 1. Control (CK): Sawdust and wood chip (0.5mm) as the medium, no mycorrhizal package added, the same as treatment 1. The seeds of Cymbidium goeringii are small and difficult to count. For the subsequent experiment, the mature and dried fruits were peeled and weighed accurately on an electronic balance (about 50mg per box). The sawdust was soaked for one night to ensure sufficient water absorption. The mycorrhizal package was completely covered when mixed with the sawdust. The relative humidity in the box was maintained at 70%-85% and the temperature was 20-22℃. The cultivation lasted for 90 days. The seed germination and protocorm formation were observed regularly. Figure 1 ).

[0045] 1.3.2 Second stage: protocorm germination and cultivation with mycorrhizal package Since the control (CK) did not form protocorms in the first stage, no seedlings were transplanted. Only the data of the four treatment groups with mycorrhizal package were counted in this stage.

[0046] The germination and cultivation container was a transparent plastic box without a lid, with an outer diameter of 45x36x14cm and an inner diameter of 41x32x13cm. The mycorrhizal package (crushed into 0.5-1cm particles) was mixed with sterilized humus soil at a mass ratio of 1:3 to serve as the cultivation substrate, which was filled into the flowerpot to a height of 2 / 3. The protocorms (diameter ≥5mm) formed after 90 days in the first stage from each treatment group were taken out of the sealed box, the attached substrate was retained, and they were transplanted into the corresponding flowerpot at a plant spacing of 3cm, covered with substrate to 1cm above the top of the protocorms, and watered thoroughly with rooting water. They were placed in a greenhouse for cultivation at a temperature of 22-25℃ and a relative humidity of 70%-85%, with a light period of 12h / d. Regular watering was performed to keep the substrate moist, and the cultivation lasted for 120 days until the protocorms differentiated into leaves and root systems (plant height ≥8cm).

[0047] 1.3.3 Third stage: transplanting of mature seedlings Two treatment groups were set up in the greenhouse cultivation stage, namely soil with mycorrhizal package (treatment group) and soil without mycorrhizal package (control, CK), both without sawdust. The soil was disinfected by spraying with a 1000-fold dilution of carbendazim and the soil pests were killed by spraying with thiamethoxam. After three days of disinfection, the soil was mixed with the mycorrhizal package (crushed into 0.5-1cm particles, no mycorrhizal package for the control) at a mass ratio of 2:1, deeply plowed to 30cm, and leveled into a ridge (ridge width 1.2m, height 20cm). Seedlings of similar size (plant height ≥8cm) from the second stage of cultivation were selected, transplanted into the corresponding ridge with soil clumps, with a plant spacing of 10cm x 15cm, and the transplanting depth was appropriate with the top of the protocorms just submerged by the soil surface. The soil was watered thoroughly with rooting water. The temperature in the greenhouse was controlled at 22-28℃, the shading net was used to shade about 75%, and regular weeding and spraying of diluted organic fertilizer were performed. The seedlings were cultivated for 60 days, and the growth of the seedlings was monitored.

[0048] 1.4 Measurement indicators and methods The number of protocorms formed and the seedling survival rate (number of surviving seedlings / number of transplanted seedlings × 100%) were regularly recorded at each stage. Plant height, leaf length, and pseudobulb diameter were measured with a ruler, and fresh weight was determined using an electronic balance. Each treatment was replicated three times, with 30 plants randomly selected each time. Data were statistically analyzed using SPSS 22.0 software, and significant differences were analyzed using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test (P < 0.05).

[0049] 2 Results and Analysis 2.1 Isolation and propagation of Agaricus albus When isolating fungi from the rhizosphere soil of Rhododendron orchid, a variety of fungal colonies were obtained on PDA plates by the dilution coating method. After picking a single colony and purifying it for culture, a strain with relatively consistent morphological characteristics was obtained, numbered 4B. The strain was subjected to ITS-rDNA sequencing. After amplification using primers ITS1 / ITS4, the sequencing results were compared in the GenBank database, and it was found that its homology with the white false agaricus (Coprinellus disseminatus) reached more than 98%. Combined with morphological observations (the colonies were white and fluffy, the hyphae were slender and grew rapidly), the strain was determined to be white false agaricus. After BLAST comparison with the GenBank database, the sequence similarity with multiple reference strains of white false agaricus (JQ713570.1, OK584436.1, ON426445.1) reached 99.8%. The phylogenetic analysis was constructed using the maximum likelihood (ML) method ( Figure 2 C), the bootstrap value was set to 1000 times, and the results showed that strain 4B and C. disseminatus clustered in the same branch (bootstrap support rate 98%).

[0050] The purified white pseudo-Agaricus was inoculated onto the PDA slant and cultured in the dark at 25°C. After 7 days, the mycelium covered the entire slant. The mycelium grew vigorously, was white in color, and had a uniform texture. Figure 2 A). After three consecutive purifications, the pure culture strain obtained maintained good activity for one month when stored at 4°C, and the mycelium recovered quickly after inoculation. During the bag propagation process, polypropylene bags inoculated with mycelial blocks of white pseudo-Amanita phalloides were cultured in a dark incubator at 25°C for about 10 days. Mycelium was visible spreading from the inoculated block to the surrounding matrix. By the 20th day of culture, the mycelium had grown throughout the bag, and a large number of sclerotia had formed inside the bag, with a dense texture ( Figure 2 B). The time it takes for mycelium to fully grow in bags made from different batches varies little, indicating that this propagation method has good stability and repeatability, and can provide sufficient and stable fungus sources for the subsequent three-stage symbiotic seedling production. 2.2 First paragraph: Seed germination and protocorm induction (sealed box culture) After 90 days of sealed box culture, the seed germination and protocorm formation of different treatment groups showed significant differences. The protocorm growth indicators of each treatment group from high to low were treatment 3 > treatment 4 > treatment 2 > treatment 1 > control (CK). The number of protocorms in treatment 3 reached 590 per 100 cm 2 , which was significantly higher than that of other treatment groups (P<0.05), 3.28 times that of treatment 1 (180 per 100 cm 2 ); the weight of single protocorm (31.7 mg) and length (0.90 cm) were also the highest in each group, which were 2.09 times and 2.14 times that of treatment 1 (15.2 mg, 0.42 cm), respectively. Treatment 1 performed the worst among all treatment groups with added fungus blocks, with each indicator slightly higher than the control (CK) without sterile blocks, and no seed germination or protocorm formation was observed in the control group during the entire experiment (Table 1).

[0051] Comprehensive analysis showed that the presence of C. geophilum significantly promoted the seed germination and protocorm formation of C. japonicum, and the size of sawdust blocks had a significant effect on symbiosis. The 3 cm sawdust block treatment provided the most suitable microenvironment for the symbiosis between C. geophilum and C. japonicum seeds, while the sawdust ash block had the worst effect due to the lack of fungus blocks and its own structure which was not conducive to ventilation and nutrient transfer.

[0052] Table 1 Protocorm growth indicators of different treatment groups after 90 days (per 100 cm 2 ) Note: The data in Table 1 are mean ± standard deviation, and different lowercase letters after the same column data indicate significant differences between treatments (P<0.05).

[0053] 2.3 Second paragraph: Protocorm transplantation and growth (pot domestication) In this stage, protocorms with a diameter ≥5 mm formed in the first stage were transplanted ( Figure 4 ), and the cultivation substrate was a mixture of the corresponding fungus blocks (crushed into 0.5-1 cm particles) and sterilized humus soil at a mass ratio of 1:3, without additional sawdust. The growth differences of seedlings in each group were mainly due to the initial size differences of protocorms formed in the first stage.

[0054] Table 2 Growth indicators of seedlings in different treatment groups after 120 days Note: The data in Table 2 are mean ± standard deviation, and different lowercase letters after the same column data indicate significant differences between treatments (P<0.05).

[0055] From Table 2, the seedling growth index of treatment 3 was significantly better than other treatment groups (P<0.05), which was consistent with the initial size of protocorm formed in the first stage of culture (weight 31.7 mg, length 0.9 cm) significantly better than other groups. Treatment 1 had the smallest protocorm formed in the first stage (weight 19.3 mg, length 0.58 cm), and the fresh weight and plant height were the lowest after transplanting, only 37.5% and 51.7% of treatment 3. The seedling growth index of treatment 2 and treatment 4 was between treatment 3 and treatment 1, and was significantly positively correlated with the size of the protocorm in the first stage. The control (CK) did not transplant seedlings because no protocorm was formed in the first stage. The results showed that the size of the protocorm formed in the first stage of culture was the key factor determining the growth of seedlings in the second stage, and the 3 cm sawdust treatment promoted the efficient development of protocorms, laying a foundation for subsequent growth.

[0056] 2.4 Third stage: seedling planting and greenhouse cultivation In this stage, two treatment groups were set up, soil with added fungus package blocks (treatment group) and soil without added fungus package blocks (control CK), and there was no sawdust in the soil. Healthy seedlings with good growth and similar size (plant height ≥ 8 cm) in treatment 3 in the second stage were selected for transplanting, 100 seedlings per group. After 60 days of greenhouse cultivation, the seedling growth index and survival rate of the two groups were significantly different (P<0.05). Figure 5 ).

[0057] From Figure 5 it can be seen that the plant height of the seedlings in the treatment group with added fungus package blocks was (19.6±1.3) cm, which was significantly higher than that of the control (13.3±0.9) cm (P<0.05), and the plant height increased by 47.4%; In terms of fresh weight, the average fresh weight of seedlings with added fungus package blocks was (3.8±0.25) g, which was significantly higher than that of the control (2.1±0.18) g (P<0.05), which was 1.81 times that of the control; In terms of seedling survival rate, the survival rate of the treatment group with added fungus package blocks reached 91%, which was significantly higher than that of the control (58%) (P<0.05), and the survival rate increased by 56.9%. The results showed that even if the seedlings cultivated in treatment 3 with the best growth in the second stage were transplanted, the soil environment with added fungus package blocks could still continuously promote the growth of seedlings and improve their survival ability, while the control environment without fungus package blocks significantly limited the growth potential of seedlings.

[0058] 3 DISCUSSION The traditional aseptic seeding technique of Cymbidium depends on high-precision tissue culture equipment, requires strict control of sterile environment, and involves MS medium configuration with sucrose, agar and various hormone substances. The cost of cultivating a single seedling is as high as 3-5 yuan, and professional operators need to be systematically trained, which has a high technical threshold. In addition, the tissue culture seedlings are in a constant temperature and humidity sterile environment for a long time, and their root development is fine, the cutin layer is weak, and they lack fungal symbiosis. After being transplanted to the natural environment, they cannot adapt to the changes in soil microbial community and physicochemical properties, and the survival rate is low.

[0059] The "three-stage seedling cultivation method" developed in this study effectively overcomes the limitations of traditional aseptic tissue culture techniques of Cymbidium by introducing associated bacteria to construct a fungal-plant symbiotic system. Studies have shown that fungi degrade sawdust substrate by secreting cellulase and ligninase, continuously releasing nutrients such as glucose and amino acids, which not only reduces the dependence on culture medium (significantly saving material costs), but also simulates the natural habitat's way of nutrient supply through biological transformation. In the first stage of seed associated bacteria germination culture, from the treatment effect of different sawdust specifications, 3cm sawdust block treatment Cymbidium protocorm grows the fastest, which may be because the 3cm sawdust block has appropriate porosity and water and fertilizer retention capacity, providing a good environment for the growth and reproduction of associated bacteria, thereby promoting the material exchange between protocorms through the network action of fungal hyphae. In contrast, the sawdust block may cause the substrate to be cemented due to the fine particles, resulting in poor aeration of the substrate, which cannot provide enough nutrients and suitable microenvironment for the symbiotic system. In the second stage of protocorm mixed bacteria germination culture, unlike the dependence on sterile environment of tissue culture seedlings, the root system of seedlings cultured by fungal symbiosis has formed mycorrhizal structure. This symbiotic relationship not only improves the nutrient uptake rate, but also enhances the disease resistance of the root system through the secretion of chitinase by the mycelium. In the third stage of transplanting mature seedlings, even if the seedlings (plant height ≥8cm) grown in the second stage treatment 3 (which has the best growth status) are transplanted, the soil environment with added fungus package block can still continuously promote the growth of seedlings and improve their survival ability, while the control environment lacking fungus package block significantly limits the growth potential of seedlings, further indicating the key role of stable symbiotic relationship between associated bacteria and Cymbidium in the later development of plants. The role of associated bacteria is not limited to the seed germination or protocorm formation stage, but extends throughout the growth process of Cymbidium seedlings, providing continuous theoretical support for their transition from artificial cultivation environment to natural soil environment, which further highlights the core value of fungal symbiosis in the "three-stage seedling method".

[0060] 4 Conclusion The "three-stage seedling cultivation method" developed in this study breaks through the limitations of traditional Cymbidium ensifolium tissue culture technology, achieving the synergistic optimization of economic feasibility, technical generalizability, and ecological adaptability. In terms of industrial application, this method has significant advantages: (1) Economic feasibility - sawdust substrate can be obtained from forestry waste factories, and fungus bag blocks can be mass-produced through simple fermentation tanks (cost about 5000 yuan). The cost of cultivating a single seedling is reduced to 1.2-1.5 yuan, which is only 30-40% (1 / 3-2 / 5) of the cost of tissue culture seedlings. With an annual production of 10,000 m 2 , the cost savings are 425-597 million yuan; (2) Technical generalizability - the method is simple to operate, and the entire process does not require a sterile operating room. Farmers can master the key technology after simple training, making it possible for local cultivation in remote areas; (3) Ecological adaptability - the root system of the seedlings produced through symbiotic cultivation is more robust, and the survival rate after transplantation is significantly improved, solving the core problem of poor environmental adaptability of tissue culture seedlings.

[0061] These innovative breakthroughs not only provide a commercially viable alternative for the large-scale production of Cymbidium ensifolium, but the technical principles can also be applied to the conservation and commercial cultivation of other rare orchid species. Future research can further optimize the combination of symbiotic strains and environmental control parameters to continuously improve the stability and applicability of the technology.

Claims

1. A three-stage seedling raising method for azalea orchid, characterized by: Follow these steps: (1) Cultivation of mycelial blocks of Coprinus albopictus: Inoculate Coprinus albopictus onto the slant of PDA culture medium and culture in the dark at a constant temperature of 23-27°C for 6-8 days. After the mycelium has grown all over the plate, pick the pure mycelium at the edge and transfer it to a new PDA culture medium. Purify continuously for 2-4 times to obtain mycelial blocks of Coprinus albopictus, which are then stored at 3-5°C for later use. (2) Preparation of sawdust-bran mixed matrix: grind broadleaf sawdust through a 1.5-2.5 mm sieve, add bran, gypsum powder and sucrose and mix. The mass ratio of broadleaf sawdust, bran, gypsum powder and sucrose is 7-9:0.8-0.12:0.4-0.6:0.4-0.

6. Add water to adjust the moisture content to 60%-65% to obtain a sawdust-bran mixed matrix, which is set aside. (3) Preparation of the white false agaricus fungus bag: 400-600 g of sawdust-bran mixed matrix is ​​packed into a bag, compacted and sealed with cotton plugs. After sterilization, the bag is cooled to room temperature and then inoculated with white false agaricus mycelium blocks in a clean bench. Each bag is inoculated with 2-4 blocks, each of which has a diameter of 9-11 mm. The bags are cultured in the dark at 23-27 ° C for 18-22 days until the mycelium grows all over the bag and forms sclerotia. The white false agaricus fungus bag is obtained and set aside. (4) Preparation of humus soil: Take humus soil with a pH of 5.5-6.5 and sterilize it under high pressure at 121℃ for 2 hours to obtain sterilized humus soil for later use; (5) Seed-associated fungus germination culture: Four layers of culture substrate are laid in the germination culture container, namely the bottom layer, the middle layer, the upper layer and the surface layer. The bottom layer is laid with sterilized humus soil with a thickness of 3.5-4.5 cm. The middle layer is laid on the sterilized humus soil of the bottom layer. The structure of the middle layer is designed to first use half of the sawdust blocks as a base, and then distribute the white pseudo-coprinus agaricus mushroom bags in a grid shape with a spacing of about 4.5-5.5 cm on the sawdust blocks, and then completely cover the remaining sawdust blocks. The thickness of the middle layer is 4-9 cm, and the volume ratio of the sawdust blocks to the white pseudo-coprinus agaricus mushroom blocks is 0.8-1.2:0.8-1.

2. The size of the sawdust blocks is 1-2 cm thick, 1 cm wide, and 1-5 cm long. The white pseudo-coprinus agaricus mushroom blocks are obtained by crushing the white pseudo-coprinus agaricus mushroom bags into 3-4 cm 3 The middle layer is covered with an upper layer of sterilized humus soil with a thickness of 0.8-1.2 cm, and the azalea seeds are sown on the sterilized humus soil on the upper layer; finally, the surface layer is covered with sterilized humus soil with a thickness of 0.8-1.2 cm. The sterilized humus soil covers the seeds, and the seeds are cultured for 90 days under the conditions of relative humidity of 70%-85% and temperature of 20-22°C to obtain the germinated azalea protocorms; (6) Protocorm germination cultivation with fungi: Crush the white pseudo-coprinus agaricus mushroom bags into 0.5-1 cm particles and mix them with sterilized humus soil at a mass ratio of 1:2.5-3.5 as germination cultivation medium, and put them into the germination cultivation container to a height of 0.8-1.0 cm. Select the azalea orchid protocorms with a diameter of ≥5 mm and plant them together with the germination culture medium at a spacing of 2.5-3.5 cm into the germination cultivation container, cover the protocorms with the germination culture medium to completely cover the protocorms and be 0.8-1.2 cm higher than the protocorms, water them thoroughly to establish roots, and place them in a greenhouse at 22-25℃ and a relative humidity of 70%-85% for cultivation, with a light duration of 12h / d, for 110-130 days, and water them every 1-3 days to keep the medium moist. Wait until the protocorms differentiate into leaves and roots, and obtain mature azalea orchid seedlings with a plant height of ≥8 cm; (7) Transplanting seedlings: Prepare the soil in the greenhouse in advance, dilute carbendazim 800-1200 times and spray it on the greenhouse soil for sterilization and disinfection, then use clothianidin to kill pests in the soil. After disinfection for 2-4 days, crush the white false agaricus mushroom capsules into 0.5-1cm particles and mix them with the soil. The mass ratio of soil to white false agaricus mushroom capsules is 2:0.8-1.

2. After deep plowing 25-35cm, level the soil to make a ridge. The ridge is 1-1.5m wide and 18-25cm high. Select azalea orchid with a height of ≥8cm and transplant it with soil clumps to the ridge surface. The spacing between rows and plants is 10cm×15cm. The transplanting depth should be such that the top of the bulb is just submerged in the soil surface. Water thoroughly to allow the roots to take root. The temperature in the greenhouse is controlled at 22-28℃. Use a shade net to shade about 75%. Cultivate for 55-65 days and monitor the growth of the seedlings.

2. The three-stage seedling raising method of azalea according to claim 1, wherein: In the step (1), the mycelial block of Coprinus comatus is cultured as follows: Coprinus comatus is inoculated onto the slant of a PDA culture medium, and cultured in the dark at a constant temperature of 25°C for 7 days. After the mycelium has grown all over the plate, pure mycelium at the edge is picked and transferred to a new PDA culture medium, and purified three times continuously to obtain the mycelial block of Coprinus comatus, which is then stored at 4°C for later use.

3. The three-stage seedling raising method for azalea orchid according to claim 1 or 2, wherein: In the step (1), the PDA culture medium is prepared by adding 200 g / L potato, 20 g / L glucose, and 20 g / L agar to a volume of 1 L with distilled water, and then sterilized under high pressure at 121° C. for 20 min.

4. The three-stage seedling raising method of azalea according to claim 1, wherein: In the step (2), the mass ratio of broadleaf sawdust, bran, gypsum powder and sucrose is 8:1:0.5:0.

5.

5. The three-stage seedling raising method of azalea according to claim 1, wherein: In the step (3), 500 g of the sawdust-bran mixed matrix is ​​packed into bags, compacted and sealed with cotton plugs, sterilized at 121° C. for 2 h, cooled to room temperature, and then inoculated with white pseudo-coprinus mycelium blocks in a clean bench, 3 blocks per bag, each of which has a diameter of 10 mm. The bags are cultured in the dark at 25° C. for 20 days until the mycelium grows full of fungus bags and forms sclerotia, thereby obtaining white pseudo-coprinus mycelium bags for later use.

6. The three-stage seedling raising method for azalea orchid according to claim 1 or 5, characterized in that: In the step (3), the culture bag used for the sawdust-bran mixed matrix is ​​a polypropylene bag, and the specification of the polypropylene bag is 17 cm×33 cm.

7. The three-stage seedling raising method for azalea according to claim 1, wherein: In the step (5), the seed is cultured with fungi for germination: four layers of culture substrate are laid in the germination culture container, namely the bottom layer, the middle layer, the upper layer and the surface layer. The bottom layer is paved with sterilized humus soil with a thickness of 4 cm. The middle layer is laid on the sterilized humus soil of the bottom layer. The structure of the middle layer is designed to first use half of the sawdust blocks as a base, and the white pseudo-coprinus mushroom bags are distributed on the sawdust blocks in a grid shape with a spacing of about 5 cm, and then the remaining sawdust blocks are completely covered. The thickness of the middle layer is 5-8 cm, the volume ratio of the sawdust blocks to the white pseudo-coprinus mushroom bag blocks is 1:1, the size of the sawdust blocks is 1-2 cm thick, 1 cm wide, and 1-5 cm long, and the white pseudo-coprinus mushroom bag blocks are obtained by crushing the white pseudo-coprinus mushroom bags into 3-4 cm 3 The middle layer is covered with an upper layer of sterilized humus soil, the thickness of the upper layer is 1 cm, and the azalea seeds are sown on the sterilized humus soil on the upper layer; Finally, cover the surface with sterilized humus soil with a thickness of 1 cm. Cover the seeds with sterilized humus soil and culture them for 90 days under the conditions of relative humidity of 70%-85% and temperature of 20-22°C to obtain the germinated azalea protocorms.

8. The three-stage seedling raising method for azalea according to claim 1, wherein: In the step (6), the protocorms are cultured with bacteria for germination: the white pseudo-coprinus agaricus mushroom bags are crushed into 0.5-1 cm particles and mixed with sterilized humus soil in a mass ratio of 1:3 as a germination culture medium, and the particles are placed in a germination culture container to a height of 0.8-0.9 cm. The protocorms of azalea orchid with a diameter of ≥5 mm are selected and planted together with the germination culture medium at a spacing of 3 cm into the germination culture container, and the germination culture medium is covered to completely cover the protocorms and be 1 cm higher than the protocorms. The seeds are watered thoroughly for rooting, and the seeds are placed in a greenhouse at 22-25°C and a relative humidity of 70%-85% for cultivation, with a light duration of 12 h / d, for 120 days, and watered every 1-3 days to keep the medium moist. The protocorms are differentiated into leaves and roots to obtain mature azalea orchid seedlings with a plant height of ≥8 cm.

9. The three-stage seedling raising method for azalea according to claim 1, characterized in that: In the step (7), seedling transplanting: prepare the soil in advance in the greenhouse, dilute carbendazim 1000 times and spray it on the greenhouse soil for sterilization and disinfection, then spray the soil pests with clothianidin, and after disinfection for 3 days, crush the white false agaricus mushroom capsules into 0.5-1 cm particles and mix them with soil, the mass ratio of soil to white false agaricus mushroom capsules is 2:1, deep plow 30 cm and then level and make a bed, the bed width is 1.2 m and the height is 20 cm, select azalea orchids with a height of ≥8 cm and transplant them with soil clumps to the bed surface, the plant spacing is 10 cm×15 cm, the transplanting depth is preferably such that the top of the bulb is just submerged by the soil surface, and water thoroughly to establish roots; the temperature in the greenhouse is controlled at 22-28 ° C, and a shade net is used to shade about 75%. Cultivate for 60 days and monitor the growth of the seedlings.

10. The three-stage seedling raising method of azalea according to claim 1, characterized in that: In the step (7), after the seedlings are transplanted, field management is carried out, weeding is performed, and diluted organic fertilizer is sprayed.

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