Cymbidium orchid non-humus soil seedling cultivation and high seedling emergence rate transplanting method

Abstract

The application discloses a kind of dujuanlan non-humus seedling cultivation and high germination rate transplanting method, belong to plant cultivation technical field.The application uses waste mushroom residue and wood ash composite substrate to combine coprophilous fungi to symbiotic seedling, without humus, greatly reduce technical threshold and cost, seedling symbiotic flora development perfect, strong adaptability.Transplanting stage adopts wood block hydrophobic layer, double-layered strain layer and pine needle covering and other multi-layer three-dimensional structure, germination rate reaches more than 44%, and transplanting survival rate reaches more than 96%, significantly better than traditional land cultivation and tissue culture seedling transplanting.The whole process uses agricultural waste (waste mushroom residue, waste mushroom stick) as main raw material, realizes resource recycling, green and environmental protection and low cost.The whole process is standardized, easy to operate, suitable for large-scale promotion, and has ecological and economic benefits.

Description

Technical Field

[0001] This invention relates to the field of plant cultivation technology, and in particular to a method for cultivating rhododendron seedlings without humus soil and transplanting them with a high germination rate. Background Technology

[0002] *Cremastra*, a perennial terrestrial herb belonging to the genus *Cremastra* in the family Orchidaceae, is known as *Shan Cigu*, a traditional Chinese medicine. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it was listed as a lower-grade herb and is included in the 2020 edition of the *Chinese Pharmacopoeia*. *Shan Cigu* is sweet and slightly pungent in taste, cool in nature, and slightly toxic. It possesses properties of clearing heat and detoxifying, resolving phlegm and dissipating nodules, and reducing swelling. Clinically, it is mainly used for carbuncles, boils, scrofula, lymph node tuberculosis, and snake and insect bites. Modern pharmacological studies have shown that its active ingredients mainly include phenanthrene, bibenzyl, and flavonoids, which have important application value in the development of anti-tumor drugs. *Cremastra* naturally grows in shady and damp environments under forests at altitudes of 500-3000m, preferring cool, shady, and well-drained habitats. It flowers from May to June and fruits from September to December.

[0003] Due to its extremely high medicinal value and persistently high market price, wild Rhododendron orchids have suffered from long-term predatory harvesting, coupled with the continuous destruction of their natural habitats, leading to a sharp decline in wild populations. It has been listed in the "National Key Protected Wild Plants List" (2021) as an endangered protected plant. Large-scale artificial cultivation has become the only feasible way to ensure the supply of *Cremastra appendiculata* medicinal materials and protect wild resources. However, as a typical orchid, Rhododendron orchids have slender, spindle-shaped seeds without endosperm, resulting in incomplete embryo development. The seed coat consists of a double layer of cells, and the outer seed coat is not easily permeable to water. Under natural conditions, germination requires infection by specific symbiotic fungi, and the natural germination rate is usually less than 10%, severely limiting the efficiency of artificial propagation.

[0004] Currently, existing artificial propagation methods are mainly divided into two categories. The first category is tissue culture seedling cultivation technology, which requires multiple processes such as seed germination induction, protocorm proliferation and differentiation, and rooting and seedling strengthening. It requires high investment in specialized equipment such as ultra-clean workbenches and constant temperature incubation rooms, and has a high technical threshold, making it difficult for ordinary growers to master. At the same time, tissue culture seedlings are separated from the natural microbial environment for a long time, resulting in a long recovery period after transplanting to the field, weak environmental adaptability, and a generally low survival rate (generally 50%-65%). Moreover, it continuously consumes wild germplasm resources as explant sources, which is not conducive to large-scale sustainable promotion. The second category is traditional ground planting seedling cultivation, which involves directly sowing azalea seeds into the soil without adding targeted symbiotic bacteria and suitable growth substrates. The soil is prone to compaction, leading to insufficient aeration, and waterlogging during the rainy season, resulting in seed and seedling rot. The germination rate is generally low (20%-30%), and the seedlings are uneven in appearance and growth, making later field management difficult.

[0005] On the other hand, my country's edible mushroom industry has a huge annual output, generating a large amount of waste mushroom residue and substrate (such as oyster mushroom substrate and shiitake mushroom residue). These agricultural wastes still contain rich organic matter, protein, cellulose, and various trace elements. Random dumping not only occupies land resources but also easily breeds miscellaneous bacteria and pollutes the surrounding water environment. Currently, the resource utilization of waste mushroom residue mainly focuses on organic fertilizer preparation, feed additives, and fuel utilization, resulting in relatively simple utilization methods and generally low added value. Using waste mushroom residue as a raw material for orchid cultivation substrate can solve the waste disposal problem and reduce cultivation costs, offering significant ecological and economic benefits.

[0006] In summary, existing rhododendron cultivation techniques suffer from four major bottlenecks: a single seedling cultivation model (high tissue culture costs and low germination rates with traditional field planting), an imperfect transplanting system (poor compatibility between seedlings and field microbial communities, resulting in unstable germination rates), the large-scale collection of forest humus during artificial cultivation to accommodate the wild habits of rhododendrons, which damages the ecological environment, and the ineffective recycling of agricultural waste microbial residue. Therefore, it is necessary to develop an integrated method that organically combines humus-free symbiotic seedling cultivation with structured field transplanting, and deeply couples the resource utilization of agricultural waste with the efficient propagation of rhododendrons, to simultaneously solve the core problems of low seedling cultivation efficiency, poor transplant survival rates, and high production costs. Summary of the Invention

[0007] The purpose of this invention is to provide a method for cultivating Rhododendron seedlings without humus soil and transplanting them with a high germination rate, thereby solving the problems existing in the prior art. This invention deeply couples the symbiotic germination-inducing technology of Coprinus comatus fungi with the resource utilization of agricultural waste fungal residue, constructing an integrated technology system for high-yield field transplanting of protocorms through humus-free symbiotic cultivation and stratified cultivation of Rhododendron seedlings in a suitable environment. This solves the problem of ecological damage caused by the traditional need to collect large amounts of forest humus soil for Rhododendron cultivation, improves the success rate of Rhododendron seedling cultivation and transplanting, and promotes green development.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for cultivating azalea seedlings without humus soil and transplanting them with a high germination rate, comprising the following steps: (1) Humus-free seedling cultivation The waste residue from edible fungi is mixed with wood ash to obtain a mixed matrix; The activated Coprinus fungi were propagated and cultured to obtain cultivated strains; The cultivation strain is inoculated onto the surface of the mixed substrate, and then azalea seeds are sown on top of the cultivation strain. After cultivation, seedlings containing protocorms and free of humus are obtained. (2) Field transplanting and planting Prepare the field land, build a grid planting area, lay wooden blocks in the grid planting area, and carry out the first disinfection treatment as the wooden block layer; Cut the cultured spawn into pieces and spread the cut spawn on top of the wood block layer to form a large spawn layer; A composite substrate made of edible fungi waste residue and wood ash is laid on top of the large-block inoculum layer as a substrate layer. The culture strain is crushed and then sown on top of the substrate layer to form a crushed culture strain layer. The seedlings containing the original bulbs in humus-free soil were sown on top of the broken inoculum layer. Edible fungi waste substrate and fine soil were then laid on top of the seedlings containing the original bulbs in humus-free soil. The surface of the fine soil was then subjected to a second disinfection treatment. Leaves were then laid on top of the disinfected fine soil. After cultivation, azalea seedlings were obtained.

[0009] Further, in step (1), the mass ratio of the edible fungus waste residue to wood ash is 80-90:10-20; the moisture content of the mixed substrate is 45%-55%.

[0010] Further, in step (1), the method of propagation and cultivation is to inoculate the activated Coprinus fungi into the culture medium for edible fungi cultivation and cultivate them at 27-29℃ and 65%-75% relative humidity until the mycelium fully grows on the culture medium. The formula for the culture medium for edible fungi cultivation is: 78% cottonseed hulls, 20% wheat bran, 1% gypsum and 1% lime, with a water content of 60%-65%.

[0011] Further, in step (1), the thickness of the mixed substrate is 8-12 cm; the inoculation amount of the cultivated microbial strain is 0.01-0.02 m. 3 100g of culture spawn was inoculated into the mixed substrate; the azalea seeds were sown at a rate of 0.01-0.02m³. 3 Sow seeds from 1 / 4 to 1 / 2 of a capsule in the mixed substrate.

[0012] Further, in step (1), the cultivation temperature is 20-28℃, the relative humidity is 60%-75%, the shading degree is 75%-85%, and the cultivation is carried out in the dark and ventilated for 40-60 days; during the cultivation period, the substrate moisture content is controlled at 50%±5%.

[0013] Further, in step (2), the method of repairing the field land is as follows: repair according to the original terrain of the planting plot, remove stones, weeds, dead branches, grass roots and other debris from the soil, rake the soil to make it level and fine, and sort out the field drainage ditches to ensure smooth drainage of the plot and prevent water accumulation after rain; the slope of the repaired plot is controlled at 3°-8° to ensure natural drainage, and drainage ditches are opened around the site with a depth of 20-30cm, which are connected to natural waterways to ensure that excess water can be discharged in time during heavy rain. The width of the square planting area is 0.8-1.2m and the length is 8-12m; the spacing between two adjacent square planting areas is 8-12cm; the square planting area uses a hard wooden template with a width of 10cm and a thickness of 2cm, which is built on the leveled plot; the spacing serves as a field operation passage, taking into account both drainage and daily management needs. The wood block layer consists of wood blocks that are 8-10cm long, 2-3cm wide, and 0.4-0.6cm thick, laid evenly at a rate of 8kg / m². 2 The thickness of the wood blocks is 3-5cm; the wood blocks include broad-leaved wood blocks, which are naturally dried to a moisture content of 15%-20% before use; the wood blocks are loosely arranged with uniform gaps to form a hydrophobic layer that can quickly drain excess water; The large inoculum layer consists of cultivated inoculum 4-6 cm wide and 1-2 cm thick, spread at a rate of 3.5 kg / m². 2 Large-scale cultivation of microbial strains can be established above the wood block layer, and the hyphae can extend downwards along the gaps between the wood blocks to the wood block layer and upwards to the substrate layer, thus constructing a basic symbiotic microbial community system. The substrate layer is made of a composite substrate obtained by mixing edible fungus waste residue and wood ash in a mass ratio of 80-90:10-20, with a thickness of 0.8-1.2cm. The fragmented inoculum layer is formed by spreading cultivated inoculum with a particle size of 0.5-1.5 cm at a spreading rate of 5 kg / m³. 2 The fragmented fungal blocks scattered on the substrate surface increase the contact area with the protocorm and strengthen the symbiotic relationship. The design of the total amount of inoculum in the large inoculum layer and the fragmented inoculum layer fully ensures the density of effective symbiotic microbial communities per unit area; The sowing rate of the humus-free seedlings containing protocorms is per 6m³. 2 Spread at 0.01-0.02m 3 Nutrient-free seedlings containing protocorms; The thickness of the edible mushroom waste substrate is 0.4-0.6cm; the edible mushroom waste substrate is shiitake mushroom waste substrate; shiitake mushroom waste substrate is lightweight and highly breathable, and when laid on it, it can form a protective layer on top of the seedlings, blocking direct sunlight and slowly releasing trace elements. The thickness of the fine soil layer is 0.5-1cm; The leaves include pine needles and broad-leaved leaves, with a thickness of 2-4 cm. The pine needles and broad-leaved leaves are mixed in a volume ratio of 1:1 to 2:1. The covering layer can provide shade, heat preservation, and water retention. During the decomposition process, the pine needles and leaves continuously release organic matter and trace nutrients, while providing a suitable micro-ecological environment for surface symbiotic fungi, simulating the growth environment of wild rhododendrons covered with dead branches and fallen leaves.

[0014] Furthermore, in step (2), the cultivation temperature is 20-28℃, the soil moisture is maintained at 50%-55% for 15 days after sowing, and the soil moisture is increased to 55%-60% after 15 days to promote the germination of the original bulbs; the shade of the shade shed is not less than 80%; weeds are managed by manual weeding at regular intervals, and the use of chemical herbicides is prohibited; pest and disease control is carried out at least once a month in the field, and preventive control is carried out for common diseases of orchid plants (such as gray mold and white mold) and pests (such as aphids and spider mites), and biological agents are given priority.

[0015] Furthermore, the method for the first disinfection treatment is as follows: spray a mixture of broad-spectrum fungicide and insecticide into the grid planting area, ensuring that the surface wood blocks are fully moistened but without any flowing water, and let it stand for 12-24 hours; The second disinfection treatment method is as follows: spray a mixture of broad-spectrum bactericide and insecticide onto the surface of the fine soil, with the spraying amount being 1 / 3 to 1 / 2 of the amount sprayed in the first disinfection treatment.

[0016] Optionally, the broad-spectrum fungicide is 800-1000 times dilution of carbendazim or thiophanate-methyl wettable powder, and the insecticide is 2000-3000 times dilution of high-efficiency cyhalothrin emulsifiable concentrate.

[0017] Furthermore, the edible fungi waste residue includes shiitake mushroom waste residue or oyster mushroom waste substrate. Oyster mushroom waste substrate has a high organic matter content (crude protein content of approximately 8%-12% and crude fiber content of approximately 15%-25%), and when combined with wood ash, it can optimize the substrate pH value (adjusting it to 6.5-7.0), while also possessing air permeability, fertilizer retention, and water retention properties, providing sufficient nutrients for the growth of spawn and protocorms. Shiitake mushroom waste residue is lightweight, highly permeable, and can slowly release trace elements.

[0018] Furthermore, the *Coprinus* fungus is *Coprinus granulosus*.

[0019] The present invention discloses the following technical effects: (1) The innovative humus-free seedling cultivation model significantly improves the quality of seedlings. This invention employs a symbiotic cultivation technique using a composite substrate of waste fungal residue and wood ash, combined with *Coprinus* fungi. It fully simulates the symbiotic germination process of *Azalea* seeds in their natural habitat, eliminating the need for expensive tissue culture equipment and specialized technicians, significantly reducing the technical threshold and investment costs. In the humus-free cultivation system, *Coprinus* fungal hyphae invade the *Azalea* seed embryo, decomposing organic macromolecules such as cellulose and lignin in the substrate, converting them into small-molecule nutrients required for seed germination and protocorm growth, thus achieving targeted cultivation of robust protocorms. Seedlings cultivated using this method exhibit well-developed symbiotic fungal communities and significantly stronger adaptability than tissue-cultured seedlings.

[0020] (2) The three-dimensional layered transplanting structure greatly improves the germination rate and survival rate. This invention employs a multi-layered, three-dimensional structure in the transplanting stage of Rhododendron seedlings, consisting of a bottom layer of permeable wooden blocks, a double layer of mycelium, a multi-layered substrate, a thin layer of topsoil, and a shading layer of pine needles and leaves. The bottom layer of wooden blocks provides rapid drainage, preventing waterlogging and seedling rot. The first layer of large pieces of mycelium establishes a basic symbiotic microbial community, while the second layer of broken mycelium increases the contact area with the original bulb. The double-layered inoculation creates a high-density, multi-layered symbiotic microbial network, shortening the distance between the mycelium and the original bulb and strengthening the symbiotic relationship. The combination of oyster mushroom and shiitake mushroom waste for covering provides excellent aeration, water retention, and fertilizer retention. The thin layer of topsoil and pine needles and leaves prevents excessive soil layer pressure on the original bulb while providing a microenvironment for shading, moisture retention, and warmth. Under these combined effects, the seedling emergence rate reaches over 44%, and the transplant survival rate reaches over 96%, significantly higher than traditional ground planting (emergence rate approximately 26%, survival rate approximately 58%) and tissue culture seedling transplanting (survival rate approximately 65%). The wood block layer has a dual-function design: firstly, a physical hydrophobic function—the natural gaps between the wood blocks form capillary drainage channels, which can quickly drain excess water and prevent waterlogging in the lower soil layer, which could lead to oxygen deficiency and rotting of the protocorm; secondly, a biological attachment function—the rough surface and loose structure of the wood blocks provide an ideal attachment carrier for the downward extension of Coprinus mycelium, allowing the mycelium to fully expand vertically along the gaps between the wood blocks, constructing a three-dimensional mycelial network covering the entire transplant profile, continuously providing nutrients to the protocorm and roots of the Rhododendron, while effectively inhibiting the growth of pathogenic microorganisms of Rhododendron in nature.

[0021] (3) Recycling agricultural waste to achieve green and low-carbon production The humus-free cultivation substrate of this invention uses waste mushroom residue as the main raw material. During the field transplanting stage, agricultural waste such as waste mushroom sticks of oyster mushrooms and waste mushroom residue of shiitake mushrooms are used to construct a multi-layer substrate system, realizing the resource-efficient reuse of waste mushroom residue from edible mushrooms. This solves the problem of environmental pollution caused by the random dumping of waste residue from the edible mushroom industry. At the same time, it significantly reduces the raw material procurement cost of the azalea cultivation substrate, achieving a win-win situation for both ecological and economic benefits.

[0022] (4) The process is standardized, highly operable, and suitable for large-scale promotion. The operation methods, material ratios, and dosage specifications of all processes in this invention are quantitatively defined, making the operation simple and highly repeatable. No professional tissue culture technicians are required; ordinary growers can master and independently complete all operations after simple training. The grid-based planting model facilitates field management, targeted pest and disease control, and zoned harvesting, making it suitable for large-scale artificial cultivation in the field. The entire process uses Coprinus fungi as the symbiotic fungus, ensuring that the fungal community is consistent with that of seedlings grown in humus-free soil and transplanted to the field. There is no transition period for fungal community adaptation in the seedlings, resulting in a short recovery period, uniform emergence, and consistent growth in the later stages, facilitating standardized medicinal herb production management. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The seedlings containing protocorms and grown in humus-free soil were obtained in step one of Example 1. Figure 2 This is a schematic cross-sectional view of the stratified structure of the grid planting area in step two of Example 1. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0031] Example 1 Step 1: Cultivation of seedlings in humus-free soil 1.1 Substrate Pretreatment: Take fresh shiitake mushroom waste residue, mechanically crush it, and pass it through a 5mm sieve. Mix it thoroughly with wood ash at a mass ratio of 85:15. Gradually spray clean water into the mixture while continuously stirring, adjusting the overall moisture content to 50%. Dispense the prepared mixed substrate into high-temperature resistant sterilization bags and autoclave at 121℃ and 0.1MPa for 2 hours. After sterilization, allow it to cool naturally to room temperature (approximately 25℃) for later use.

[0032] 1.2 Propagation of *Coprinus* strains: *Coprinus granulosus* (CGMCC 5.671), activated on PDA slant medium, was inoculated into sterilized conventional edible mushroom cultivation medium (formula: 78% cottonseed hulls, 20% wheat bran, 1% gypsum, 1% lime, moisture content 60%-65%) in a sterile operating table. The inoculation amount was approximately 10g of original culture per bag. After inoculation, the bags were transferred to a 28℃ constant temperature incubation room, maintaining a relative humidity of 65%-75%, and incubated for 18-22 days until the mycelium completely covered the medium, yielding a sufficiently active spawn.

[0033] 1.3 Symbiotic Humus-Free Soil Cultivation: Evenly fill the sterilized mixed substrate into seedling containers (35cm × 50cm). Pre-line the inside of the containers with perforated plastic sheeting, covering the bottom and surrounding edges. Make a hole every 10cm, with a diameter of 3-5cm, to prevent seedling roots from penetrating the container and causing root entrapment, while allowing for smooth drainage of water and fertilizer, protecting the substrate structure, and ensuring the integrity of the root ball during transplanting. The substrate filling thickness should be 10cm. Evenly inoculate the substrate surface with 100g of inoculum per bag, then evenly sow 1 / 3 of the seeds of a mature, unopened rhododendron capsule (approximately 2000-3000 seeds) above the inoculum layer. See the schematic diagram of the final seedling container structure. Figure 1 .

[0034] Cultivation environment parameters: temperature 24℃, relative humidity 68%, shading 80%, cultivated in the dark and well-ventilated environment for 50 days. During cultivation, check the substrate moisture content every 7 days. If the substrate shows signs of drying, replenish moisture by misting to maintain a moisture content of 50% ± 5%. After 50 days, the azalea seeds differentiate into plump and robust protocorms, yielding seedlings containing protocorms in humus-free soil (see...). Figure 1 ).

[0035] Step 2: Field transplanting and planting 2.1 Land leveling: Select a shady, well-drained site, remove stones, weeds, dead branches, grass roots and other debris from the field, rake the soil to a fine texture, and dig drainage ditches 25cm deep around the site.

[0036] 2.2 Grid construction: Using wooden templates with a width of 10cm and a thickness of 2cm, construct long strip grids with a single width of 1m, a grid length of 10m, and a spacing of 10cm between adjacent grids.

[0037] 2.3 Wood Block Laying: Lay small broadleaf wood blocks, 9cm long, 2.5cm wide, and 0.5cm thick, within the grid, using 8kg / m². 2 Natural gaps are maintained between the wooden blocks.

[0038] 2.4 First disinfection: Mix 800 times dilution of 50% carbendazim wettable powder with 2500 times dilution of 2.5% high-efficiency cyhalothrin emulsifiable concentrate, spray evenly on the surface of the wooden blocks in the grid, and let stand for 18 hours.

[0039] 2.5 First inoculation: Take the fully propagated spawn, manually cut it into blocks 5cm wide and 1.5cm thick, and evenly spread them on top of the sterilized wood block layer. The dosage is 3.5kg / m². 2 .

[0040] 2.6 Substrate laying: Mix the waste mushroom logs of oyster mushrooms with wood ash at a mass ratio of 85:15 to form a composite substrate, crush it to a particle size of 1mm using a pulverizer, and evenly lay it on top of the first batch of mushroom blocks to a thickness of 1cm.

[0041] 2.7 Second inoculation: Take another batch of cultured microorganisms, crush them into pieces about 1 cm in diameter using a pulverizer, and evenly spread them on the surface of the composite substrate at a rate of 5 kg / m². 2 .

[0042] 2.8 Seedling sowing: Mix the humus-free seedlings containing protocorms obtained in step one with the substrate in the seedling container evenly, sowing at a rate of 6m². 2 Sow the seedlings for one seedling container (step one) evenly on the surface of the broken fungal blocks.

[0043] 2.9 Covering and covering with soil: Take the crushed shiitake mushroom waste substrate (particle size 4-8mm) and spread it evenly on top of the seedling layer to a thickness of 0.5cm; take fine field soil (particle size 2-3mm) and cover it evenly on top of the shiitake mushroom waste substrate to a thickness of 0.8cm. Humus soil is not required.

[0044] 2.10 Secondary disinfection: Mix 800 times dilution of carbendazim with 2500 times dilution of high-efficiency cyhalothrin, and then lightly spray the soil surface. The amount of solution used is half of the amount used in the first disinfection.

[0045] 2.11 Surface Covering: A 3cm thick layer of mixed pine needles and broadleaf leaves (pine needles:broadleaf leaves = 1.5:1 by volume) is evenly spread on top of the soil after secondary disinfection. See the schematic diagram of the stratified structure of the grid planting area in the field. Figure 2 .

[0046] 2.12 Field Management: The ambient temperature should be controlled at 24℃ throughout the process; the soil moisture should be maintained at 52% for the first 15 days after sowing, and water should be replenished every 3 days by misting; after 15 days of sowing, the soil moisture should be increased to 56%, and water should be replenished every 5 days; a shade structure should be built to ensure 82% shading; weeds in and around the grid should be removed manually every 10 days; a field inspection should be carried out once a month, focusing on checking the germination status of the original bulbs and the occurrence of diseases and pests.

[0047] Comparative Example 1 (Traditional Soil Direct Seeding Cultivation) Select a shady, well-drained area of ​​the same plot, level the soil, and directly sow an equal amount of azalea seeds. Water and shade the plants as usual, without adding symbiotic bacteria or laying substrate or wood blocks. All other environmental conditions are the same as in Example 1.

[0048] Comparative Example 2 (Conventional Transplanting of Tissue Culture Seedlings) Existing Rhododendron tissue culture seedlings (purchased from Yunnan Haochen Agricultural Co., Ltd.) were used. After leveling the same plot, the seedlings were directly transplanted into the soil. Conventional field management was carried out, without stratification or supplementation of symbiotic bacteria. All other environmental conditions were the same as in Example 1.

[0049] Table 1 shows the comparison of specific cultivation parameters and cultivation effects between Example 1 and Comparative Examples 1 and 2.

[0050] Table 1 Comparison of cultivation parameters and results between Example 1 and the comparative example Example 2 The differences from Example 1 are as follows: Step 1: Cultivation of seedlings in humus-free soil 1.1 The substrate moisture content was adjusted to 48%, and sterilization was carried out under normal pressure (105℃, 11h); the remaining operations were the same as in Example 1.

[0051] 1.2 The cultivation temperature was controlled at 22℃, the relative humidity was 65%, the shading degree was 78%, and the cultivation period was 45 days. The remaining operations were the same as in Example 1.

[0052] Step 2: Field transplanting and planting 2.1 The specifications of the small wooden blocks are 8cm in length, 2cm in width, and 0.5cm in thickness; the first disinfection and static placement time is 12h; the remaining operations are the same as in Example 1.

[0053] 2.2 The thickness of the topsoil cover is 0.5cm; the thickness of the topsoil cover layer of mixed pine needles and leaves is 2cm; the rest of the operation is the same as in Example 1.

[0054] 2.3 The field temperature was controlled at 22℃; the soil moisture was kept at 50% for the first 15 days after sowing, and then increased to 53% after 15 days; the soil moisture was checked and replenished every 5 days, and the remaining operations were the same as in Example 1.

[0055] Table 2 shows the comparison of specific cultivation parameters and cultivation effects between Example 2 and Comparative Examples 1 and 2.

[0056] Table 2 Comparison of cultivation parameters and results between Example 2 and the comparative example Example 3 The differences from Example 1 are as follows: Step 1: Cultivation of seedlings in humus-free soil 1.1 The substrate moisture content was adjusted to 53%, and it was sterilized at 121℃ for 1.8h; the remaining operations were the same as in Example 1.

[0057] 1.2 The cultivation temperature was controlled at 27℃, the relative humidity was 72%, the shading degree was 83%, and the cultivation period was 58 days. The remaining operations were the same as in Example 1.

[0058] Step 2: Field transplanting and planting 2.1 The specifications of the small wooden blocks are 10cm long, 3cm wide and 0.5cm thick; the first disinfection and static placement time is 24h; the rest of the operation is the same as in Example 1.

[0059] 2.2 The topsoil layer thickness is 1cm; the topsoil layer of mixed pine needles and leaves is 4cm thick; the remaining operations are the same as in Example 1.

[0060] 2.3 The field temperature should be controlled at 27℃; the soil moisture should be maintained at 55% for 15 days after sowing, and then increased to 60% after 15 days; the watering frequency should be adjusted according to the weather conditions, and the rest of the operation is the same as in Example 1.

[0061] Table 3 shows the comparison of specific cultivation parameters and cultivation effects between Example 3 and Comparative Examples 1 and 2.

[0062] Table 3 Comparison of cultivation parameters and results between Example 3 and the comparative example The seed / seedling germination rate, transplant survival rate, and germination uniformity of each embodiment and comparative example were statistically analyzed as follows: Examples 1-3: The germination rate was no less than 44% (47% in Example 1, 44% in Example 2, and 45% in Example 3), and the transplant survival rate was no less than 92% (94% in Example 1, 92% in Example 2, and 93% in Example 3). The germination was uniform, the seedlings were vigorous, the leaves were of normal color, and the root system was well developed.

[0063] Comparative Example 1 (traditional direct seeding in soil): The germination rate was only about 26%, and the seedlings grew unevenly after emergence. Some seedlings had yellow leaves and poor root development, with a survival rate of about 58%.

[0064] Comparative Example 2 (conventional transplanting of tissue culture seedlings): The survival rate of transplanting was about 65%, the recovery period was as long as 15-20 days, the emergence was delayed and uneven, and some plants showed weak seedlings and dead seedlings in the later stage.

[0065] Experimental results show that the integrated method of humus-free symbiotic seedling cultivation and layered structural transplanting provided by this invention can significantly improve the germination rate and transplant survival rate of Rhododendron simsii, demonstrating clear technical advantages and suitability for large-scale artificial cultivation. Furthermore, the Coprinus fungi, fungicides, and insecticides used in this invention are all conventional strains and agents used in orchid cultivation; the waste mushroom residue, oyster mushroom waste substrate, shiitake mushroom waste substrate, wood ash, pine needles, and leaves used are all common agricultural materials, readily available and inexpensive. This invention does not introduce any special highly toxic materials or rare strains, ensuring safe production operations and meeting the relevant requirements for standardized cultivation of Chinese medicinal herbs and resource utilization of agricultural waste.

[0066] Example 4 This embodiment also includes the following experimental groups to investigate the effect of field transplanting on the cultivation effect of Rhododendron: Experimental Group A: No grid planting area was constructed; Experimental Group B: The composite substrate of "waste mushroom substrate and wood ash" was replaced with "humus". Experimental Group C: The laying steps and positions of the large inoculum layer and the broken inoculum layer were interchanged; Experimental group D: Remove the spent mushroom substrate; Experimental Group E: All steps are the same as in Example 1.

[0067] The other field emergence steps for each experimental group, as well as the cultivation of seedlings without humus, propagation of *Coprinus* strains, and symbiotic cultivation without humus, were the same as in Example 1. Each experimental group underwent field cultivation for one year, and the results are shown in Table 4.

[0068] Table 4 Comparison of results from different experimental groups The results are analyzed as follows: The seedling emergence rate was ranked as follows: E > C > D > A > B. Group E, with its complete process, had the highest seedling density, indicating that the synergistic effect of each step in the original process was most conducive to the field germination and seedling formation of *Azalea*. Group B, using humus soil instead of the *Pleurotus ostreatus* waste substrate + wood ash composite substrate, had the lowest seedling emergence rate. This indicates that the combination of *Pleurotus ostreatus* waste substrate and wood ash substrate is more suitable for the symbiotic system of *Azalea* and *Coprinus comatus* symbiotic fungi compared to humus soil alone. Humus soil alone cannot meet the root microenvironment's nutrient, aeration, and microbial needs, significantly inhibiting seedling emergence. In Group C, the order of inoculation was reversed twice; in Group D, the covering of *Viola yedoensis* waste substrate was omitted. The seedling emergence rate was lower than E, but significantly better than A and B, proving that reversing the inoculation order and omitting the substrate covering weakens the seedling emergence effect, but the negative impact is weaker than substrate replacement and the absence of a grid structure. In Group A, without a grid structure, the seedling emergence rate was significantly lower than E, C, and D. The grid structure can optimize the field microenvironment (humidity, shading, soil compaction prevention, and reduction of bulb compression and rotting), while removing the grid structure will reduce the germination rate in the field.

[0069] Plant height ranking: E ​​> C > B > D > A. The seedlings in control group E were significantly taller than all other groups, indicating that the complete inoculum treatment process was most beneficial for aboveground vegetative growth. In experimental group C, after adjusting the inoculum application order, the plant height was second only to E, suggesting that changing the inoculum application order had a relatively small negative impact on plant elongation. Group A had the lowest seedling height without the grid structure. The grid structure regulates field temperature and humidity and reduces rainwater erosion; its absence significantly limited vegetative growth. In group B, after substrate replacement, the seedling height was at a moderate level, but combined with the pseudobulb data, it can be seen that while humus alone can maintain some aboveground stem and leaf growth, it is not conducive to underground pseudobulb enlargement.

[0070] The pseudobulb diameter was ranked as follows: E > C > D > A > B. Group E showed the best pseudobulb enlargement effect, representing the treatment closest to commercial specifications. Group B had the smallest pseudobulb diameter, the lowest among all groups. This clearly demonstrates that the composite substrate of spent oyster mushroom substrate and wood ash is the key condition for promoting pseudobulb enlargement and bulb formation; replacing it with humus soil severely restricts the development of underground organs. In Group C, the pseudobulb growth performance after changing the inoculation order was better than that of Groups A, D, and B, indicating that while the inoculation order has an impact, it is not the primary factor restricting pseudobulb enlargement. Removing the shiitake mushroom substrate cover in Group D and eliminating the grid structure in Group A both hindered pseudobulb enlargement. Covering with spent shiitake mushroom substrate provides a continuous supply of microorganisms and organic matter to the rhizosphere, and the grid structure stabilizes soil moisture and air conditions; the absence of both processes is detrimental to the development of underground pseudobulbs.

[0071] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for cultivating Rhododendron seedlings without humus soil and transplanting them with a high germination rate, characterized in that, Includes the following steps: (1) Humus-free seedling cultivation The waste residue from edible fungi is mixed with wood ash to obtain a mixed matrix; The activated Coprinus fungi were propagated and cultured to obtain the cultured strain; The cultivation strain is inoculated onto the surface of the mixed substrate, and then azalea seeds are sown on top of the cultivation strain. After cultivation, seedlings containing protocorms and free of humus are obtained. (2) Field transplanting and planting Prepare the field land, build a grid planting area, lay wooden blocks in the grid planting area, and carry out the first disinfection treatment as the wooden block layer; Cut the cultured spawn into pieces and spread the cut spawn on top of the wood block layer to form a large spawn layer; A composite substrate made of edible fungus waste residue and wood ash is laid on top of the large-block inoculum layer as a substrate layer. The culture strain is crushed and then sown on top of the substrate layer to form a crushed culture layer. The humus-free seedlings containing protocorms are sown above the layer of broken fungal inoculum; On top of the seedlings containing the original bulbs and humus-free soil, edible fungi waste substrate and fine soil are laid in sequence, and the surface of the fine soil is subjected to a second disinfection treatment; leaves are laid on top of the disinfected fine soil, and after cultivation, azalea seedlings are obtained.

2. The method for cultivating Rhododendron seedlings without humus soil and transplanting them with a high germination rate according to claim 1, characterized in that, In step (1), the mass ratio of the edible fungus waste residue to wood ash is 80-90:10-20; the moisture content of the mixed substrate is 45%-55%.

3. The method for cultivating Rhododendron seedlings without humus soil and transplanting them with a high germination rate according to claim 1, characterized in that, In step (1), the propagation and cultivation method is to inoculate the activated Coprinus fungi into the edible fungus cultivation medium and cultivate them at 27-29℃ and 65%-75% relative humidity until the mycelium fully grows on the medium. The culture medium for edible fungi cultivation is formulated as follows: 78% cottonseed hulls, 20% wheat bran, 1% gypsum and 1% lime, with a water content of 60%-65%.

4. The method for cultivating Rhododendron seedlings without humus soil and transplanting them with a high germination rate according to claim 1, characterized in that, In step (1), the thickness of the mixed substrate is 8-12 cm; the inoculation amount of the culture medium is 0.01-0.02 m. 3 100g of culture spawn was inoculated into the mixed substrate; the azalea seeds were sown at a rate of 0.01-0.02m³. 3 Sow seeds from 1 / 4 to 1 / 2 of a capsule in the mixed substrate.

5. The method for cultivating Rhododendron seedlings without humus soil and transplanting them with a high germination rate according to claim 1, characterized in that, In step (1), the cultivation temperature is 20-28℃, the relative humidity is 60%-75%, the shading degree is 75%-85%, and the cultivation is carried out in the dark and ventilated for 40-60 days; during the cultivation period, the substrate moisture content is controlled at 50%±5%.

6. The method for cultivating Rhododendron seedlings without humus soil and transplanting them with a high germination rate according to claim 1, characterized in that, In step (2), the width of the square planting area is 0.8-1.2m and the length is 8-12m; the distance between two adjacent square planting areas is 8-12cm. The wood block layer consists of wood blocks that are 8-10cm long, 2-3cm wide, and 0.4-0.6cm thick, laid evenly at a rate of 8kg / m². 2 The thickness of the paving is 3-5cm; the wood blocks include broad-leaved wood blocks. The large inoculum layer consists of cultivated inoculum 4-6 cm wide and 1-2 cm thick, spread at a rate of 3.5 kg / m². 2 ; The substrate layer is made of a composite substrate obtained by mixing edible fungus waste residue and wood ash in a mass ratio of 80-90:10-20, with a thickness of 0.8-1.2cm. The fragmented inoculum layer is formed by spreading cultivated inoculum with a particle size of 0.5-1.5 cm at a spreading rate of 5 kg / m³. 2 ; The sowing rate of the humus-free seedlings containing protocorms is per 6m³. 2 Spread at 0.01-0.02m 3 Nutrient-free seedlings containing protocorms; The thickness of the edible mushroom waste substrate is 0.4-0.6 cm; the thickness of the fine soil is 0.5-1 cm. The leaves include pine needles and broad-leaved leaves, and the thickness of the layer is 2-4cm.

7. The method for cultivating Rhododendron seedlings without humus soil and transplanting them with a high germination rate according to claim 1, characterized in that, In step (2), the cultivation temperature is 20-28℃, the soil moisture is maintained at 50%-55% for 15 days after sowing, and the soil moisture is increased to 55%-60% after 15 days. A shade shed is built to ensure that the shading degree is not less than 80%.

8. The method for cultivating Rhododendron seedlings without humus soil and transplanting them with a high germination rate according to claim 1, characterized in that, The first disinfection treatment method is as follows: spray a mixture of broad-spectrum fungicide and insecticide into the grid planting area, ensuring that the surface wood blocks are fully moistened but without flowing water, and let it stand for 12-24 hours. The second disinfection treatment method is as follows: spray a mixture of broad-spectrum bactericide and insecticide onto the surface of the fine soil, with the spraying amount being 1 / 3 to 1 / 2 of the amount sprayed in the first disinfection treatment.

9. The method for cultivating Rhododendron seedlings without humus soil and transplanting them with a high germination rate according to claim 1, characterized in that, The edible fungi waste residue includes shiitake mushroom waste residue and oyster mushroom waste substrate.

10. The method for cultivating Rhododendron seedlings without humus soil and transplanting them with high germination rate according to claim 1, characterized in that, The fungus mentioned is *Coprinus comatus*.

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