A cultivation method for Dendrobium nobile with moss companion growth

Abstract

This invention discloses a cultivation method for Dendrobium nobile with moss companions. This method utilizes different moss species to accompany Dendrobium nobile, aiming to improve its yield and quality. The dominant moss species for companion cultivation are identified, primarily *Moss sphaerocephala* and *Moss juniperae*. The companion cultivation of *Moss sphaerocephala* and *Moss juniperae* increases agronomical indicators and yield of one-year-old Dendrobium nobile, such as plant height, stem diameter, and single-plant weight. It also increases the content of stem extracts and dendrobine in two-year and three-year-old Dendrobium nobile, while having little impact on moisture and ash content. This method can enhance the levels of macronutrients in the stems of Dendrobium nobile.

Description

Technical Field

[0001] This invention relates to a method for cultivating Dendrobium nobile with moss companions, belonging to the technical field of plant cultivation. Background Technology

[0002] Dendrobium nobile, a perennial epiphytic herb belonging to the genus Dendrobium in the family Orchidaceae, is used medicinally for its dried stems. It is a rare and precious traditional Chinese medicine under second-class protection in China. Rich in polysaccharides, alkaloids, and other active substances, it possesses high medicinal value, exhibiting effects such as enhancing immunity, lowering blood sugar, and anti-tumor properties. The artificial cultivation of Dendrobium nobile in Chishui City, Guizhou Province, China, has a long history. Relying on its unique Danxia landform geological conditions and hot and humid climate, Chishui City has gradually become a major source of raw medicinal materials for the market. Currently, the area of ​​Dendrobium nobile cultivated under forest cover with Danxia landforms in Chishui reaches 96,000 mu (approximately 6,400 hectares), accounting for over 90% of the national Dendrobium nobile planting area and commercial volume. Dendrobium nobile has demanding growth requirements, preferring high temperature, high humidity, and cool environments. It thrives at altitudes of 300–1000 m, often epiphytically on rocks or tree trunks with abundant humus and moss, typically on cliffs or steep slopes. It usually requires shade from trees, proximity to water sources, and ample indirect sunlight, resulting in relatively slow growth. Simulated wild cultivation on Danxia landforms generally takes more than three years from planting to harvest, with a long management cycle, unstable yields, and significant investment of human, material, and financial resources, which is currently a key factor restricting the development of the Dendrobium nobile industry. The applicant's research has found a close relationship between the growth of Dendrobium nobile and the microenvironment of Danxia landforms, with different moss species having varying impacts on its growth. This invention uses different moss species as companion plants for Dendrobium nobile to improve its yield and quality, identifying the dominant moss species for companion cultivation, aiming to provide a scientific basis for the rational cultivation and quality evaluation of Dendrobium nobile. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a method for cultivating Dendrobium nobile with moss. The purpose of this invention is to improve the yield and quality of Dendrobium nobile by using different types of moss to grow it together.

[0004] To solve the above technical problems, the present invention adopts the following technical solution:

[0005] A method for cultivating Dendrobium nobile with moss companion growth, the method comprising the following steps:

[0006] (1) Site selection: Select bare Danxia rocks at an altitude of 300m-1000m, shaded by broad-leaved trees;

[0007] (2) Land preparation: Remove shrubs, weeds, dead branches and leaves, and soil;

[0008] (3) Transplanting time: Spring transplanting is done from April to May, and autumn transplanting is done from September to October. Transplanting should be done on cloudy days.

[0009] (4) Transplanting: Select two-year-old Dendrobium nobile seedlings that are free from damage and disease. Plant them at a spacing of 30cm×30cm. Attach the fibrous roots and base of the Dendrobium nobile seedlings to the stone surface and fix them with wire clips. Let the roots spread out naturally. Fix the wire clips 1.5cm to 2.5cm above the base of the main stem of the seedling to ensure that the seedlings are stable on the stone surface and obtain well-fixed Dendrobium nobile tissue culture seedlings.

[0010] (5) Preparation of cow dung slurry: Take well-rotted cow dung, dilute it with water, and set aside;

[0011] (6) Apply cow dung paste to the base of the fixed Dendrobium nobile tissue culture seedlings, covering an area of ​​80-120cm. 2 The cow dung slurry extends from the root area and is applied to the stone surface, covering an area of ​​800-1000 cm². 2 ;

[0012] (7) Apply live moss evenly and lightly to the roots of the plant and the stone surface that has been coated with cow dung slurry. Spray water evenly to wet the roots of Dendrobium nobile, the stone surface and the moss. After the seedlings have recovered, clean the dead branches and fallen leaves on the Dendrobium nobile plant and around its growth every 10 to 20 days to ensure the normal growth of Dendrobium nobile and moss. Plants transplanted in spring can be harvested in October or November of the following year, and plants transplanted in autumn can be harvested in October or November of the following year.

[0013] In step (1) above, the relative humidity of the broad-leaved forest is 75% to 80%, and the shading degree is 55% to 70%.

[0014] In the aforementioned step (3), spring transplanting is carried out in April to May, and autumn transplanting is carried out in September to October. Transplanting is carried out on cloudy days and the transplanting temperature is above 10℃.

[0015] In step (5) above, the cow dung slurry is prepared by diluting well-rotted cow dung with water at a mass ratio of 1:2 to 4 and keeping it for later use.

[0016] Specifically, in the aforementioned step (5), the preparation of cow dung slurry involves diluting well-rotted cow dung with water at a mass ratio of 1:3 for later use.

[0017] In step (6) above, cow dung slurry is brushed onto the roots of the fixed Dendrobium nobile tissue culture seedlings, with the root area covering 90-110cm. 2 The cow dung slurry extends from the root area to cover an area of ​​850-1050 cm² on the stone surface. 2 .

[0018] Specifically, in step (6) above, cow dung slurry is brushed onto the roots of the fixed Dendrobium nobile tissue culture seedlings, with the root area covering 100cm. 2 The cow dung slurry extends from the root area and is applied to the stone surface, covering an area of ​​900 cm². 2 .

[0019] In the aforementioned step (7), the moss is *Moss sphaerocephala* and *Moss juniper*, and the mass ratio of *Moss sphaerocephala* to *Moss juniper* is 0.5-3:3-0.5.

[0020] Specifically, in the aforementioned step (7), the moss is *Moss sphaerocephala* and *Moss juniper*, and the mass ratio of *Moss sphaerocephala* to *Moss juniper* is 1-3:3-1.

[0021] In step (7) above, the moss also includes one or more of the following: Selaginella thin-walled, Selaginella short-ribbed, Bryophytum bilatum, Bryophytum longleaf, Bryophytum glacifolium, Bryophytum tufted, or Bryophytum curcumae.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention utilizes different species of mosses as companion plants to *Dendrobium nobile* to improve its yield and quality. Through experiments involving covering the roots of *Dendrobium nobile* with *Mossula verrucosum*, 1 / 2 *Mossula verrucosum*, 1 / 2 *Mossula juniper*, and *Mossula juniper*, it was found that the companion plants of *Mossula verrucosum* and *Mossula juniper* increased agronomical indicators and yield of one-year-old *Dendrobium nobile*, such as plant height, stem diameter, and single-plant weight. They also increased the content of stem extracts and dendrobine in two-year-old and three-year-old *Dendrobium nobile*, with minimal impact on moisture and ash content. These methods also improved the levels of macronutrients in the stems of *Dendrobium nobile*. The aim is to identify the dominant moss species for companion planting with *Dendrobium nobile*, providing a scientific basis for the rational cultivation and quality evaluation of this plant. Attached Figure Description

[0024] Figure 1 Photographs of different bryophyte morphologies (1. Juniper-leaved white-haired moss; 2. Warty-light moss; 3. Lateral-branched creeping moss; 4. Curly-leaved phoenix tail moss; 5. Short-ribbed feather moss; 6. Large gray moss; 7. Calcareous gray moss; 8. Beautiful gray moss; 9. Glacier blue moss; 10. Loose-netted beautiful beak moss; 11. Feathered beautiful beak moss; 12. Dark green folded leaf moss; 13. Broad-leaved hairy mouth moss; 14. Curly-leaved hairy mouth moss; 15. Tipped hairy mouth moss; 16. Twisted moss; 17. Long-leaved twisted moss; 18. Curly-leaved wetland moss).

[0025] Figure 2 Photographs of different bryophyte morphologies (19: *Scleroderma sclerotium*; 20: *Scleroderma sclerotium*; 21: *Scleroderma sclerotium*; 22: *Scleroderma sclerotium*; 23: *Scleroderma sclerotium*; 24: *Selaginella sclerotium*; 25: *Scleroderma sclerotium*; 26: *Scleroderma bilatum*; 27: *Scleroderma sclerotium*; 28: *Scleroderma sclerotium*; 29: *Scleroderma sclerotium*; 30: *Scleroderma sclerotium*; 31: *Scleroderma sclerotium*).

[0026] Figure 3 Differences in moss species among 35 quadrats;

[0027] Figure 4 Frequency and coverage of 28 moss species;

[0028] Figure 5 Number of quadrats in which 9 types of moss grew in a mixed manner;

[0029] Figure 6 Dry matter accumulation of one-year-old stems of Dendrobium nobile in 35 quadrats;

[0030] Figure 7 Dry matter accumulation of 2-year-old stems of Dendrobium nobile in 35 quadrats;

[0031] Figure 8 Dry matter accumulation of 3-year-old stems of Dendrobium nobile in 35 quadrats;

[0032] Figure 9 Content of extracts from 3-year-old stems of Dendrobium nobile in 35 sample plots;

[0033] Figure 10 Dendrobine content in 3-year-old stems of Dendrobium nobile from 35 sample plots;

[0034] Figure 11 Growth chart of Dendrobium nobile and Pseudomonas juniperii growing together;

[0035] Figure 12 : Growth of Dendrobium without moss companion plants;

[0036] Figure 13 Image of mixed growth of *Moss sphagnum* and *Moss juniper*. Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0038] Example 1.

[0039] (1) Site selection: Select bare Danxia landforms at an altitude of 300m-1000m, shaded by broad-leaved trees, with a relative humidity of 75%-80% and a shading degree of 55%-70% in the broad-leaved forests;

[0040] (2) Land preparation: Remove shrubs, weeds, dead branches and leaves, and soil;

[0041] (3) Transplanting time: Transplanting in September 2021, on cloudy days, with the transplanting temperature above 10℃;

[0042] (4) Transplanting: Select two-year-old Dendrobium nobile tissue culture seedlings that are free from damage and pests (purchased from Chishui Xintian Traditional Chinese Medicine Industry Development Co., Ltd.). Plant them at a spacing of 30cm×30cm. Attach the fibrous roots and base of the Dendrobium nobile seedlings to the stone surface and fix them with wire clips. Let the roots spread out naturally. Fix the wire clips 1.5cm to 2.5cm above the base of the main stem of the seedling to ensure that the seedlings are stable on the stone surface and obtain well-fixed Dendrobium nobile tissue culture seedlings.

[0043] (5) Preparation of cow dung slurry: Take well-rotted cow dung and dilute it with water at a mass ratio of 1:3, and set aside;

[0044] (6) Apply cow dung paste to the base of the fixed Dendrobium nobile tissue culture seedlings, covering an area of ​​100cm around the roots. 2 The cow dung slurry extends from the root area and is applied to the stone surface, covering an area of ​​900 cm². 2 ;

[0045] (7) Apply live moss and white cypress moss evenly and lightly to the roots of the plant and the stone surface that has been coated with cow dung slurry. The mass ratio of moss and white cypress moss is 1:1. Spray water evenly to wet the roots of Dendrobium nobile, the stone surface and its moss. After the seedlings have recovered, clean the dead branches and fallen leaves on the Dendrobium nobile plant and around its growth every 10 to 20 days to ensure the normal growth of Dendrobium nobile and moss. It can be harvested in October or November of the following year.

[0046] Example 2.

[0047] (1) Site selection: Select bare Danxia landforms at an altitude of 300m-1000m, shaded by broad-leaved trees, with a relative humidity of 75%-80% and a shading degree of 55%-70% in the broad-leaved forests;

[0048] (2) Land preparation: Remove shrubs, weeds, dead branches and leaves, and soil;

[0049] (3) Transplanting time: Transplanting in September 2021, on cloudy days, with the transplanting temperature above 10℃;

[0050] (4) Transplanting: Select two-year-old Dendrobium nobile tissue culture seedlings that are free from damage and pests (purchased from Chishui Xintian Traditional Chinese Medicine Industry Development Co., Ltd.). Plant them at a spacing of 30cm×30cm. Attach the fibrous roots and base of the Dendrobium nobile seedlings to the stone surface and fix them with wire clips. Let the roots spread out naturally. Fix the wire clips 1.5cm to 2.5cm above the base of the main stem of the seedling to ensure that the seedlings are stable on the stone surface and obtain well-fixed Dendrobium nobile tissue culture seedlings.

[0051] (5) Preparation of cow dung slurry: Take well-rotted cow dung and dilute it with water at a mass ratio of 1:2, and set aside;

[0052] (6) Apply cow dung paste to the base of the fixed Dendrobium nobile tissue culture seedlings, covering an area of ​​80cm around the roots. 2 The cow dung slurry extends from the root area and is applied to the stone surface, covering an area of ​​1000 cm². 2 ;

[0053] (7) Apply live moss and white cypress moss evenly and lightly to the roots of the plant and the stone surface that has been coated with cow dung slurry. The mass ratio of moss and white cypress moss is 1:3. Spray water evenly to wet the roots of Dendrobium nobile, the stone surface and its moss. After the seedlings have recovered, clean the dead branches and fallen leaves on the Dendrobium nobile plant and around its growth every 10 to 20 days to ensure the normal growth of Dendrobium nobile and moss. Plants transplanted in spring can be harvested in October or November of the same year.

[0054] Example 3.

[0055] (1) Site selection: Select bare Danxia landforms at an altitude of 300m-1000m, shaded by broad-leaved trees, with a relative humidity of 75%-80% and a shading degree of 55%-70% in the broad-leaved forests;

[0056] (2) Land preparation: Remove shrubs, weeds, dead branches and leaves, and soil;

[0057] (3) Transplanting time: Transplanting should be carried out in October 2021, on cloudy days, with the transplanting temperature above 10℃;

[0058] (4) Transplanting: Select two-year-old Dendrobium nobile tissue culture seedlings that are free from damage and pests (purchased from Chishui Xintian Traditional Chinese Medicine Industry Development Co., Ltd.). Plant them at a spacing of 30cm×30cm. Attach the fibrous roots and base of the Dendrobium nobile seedlings to the stone surface and fix them with wire clips. Let the roots spread out naturally. Fix the wire clips 1.5cm to 2.5cm above the base of the main stem of the seedling to ensure that the seedlings are stable on the stone surface and obtain well-fixed Dendrobium nobile tissue culture seedlings.

[0059] (5) Preparation of cow dung slurry: Take well-rotted cow dung and dilute it with water at a mass ratio of 1:4, and set aside;

[0060] (6) Apply cow dung paste to the base of the fixed Dendrobium nobile tissue culture seedlings, covering a root zone of 120cm. 2 The cow dung slurry extends from the root area and is applied to the stone surface, covering an area of ​​800 cm². 2 ;

[0061] (7) Apply live moss and white cypress moss evenly and lightly to the roots of the plant and the stone surface that has been coated with cow dung slurry. The mass ratio of moss and white cypress moss is 3:1. Spray water evenly to wet the roots of Dendrobium nobile, the stone surface and its moss. After the seedlings have recovered, clean the dead branches and fallen leaves on the Dendrobium nobile plant and around its growth every 10 to 20 days to ensure the normal growth of Dendrobium nobile and moss. Plants transplanted in spring can be harvested in October or November of the same year.

[0062] Example 4.

[0063] (1) Site selection: Select bare Danxia landforms at an altitude of 300m-1000m, shaded by broad-leaved trees, with a relative humidity of 75%-80% and a shading degree of 55%-70% in the broad-leaved forests;

[0064] (2) Land preparation: Remove shrubs, weeds, dead branches and leaves, and soil;

[0065] (3) Transplanting time: Transplanting in April 2022, on cloudy days, with the transplanting temperature above 10℃;

[0066] (4) Transplanting: Select two-year-old Dendrobium nobile tissue culture seedlings that are free from damage and pests (purchased from Chishui Xintian Traditional Chinese Medicine Industry Development Co., Ltd.). Plant them at a spacing of 30cm×30cm. Attach the fibrous roots and base of the Dendrobium nobile seedlings to the stone surface and fix them with wire clips. Let the roots spread out naturally. Fix the wire clips 1.5cm to 2.5cm above the base of the main stem of the seedling to ensure that the seedlings are stable on the stone surface and obtain well-fixed Dendrobium nobile tissue culture seedlings.

[0067] (5) Preparation of cow dung slurry: Take well-rotted cow dung and dilute it with water at a mass ratio of 1:3, and set aside.

[0068] (6) Apply cow dung paste to the base of the fixed Dendrobium nobile tissue culture seedlings, covering an area of ​​100cm around the roots. 2 The cow dung slurry extends from the root area and is applied to the stone surface, covering an area of ​​900 cm². 2 ;

[0069] (7) Apply live moss, white juniper moss, and thin-walled selaginella evenly and lightly to the roots of the plant and the stone surface that has been coated with cow dung slurry. The mass ratio of moss, white juniper moss, and thin-walled selaginella is 1:1:0.1. Spray water evenly to wet the roots of Dendrobium nobile, the stone surface, and the moss. After the seedlings have recovered, clean the dead branches and fallen leaves on the Dendrobium nobile plant and around its growth every 10 to 20 days to ensure the normal growth of Dendrobium nobile and moss. It can be harvested in October or November of the following year.

[0070] Example 5.

[0071] (1) Site selection: Select bare Danxia landforms at an altitude of 300m-1000m, shaded by broad-leaved trees, with a relative humidity of 75%-80% and a shading degree of 55%-70% in the broad-leaved forests;

[0072] (2) Land preparation: Remove shrubs, weeds, dead branches and leaves, and soil;

[0073] (3) Transplanting time: May 2022, transplanting should be carried out on cloudy days, and the temperature should be above 10℃;

[0074] (4) Transplanting: Select two-year-old Dendrobium nobile tissue culture seedlings that are free from damage and pests (purchased from Chishui Xintian Traditional Chinese Medicine Industry Development Co., Ltd.). Plant them at a spacing of 30cm×30cm. Attach the fibrous roots and base of the Dendrobium nobile seedlings to the stone surface and fix them with wire clips. Let the roots spread out naturally. Fix the wire clips 1.5cm to 2.5cm above the base of the main stem of the seedling to ensure that the seedlings are stable on the stone surface and obtain well-fixed Dendrobium nobile tissue culture seedlings.

[0075] (5) Preparation of cow dung slurry: Take well-rotted cow dung and dilute it with water at a mass ratio of 1:2.5, and set aside.

[0076] (6) Apply cow dung paste to the base of the fixed Dendrobium nobile tissue culture seedlings, with the base area extending to 110cm. 2 The cow dung slurry extends from the root area and is applied to the stone surface, covering an area of ​​800 cm². 2 ;

[0077] (7) Apply live moss, white juniper moss, and short-ribbed feather moss evenly and lightly to the roots of the plant and the stone surface that has been coated with cow dung slurry. The mass ratio of moss, white juniper moss, and short-ribbed feather moss is 1:2:0.2. Spray water evenly to wet the roots of Dendrobium nobile, the stone surface, and the moss. After the seedlings have recovered, clean the dead branches and fallen leaves on the Dendrobium nobile plant and around its growth every 10 to 20 days to ensure the normal growth of Dendrobium nobile and moss. It can be harvested in October or November of the following year.

[0078] The method for cultivating moss-associated Dendrobium nobile is characterized in that: in step (7), the moss also includes one or more of the following: Selaginella tamariscina, Bryophytum brevicornu, Bryophytum bilatum, Bryophytum longifolium, Bryophytum glacifolium, Bryophytum tuftum or Bryophytum tuftum.

[0079] Example 6.

[0080] (1) Site selection: Select bare Danxia landforms at an altitude of 300m-1000m, shaded by broad-leaved trees, with a relative humidity of 75%-80% and a shading degree of 55%-70% in the broad-leaved forests;

[0081] (2) Land preparation: Remove shrubs, weeds, dead branches and leaves, and soil;

[0082] (3) Transplanting time: May 2022, transplanting should be carried out on cloudy days, and the temperature should be above 10℃;

[0083] (4) Transplanting: Select two-year-old Dendrobium nobile tissue culture seedlings that are free from damage and pests (purchased from Chishui Xintian Traditional Chinese Medicine Industry Development Co., Ltd.). Plant them at a spacing of 30cm×30cm. Attach the fibrous roots and base of the Dendrobium nobile seedlings to the stone surface and fix them with wire clips. Let the roots spread out naturally. Fix the wire clips 1.5cm to 2.5cm above the base of the main stem of the seedling to ensure that the seedlings are stable on the stone surface and obtain well-fixed Dendrobium nobile tissue culture seedlings.

[0084] (5) Preparation of cow dung slurry: Take well-rotted cow dung and dilute it with water at a mass ratio of 1:3.5, and set aside.

[0085] (6) Apply cow dung paste to the base of the fixed Dendrobium nobile tissue culture seedlings, with the base area covering 85cm. 2 The cow dung slurry extends from the root area and is applied to the stone surface, covering an area of ​​950 cm². 2 ;

[0086] (7) Apply live moss, white juniper moss and pilagon moss evenly and lightly to the roots of the plant and the stone surface that has been coated with cow dung slurry. The mass ratio of moss, white juniper moss and pilagon moss is 2:1:0.2. Spray water evenly to wet the roots of Dendrobium nobile, the stone surface and its moss. After the seedlings have recovered, clean the dead branches and fallen leaves on the Dendrobium nobile plant and around its growth every 10 to 20 days to ensure the normal growth of Dendrobium nobile and moss. It can be harvested in October or November of the following year.

[0087] This invention has undergone extensive experimental research, and the results of this experimental research are as follows:

[0088] 1. Analysis of the symbiotic relationship between bryophytes and Dendrobium nobile

[0089] 1.1 Materials and Methods

[0090] 1.1.1 Overview of the test site

[0091] This study selected the Dendrobium nobile cultivation base in Yaling Village, Wanglong Town, Chishui City, Guizhou Province, as the experimental site, where Dendrobium nobile was cultivated in a semi-wild environment under trees. Wanglong Town (105°90′N, 28°52′E) is located in the central part of Chishui City and is one of the main production areas of Dendrobium nobile in Chishui. The Dendrobium nobile cultivation base in Yaling Village (105°91′E, 28°51′N) is at an altitude of 500 m and is the largest contiguous Dendrobium nobile cultivation base in Chishui City (over 2000 mu). Wanglong Town is located in a semi-mountainous hilly area in a river valley, with a terrain that is high in the southeast and low in the northwest. It has a subtropical monsoon climate and an altitude of 228–1,256 m. According to the Chishui Meteorological Bureau, the average annual rainfall in Wanglong Town from 2012 to 2022 was 785.11–1,658.37 mm, and the average annual temperature was 17.37–18.79℃.

[0092] 1.1.2 Test Materials

[0093] Dendrobium nobile: Dendrobium nobile planted in the study area for 3 years in a semi-wild environment with Danxia stone was identified by Professor Zhao Zhi of the College of Agriculture of Guizhou University as Dendrobium nobile, a plant of the genus Dendrobium in the family Orchidaceae.

[0094] Danxia stone: The rock type is sandstone.

[0095] Moss: Moss that grows only with the roots of Dendrobium nobile.

[0096] 1.1.3 Main Instruments and Equipment

[0097] Inductively coupled plasma mass spectrometer (NexION 300D), plasma emission spectrometer (ICP-OESOptima 8000), wavelength dispersive X-ray fluorescence spectrometer (Axios-mAX), fully automated Kjeldahl nitrogen analyzer (HGK-55), gas chromatograph (Agilent 7890A).

[0098] 1.1.4 Sample Collection

[0099] The experiment involved collecting bryophyte specimens twice, in September 2021 and May 2022, within the study area. Only bryophyte species associated with the roots of *Dendrobium nobile* were collected to ensure that the collected specimens were representative of the bryophyte populations associated with *Dendrobium nobile* in the region. A total of 163 specimens were collected and brought back to the laboratory for drying. The collected specimens were identified to the species level.

[0100] Thirty-five Danxia stones were randomly selected from the study area. The selection principle was that the area of ​​a certain face of the Danxia stone was greater than 1.5m × 1.5m, and each face was set as one quadrat. A total of 35 quadrats were selected, and detailed information on the 35 quadrats is shown in Table 1. Samples of Dendrobium nobile, moss, and Danxia stone were collected from the quadrats. To minimize experimental error, samples were collected on a cloudy day on May 5, 2022, after three consecutive sunny days. The specific sampling steps were as follows: ① Dendrobium nobile sample collection: Fifteen clumps of Dendrobium nobile with basically uniform growth were selected from each quadrat. One 1-year-old, 2-year-old, and 3-year-old stem was cut from each clump, and the leaves were removed. The 15 stems were mixed into one sample and transported back to the laboratory to determine the accumulation, quality, and mineral element content of the Dendrobium nobile stem dry matter. ② Moss sample collection: Moss samples of all species were collected from the quadrats, placed in resealable bags and labeled. The coverage rate of each moss species was counted, and a total of 182 samples were collected. ③ Danxia stone sample collection: Based on the three Dendrobium nobile clumps selected from each quadrat, Danxia stone samples were collected within the designated area after the moss samples were collected. The surface layer of the Danxia stone was chipped open to about 0.5 cm with a geological hammer to obtain blocky or powdery Danxia stone samples. Roots and other debris were removed, and the obtained Danxia stone samples were placed in resealable bags and sealed. A total of 105 samples were collected, and their fresh weight and dry weight were measured. Their moisture content was calculated (Table 1).

[0101] Table 1 Basic Information of 35 Danxia Stones

[0102]

[0103] 1.1.5.1 Determination of yield, quality, and mineral element content of Dendrobium nobile

[0104] 1.1.5.1.1 Biomass determination

[0105] The dry weight of 15 stems was determined for each sample. The specific method is as follows: First, the fresh weight (g / stem) of 15 stems was weighed separately under the natural moisture content of the plant. Then, the total fresh weight of the 15 stems was weighed. Next, the stems were cut into small sections and dried in a 60℃ oven until constant weight. Their dry weight was then measured, and the dry weight percentage of the stems was calculated. Finally, the dry weight of a single stem (g / stem) was calculated based on the dry weight percentage. The formula for calculating the dry weight percentage is:

[0106] Dry weight percentage (%) =

[0107] 1.1.5.1.2 Quality Measurement

[0108] Determination of extractive content: The ethanol extractive content was determined by hot extraction method under the "Determination of Alcohol-soluble Extractives (General Chapter 2201)" section of Part IV of the 2020 Chinese Pharmacopoeia [Chinese Pharmacopoeia, 2020].

[0109] Dendrobine content determination: determined according to the gas chromatography method (General Chapter 0521) of the 2020 edition of the Chinese Pharmacopoeia [Chinese Pharmacopoeia, 2020].

[0110] 1.1.5.1.3 Determination of mineral element content

[0111] The nitrogen content was determined using a Kjeldahl nitrogen analyzer, following the Kjeldahl method for protein content determination in the 2020 edition of the Chinese Pharmacopoeia (General Chapter 0731) [Chinese Pharmacopoeia, 2020]. The contents of K, Ca, P, Mg, Na, Fe, Cu, Zn, Mn, and B were determined using an inductively coupled plasma optical emission spectrometer (ICP-OES). Reference standards for each element were provided by the China Nonferrous Metals and Electronic Materials Analysis and Testing Center.

[0112] 1.1.5.2 Identification of moss species, frequency of associated occurrences, and measurement of coverage.

[0113] Species identification: The collected specimens were identified by Professor Xiong Yuanxin and Associate Professor Cao Wei of the School of Life Sciences, Guizhou University, and the specimens were identified to the species level.

[0114] Calculation of moss companion frequency, frequency, and coverage:

[0115] Associated frequency: the number of times each type of moss appears in all quadrats; Associated frequency (%): the proportion of the number of times a type of moss appears in all quadrats; Coverage (%): estimated using the grid method, i.e., the proportion of the area of ​​each type of moss growing in a quadrat to the area of ​​that Danxia stone, expressed as a percentage; The frequency calculation formula is:

[0116] Associated frequency (%) =

[0117] 1.1.5.3 Moisture content of Danxia stone

[0118] Moisture content (%): Based on the moss moisture content determination, three clumps of Dendrobium nobile were selected. A 10cm × 10cm area was demarcated with the base of the Dendrobium nobile stem as the center. The surface layer of the Danxia stone was chipped open by a geological hammer to a depth of about 0.5cm, obtaining blocky and powdery Danxia stone samples. Roots and other impurities were removed. The obtained Danxia stone samples were sealed in resealable bags and brought back to the laboratory to be weighed fresh. Subsequently, the Danxia stone was air-dried and weighed dry. The moisture content was calculated using the following formula:

[0119] Moisture content (%) =

[0120] 1.1.6 Data Processing and Statistical Methods

[0121] SPSS 25.0 and Excel 2019 were used for statistical analysis of the data, and Origin 9.8 and CorelDRAW 2020 were used for graphing.

[0122] A comprehensive evaluation is conducted using principal component analysis and membership functions.

[0123] Principal component analysis: SPSS 25.0 software was used to standardize the data of each individual index, extract the factors with eigenvalues ​​greater than 1 as principal components, obtain the comprehensive index coefficients of each principal component through principal component analysis, and multiply them accordingly to calculate the score (C value) of the comprehensive index of each sample plot.

[0124] Membership function evaluation: Calculate the membership function value U(x) of each sample plot according to formula (1), calculate the weight of each indicator according to formula (2), and obtain the comprehensive score (D value) of each sample plot using formula (3).

[0125] U(Xij) = ;(i=1,2,3,…,n)(1)

[0126] In the formula, X is the measured value, Xmax is the maximum value, and Xmin is the minimum value.

[0127] Wi = (2)

[0128] In the formula, Wi represents the weight of the extracted i-th indicator, expressed as a percentage; Vi is the coefficient of variation of the i-th indicator (i=1, 2, 3, ..., n); and Vn is the sum of the n coefficients of variation.

[0129] Comprehensive membership function value (D value) (3)

[0130] In the formula, Wn represents the sum of the percentages of the indicators.

[0131] 1.2 Results and Analysis

[0132] 1.2.1 Survey of Associated Moss Species in the Study Area

[0133] Identification revealed that the bryophytes associated with Dendrobium nobile comprise 31 species belonging to 24 genera and 15 families. Figure 1 and Figure 2 The species composition is shown in Table 2. As can be seen from the table, among the bryophytes that coexist with Dendrobium nobile, there are 29 species of bryophytes belonging to 22 genera and 13 families, and 2 species of bryophytes belonging to 2 genera and 2 families. The proportions of bryophytes in the total number of families, genera, and species are 86.67%, 91.67%, and 93.55%, respectively, indicating that the bryophytes that coexist with Dendrobium nobile are more adaptable to the local lithophytic environment than the bryophytes.

[0134] Table 2 Composition of 31 bryophyte species

[0135]

[0136] 1.2.2 Analysis of moss species associated with 35 Danxia stones

[0137] Table 3 shows the moss species and coverage of 35 Danxia stones. As shown in Table 3, a total of 28 moss species were collected from the 35 Danxia stones. Compared with the moss species collected during the previous ecological survey of the study area, only three mosses were missing: *Bryophytum repens*, *Gymnocarpus calcareosa*, and *Gymnocarpus scaber*. This indicates that the 35 Danxia stones can represent the distribution of associated mosses in the study area. The survey results revealed that the association of mosses with *Dendrobium nobile* was always a mixed association of multiple mosses, with no single moss species found. There were significant differences in the types of associated mosses among different Danxia stones. Each Danxia stone typically contained 3–9 moss species, with 5–6 species being more common on a single stone. Figure 3 Epiphytic mosses of 3, 4, and 7 species were the most common, accounting for 34.29%, while epiphytic mosses of 8 to 9 species were the least common, accounting for only 5.71%.

[0138] Table 3. Species and Coverage of Mosses on 35 Danxia Stones

[0139]

[0140]

[0141] Note: Since various mosses grow mixed within the same plot, the total coverage is not 100%.

[0142] 1.2.3 Analysis of the frequency and coverage of co-occurrence of different moss species

[0143] The frequency and coverage of different moss species in 35 quadrats were statistically analyzed. Figure 4 ),Depend on Figure 4 It was found that the frequency of occurrence varied considerably among different moss species. Among them, *Bryum juniperii* and *Bryum verrucosum* were the most common, growing in 70% of the quadrats, with frequencies of 71.43% and 68.57%, respectively. The fourteen species with the lowest frequencies were *Bryum broadleaf*, *Bryum serratum*, *Bryum pseudobrittle-branched*, *Bryum curly-leaved*, *Bryum straightleaf*, *Bryum sclerotium*, *Bryum lateralis*, *Bryum longstalked*, *Bryum tautum*, *Bryum tautum*, *Bryum tautum*, *Bryum pinnatum*, *Bryum pinnatum*, *Bryum scutellarioides*, *Bryum scutellarioides*, *Bryum scutellarioides*, *Bryum scutellarioides*, and *Bryum tautum*, ranging from 2.86% to 11.43%. The coverage rates of different moss species also varied considerably, ranging from 1.00% to 65.87%. *Bryum verrucosum*, *Bryum curly-leaved*, and *Bryum tautum* had the highest coverage rates, exceeding 50%; while *Bryum longstalked*, *Bryum tautum*, and *Bryum pinnatum* had the lowest, at only 1.00%.

[0144] To clarify the mixed and associated relationships among different moss species, the number of quadrats in which each moss species grew in pairs was counted. Mosses with more than 5 quadrats in pairs were classified as pseudo-dominant mosses and plotted on a graph. Figure 5 The results showed that only nine mosses, including *Leucobryum japonicum*, *Lysimachia verrucoides*, *Selaginella tamariscina*, *Selaginella scabra*, *Bryum bistorata*, *Bryum longifolium*, *Bryum glacifolium*, *Bryum tuftedum*, and *Bryum tuftedum*, had more than five mixed growth quadrats. Among them, *Lysimachia verrucoides* and *Leucobryum japonicum* had the highest mixed growth frequency (20 times), significantly higher than the other mosses. The mixed growth frequency among the mosses was mainly 6 times, with a total of 9 pairs of mosses.

[0145] 1.2.4 Effects of co-occurrence of different moss species on the biomass of Dendrobium nobile

[0146] The accumulation of dry matter in Dendrobium nobile associated with different types of mosses varies. Figure 6 , Figure 7 , Figure 8 The stem mass accumulation of 1-, 2-, and 3-year-old stems was 0.056–0.301 g / stem, 0.338–1.065 g / stem, and 0.147–0.75 g / stem, respectively, with coefficients of variation of 35.98%, 24.50%, and 40.13%, respectively.

[0147] 1.2.5 The Influence of Different Species of Moss Coexistence on the Quality of Dendrobium nobile

[0148] The statistical results of the extract and dendrobine content of 35 sample plots of 3-year-old stems of Dendrobium nobile are shown in the figure. Figure 9 , Figure 10 The content of extracts from 3-year-old stems of Dendrobium nobile associated with different mosses ranged from 23.41% to 28.74%, with little difference and a coefficient of variation of 5.46%; while the content of dendrobine ranged from 0.30% to 0.81%, with significant differences and a coefficient of variation of 21.08%.

[0149] 1.2.6 Comprehensive evaluation of the yield, quality and mineral element content of Dendrobium nobile in 35 sample plots

[0150] Correlation analysis of biomass, quality, and mineral element content of *Dendrobium nobile* showed weak correlations among different moss-associated species. When comprehensively evaluating the yield and quality of 35 quadrats, the lack of significant correlations between the indicators may lead to discrepancies between the comprehensive evaluation and the evaluation based on a single indicator. Therefore, to clarify the impact of different indicators on the comprehensive evaluation of *Dendrobium nobile*, principal component analysis and membership function analysis were used to comprehensively evaluate the biomass, quality, and mineral element content of *Dendrobium nobile*, exploring the influence of different moss-associated species on the yield and quality of *Dendrobium nobile*. In this experiment, the dry matter accumulation of 1-year-old, 2-year-old, and 3-year-old stems varied among the 35 quadrats; therefore, the biomass of each quadrat was calculated based on the total dry matter accumulation of 1-, 2-, and 3-year-old stems. Among the quality indicators, the coefficient of variation for leachate content was 5.46%, showing a relatively small range of variation, while the coefficient of variation for dendrobine content was 21.08%, showing a larger range of variation. Therefore, dendrobine content was used as the quality evaluation indicator for the 35 sample plots. The coefficients of variation for the contents of the 11 mineral elements were all relatively large; therefore, all elements were used as evaluation indicators. The sample plots were ranked according to their C and D values; higher values ​​indicated higher evaluation. Furthermore, the evaluation of dry matter and other single quality indicators was ranked according to their measured values.

[0151] Table 4 shows the scores and rankings of the single-index evaluation and multi-index comprehensive evaluation of the dry matter, quality, and mineral element content of 35 sample plots of Dendrobium nobile. Principal component analysis and membership function analysis showed low similarity, with only 40% of the top 10 sample plots sharing the same data. The order of similarity between the individual evaluations of dry matter, dendrobine, and mineral elements and the multi-index comprehensive evaluation was mineral elements > dendrobine > dry matter, indicating that mineral elements have a greater weight than dry matter and dendrobine in the multi-index comprehensive evaluation.

[0152] Table 4. Top 10 sample plots in terms of comprehensive evaluation scores for dry matter, dendrobine, and mineral elements of Dendrobium nobile.

[0153]

[0154] The frequencies and coverage rates of the top 10 associated moss species in each sample plot were statistically analyzed using various evaluation methods. Moss species with an association frequency greater than 5 were identified as potential dominant species associated with *Dendrobium nobile*. The statistical results are shown in Table 5. The table shows that the dominant associated moss species for all four evaluation methods included *Lysimachia verruca* and *Lysimachia juniperina*. Among them, *Lysimachia verruca* (9 times) and *Lysimachia juniperina* (10 times), which had the strongest correlation with dry matter accumulation, had significantly higher association frequencies than those evaluated by dendrobine, mineral elements, and multiple indicators. Comparison of the coverage rates of the dominant associated moss showed that the coverage rates of *Lysimachia verruca* for dry matter and dendrobine were 67.22% and 67.50%, respectively, significantly higher than those evaluated by mineral elements and multiple indicators. However, the coverage rate of *Lysimachia juniperina* did not show significant differences among different evaluation criteria. In conclusion, the dominant bryophyte species associated with Dendrobium nobile were identified as *Lysimachia verrucosum* and *Lysimachia juniper*.

[0155] Table 5. Frequency and coverage of pseudodominant mosses (frequency > 5)

[0156]

[0157] 1.3 Conclusion

[0158] A rich variety of bryophytes coexist with Dendrobium nobile. A total of 31 species belonging to 24 genera and 15 families were identified in the study area. Among them, 29 species belong to 22 genera and 13 families of bryophytes, and 2 species belong to 2 genera and 2 families of bryophytes. Bryophytes play a dominant role in the association with Dendrobium nobile, with mixed association of 5-6 species being quite common. Different bryophyte species have a significant impact on the dry matter accumulation, quality, and mineral element content of Dendrobium nobile. Principal component analysis and membership function analysis identified *Lysimachia verruca* and *Lysimachia juniperina* as the dominant bryophytes associated with Dendrobium nobile, and these two bryophytes coexist with Dendrobium nobile through mixed growth.

[0159] 2. Verification experiment of *Moss lichen* and *Moss juniper*

[0160] 2.1. Materials and Methods

[0161] 2.1.1 Overview of the test site

[0162] This study selected the Dendrobium nobile cultivation base in Yaling Village, Wanglong Town, Chishui City, Guizhou Province, as the experimental site, which is a representative contiguous area for Dendrobium nobile cultivation. The Dendrobium nobile was cultivated in a semi-wild environment under trees. Wanglong Town (105°90′N, 28°52′E) is located in the central part of Chishui City and is one of the main production areas of Dendrobium nobile in Chishui. The Dendrobium nobile cultivation base in Yaling Village (105°91′E, 28°51′N) is at an altitude of 500m and is the largest contiguous area for Dendrobium nobile cultivation in Chishui City (more than 2,000 mu). Wanglong Town is located in a semi-mountainous hilly area in a river valley, with a terrain that is high in the southeast and low in the northwest. It has a subtropical monsoon climate and an altitude of 228 to 1256 meters. According to the Chishui Meteorological Bureau, the average annual rainfall in Wanglong Town from 2012 to 2022 was 785.11 to 1658.37 mm, and the average annual temperature was 17.37 to 18.79℃.

[0163] 2.1.2 Test Materials

[0164] Dendrobium nobile: Dendrobium nobile planted in the study area for 3 years in a semi-wild environment with Danxia stone was identified by Professor Zhao Zhi of the College of Agriculture of Guizhou University as Dendrobium nobile, a plant of the genus Dendrobium in the family Orchidaceae.

[0165] 2.1.3 Experimental Design

[0166] In February 2023, the dead branches, fallen leaves, and moss around the roots of Dendrobium nobile cultivated on rocks were cleaned up. On a cloudy or rainy day, Moss sphagnum and Moss juniperae were spread on the roots of Dendrobium nobile. A 10cm × 10cm area was marked with the base of the Dendrobium stem as the center and covered with moss. There were four treatments: Moss sphagnum, 1 / 2 Moss sphagnum and 1 / 2 Moss juniperae, Moss juniperae, and a control (without moss covering). Each treatment had 50 baskets, for a total of 200 baskets.

[0167] 2.1.4 Main Instruments and Equipment

[0168] Inductively coupled plasma atomic emission spectrometer (ICP-OES Optima 8000), fully automated Kjeldahl nitrogen analyzer (HGK-55), gas chromatograph (Agilent 7890A).

[0169] 2.2. Results and Analysis

[0170] 2.2.1 Investigation of moss on the roots of Dendrobium nobile treated with *Moss sphagnum* and *Moss juniper*

[0171] A survey of the moss on the roots of 200 *Dendrobium nobile* plants revealed that all mosses associated with *Dendrobium nobile* were mixed. Even with single treatments of *Lysimachia vulgaris* and *Leucobryum juniperii*, the number of moss species on the roots of *Dendrobium nobile* remained between 4 and 8. *Lysimachia vulgaris* and *Leucobryum juniperii* were frequently associated with *Selaginella tamariscina*, *Bryum shortribum*, *Bryum bilatum*, *Bryum longifolium*, *Bryum glacifolium*, *Bryum tuftum*, and *Pteris curvatum*, reaching over 90%. *Lysimachia vulgaris* mixed with *Bryum curvatum* and *Bryum curvatum* had a relatively high coverage rate, approaching 60%; *Leucobryum juniperii* mixed with *Selaginella tamariscina* and *Bryum longifolium* had a relatively high coverage rate, reaching 50%. The moss water content varied considerably, ranging from 12.84% to 212.35%; the dry biomass of the mosses ranged from 0.49 g / plant to 6.02 g / plant.

[0172] 2.2.2 Effects of *Moss sphagnum* and *Moss juniper* treatments on the growth and yield of *Dendrobium nobile*

[0173] Different moss species associated with Dendrobium nobile showed differences in the number of leaves, stem diameter, stem length, number of stem nodes, and single-branch dry weight of one-year-old Dendrobium nobile. The plant height, stem diameter, number of leaves, number of stem nodes, and single-branch dry weight of the treatments of *Mossula versicolor*, 1 / 2 *Mossula versicolor*, 1 / 2 *Mossula juniper*, and *Mossula juniper* were significantly higher than those of the control treatment.

[0174] Table 6. Number of leaves, stem diameter, stem length, number of stem nodes, and single stem weight of annual Dendrobium nobile.

[0175]

[0176] 2.2.3 Effects of *Mossula verrucosum* and *Mossula juniper* treatments on the quality of *Dendrobium nobile*

[0177] Different types of mosses associated with Dendrobium nobile had little effect on the moisture and ash content of two-year-old and three-year-old Dendrobium nobile, all meeting the standards of the 2020 edition of the Chinese Pharmacopoeia. The extracts and dendrobine contents of the treatments of *Mossula verrucosum*, 1 / 2 *Mossula verrucosum*, 1 / 2 *Mossula juniper*, and *Mossula juniper* were significantly higher than the control treatment, all meeting the standards of the 2020 edition of the Chinese Pharmacopoeia.

[0178] Table 7. Effects of *Moss sphagnum* and *Moss juniper* treatments on the dendrobine content in two-year-old *Dendrobium nobile* stems (%)

[0179]

[0180] Table 8. Effects of *Mossula verruca* and *Mossula juniper* treatments on the dendrobine content in three-year-old *Dendrobium nobile* stems (%)

[0181]

[0182] Table 9. Effects of *Moss sphagnum* and *Moss juniper* treatments on macronutrients in the stems of *Dendrobium nobile*.

[0183]

[0184] 2.3 Conclusion:

[0185] Two dominant symbiotic mosses, *Mossula verrucosum* and *Moss juniperae*, were investigated. An experiment was conducted by covering the roots of *Dendrobium nobile* with *Mossula verrucosum*, half of which was combined with half of *Mossula verrucosum* and half of *Moss juniperae*. The results showed that the presence of *Mossula verrucosum* and *Moss juniperae* increased agronomical indicators and yield of one-year-old *Dendrobium nobile* plants, such as plant height, stem diameter, and individual plant weight. It also increased the content of stem extracts and dendrobine in two-year and three-year-old *Dendrobium nobile* plants, while having little effect on moisture and ash content. Furthermore, they increased the levels of macronutrients in some *Dendrobium nobile* stems, further validating that *Mossula verrucosum* and *Moss juniperae* are dominant symbiotic mosses of *Dendrobium nobile*.

Claims

1. A method for cultivating Dendrobium nobile with moss companion growth, characterized in that: The planting method is carried out according to the following steps: (1) Site selection: Select bare Danxia landforms at an altitude of 300m-1000m, shaded by broad-leaved trees, with a relative humidity of 75%-80% and a shading degree of 55%-70% in the broad-leaved forests; (2) Land preparation: Remove shrubs, weeds, dead branches and leaves, and soil; (3) Transplanting time: Spring transplanting is done in April to May, and autumn transplanting is done in September to October. Transplanting should be done on cloudy days and the temperature should be above 10℃. (4) Transplanting: Select two-year-old Dendrobium nobile seedlings that are free from damage and disease. Plant them at a spacing of 30cm×30cm. Attach the fibrous roots and base of the Dendrobium nobile seedlings to the stone surface and fix them with wire clips. Let the roots spread out naturally. Fix the wire clips 1.5cm to 2.5cm above the base of the main stem of the seedling to ensure that the seedlings are stable on the stone surface and obtain well-fixed Dendrobium nobile tissue culture seedlings. (5) Preparation of cow dung slurry: Take well-rotted cow dung and dilute it with water at a mass ratio of 1:3, and set aside; (6) Apply cow dung paste to the roots of the fixed Dendrobium nobile tissue culture seedlings, covering an area of ​​90-110cm. 2 The cow dung slurry extends from the root area to cover an area of ​​850-1050 cm² on the stone surface. 2 ; (7) Apply live moss evenly and lightly to the roots of the plant and the stone surface that has been coated with cow dung slurry. Spray water evenly to wet the roots of Dendrobium nobile, the stone surface and its moss. After the seedlings have recovered, clean the dead branches and fallen leaves on the Dendrobium nobile plant and around its growth every 10 to 20 days to ensure that Dendrobium nobile and moss grow normally. Plants transplanted in spring can be harvested in October to November of the second year, and plants transplanted in autumn can be harvested in October to November of the third year. The moss is *Moss sphaerocephala* and *Moss juniper*, and the mass ratio of *Moss sphaerocephala* to *Moss juniper* is 0.5–3:3–0.

5.

2. The cultivation method of Dendrobium nobile with moss companion as described in claim 1, characterized in that: In step (6), cow dung slurry is brushed onto the roots of the fixed Dendrobium nobile tissue culture seedlings, with the root area covering 100cm. 2 The cow dung slurry extends from the root area and is applied to the stone surface, covering an area of ​​900 cm². 2 .

3. The cultivation method of Dendrobium nobile with moss companion as described in claim 1, characterized in that: In step (7), the moss is *Moss sphaerocephala* and *Moss juniper*, and the mass ratio of *Moss sphaerocephala* to *Moss juniper* is 1-3:3-1.

4. The cultivation method of Dendrobium nobile with moss companion as described in claim 1, characterized in that: In step (7), the moss also includes one or more of the following: Selaginella thin-walled, Selaginella short-ribbed, Bryophytum bilatum, Bryophytum longleaf, Bryophytum glacifolium, Bryophytum tuftedum, or Bryophytum curcumae.

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