Method for promoting growth of an anthurium andraeanum seedling

Abstract

This application provides a method for promoting the growth of *Anoectochilus roxburghii* seedlings. The method involves irrigating the seedlings with a culture solution of mycorrhizal fungi from *Anoectochilus roxburghii*, allowing the mycorrhizal fungi culture solution to permeate the stems and leaves. This method significantly antagonizes pathogens causing stem rot in *Anoectochilus roxburghii* seedlings.

Description

Technical Field

[0001] This invention relates to the field of plant cultivation technology, and in particular to a method for promoting the growth of *Anoectochilus roxburghii* seedlings. Background Technology

[0002] Anoectochilus roxburghii is an orchid with high medicinal and ornamental value. However, over-harvesting has led to the depletion of wild resources, necessitating large-scale artificial propagation. But with the expansion of the Anoectochilus roxburghii industry, the problem of pathogenic contamination in tissue-cultured seedlings has become prominent, such as stem rot. For example, Chinese invention patent application number 201210430120.6 uses a wood substrate instead of leaf mold for cultivation, thereby reducing leaf rot in Anoectochilus roxburghii tissue-cultured seedlings. However, since countless pathogens exist in the environment, simply reducing the pathogen content in the cultivation soil to reduce contamination of Anoectochilus roxburghii tissue-cultured seedlings is insufficient to alleviate disease in these seedlings. Mycorrhizal fungi have limited effectiveness in controlling stem rot in *Anoectochilus roxburghii*. For example, Chinese invention patent application number 201710702306.5 isolated endophytic fungi from *Anoectochilus roxburghii* that significantly antagonize stem rot pathogens, effectively promoting the large-scale propagation of *Anoectochilus roxburghii*. However, there are few reports on the discovery of fungi that significantly antagonize stem rot pathogens in *Anoectochilus roxburghii* seedlings. Therefore, the discovery of fungi that significantly antagonize stem rot pathogens in *Anoectochilus roxburghii* seedlings remains a persistent pursuit for those skilled in the art. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for promoting the growth of *Anoectochilus roxburghii* seedlings that can significantly antagonize pathogens causing stem rot in *Anoectochilus roxburghii* seedlings.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] A method for promoting the growth of Anoectochilus roxburghii seedlings involves watering the seedlings with a culture solution of mycorrhizal fungi from the orange-yellow jade phoenix flower, so that the culture solution soaks the stems and leaves of the seedlings.

[0006] In one embodiment, the seedlings of *Anoectochilus roxburghii* are irrigated with a culture solution of mycorrhizal fungi of *Anoectochilus roxburghii*, including the following steps:

[0007] Obtain the aforementioned *Anoectochilus roxburghii* seedlings;

[0008] The seedlings of *Anoectochilus roxburghii* were sprayed with the mycorrhizal fungal culture solution so that the culture solution adhered to the stems and leaves of the seedlings.

[0009] In one embodiment, the amount of mycorrhizal fungal culture medium used is 5 ml to 15 ml.

[0010] In one embodiment, the mycorrhizal fungal culture medium is obtained through the following steps:

[0011] Picking wild orange-yellow jade phoenix flowers;

[0012] The rootstock of the orange-yellow jade phoenix flower is treated to clean the surface of the rootstock and obtain new nutrient roots that form mycorrhizae.

[0013] The newly formed nutrient roots are scraped to extract the fungal colonies from them, thus obtaining the orange-yellow jade phoenix flower fungal community.

[0014] The orange-yellow jade phoenix flower fungus community was proliferated and cultured to obtain the mycorrhizal fungi culture medium.

[0015] In one embodiment, the newly formed nutrient roots are subjected to a mycelial scraping operation, specifically as follows:

[0016] The newly formed nutrient roots were placed in a sterile water petri dish, and a single mycelial mass of the newly formed nutrient roots was scraped off using a sterilized inoculation needle.

[0017] In one embodiment, the orange-yellow jade phoenix flower microbial community is subjected to a proliferation culture operation, specifically as follows:

[0018] The orange-yellow jade flower microbial community was inoculated into PDB medium, and then the PDB medium was placed in a shaker at a temperature of 25℃~28℃ for culture.

[0019] In one embodiment, the rotational speed of the shaker is 110 r / min to 120 r / min.

[0020] In one embodiment, the PDB culture medium is in the form of 100ml to 150ml.

[0021] In one embodiment, the PDB culture medium is placed in a shaker at 25°C to 28°C and cultured for at least 3 days.

[0022] In one embodiment, the PDB culture medium is placed in a shaker at a temperature of 25°C to 28°C for cultivation, and then the PDB culture medium is broken up and mixed so that the clumps of mycelia in the PDB culture medium are evenly dispersed in the PDB culture medium to obtain the mycorrhizal fungi culture solution.

[0023] Compared with the prior art, the present invention has at least the following advantages:

[0024] The method for promoting the growth of *Anoectochilus roxburghii* seedlings of this invention involves irrigating the stems and leaves of *Anoectochilus roxburghii* seedlings with a culture solution of mycorrhizal fungi from *Anoectochilus roxburghii*. This allows the mycorrhizal fungi culture solution to directly act on the stems and leaves of the *Anoectochilus roxburghii* seedlings, promoting the entry of endophytic fungi from *Anoectochilus roxburghii* into the interior of the seedlings. This effectively enhances the stress resistance of the *Anoectochilus roxburghii* seedlings, especially showing a good antagonistic effect against pathogens causing stem rot, thereby significantly improving the survival rate of the *Anoectochilus roxburghii* seedlings. Detailed Implementation

[0025] It should be noted that when an element is referred to as being "fixed to" another element, it can be directly attached to the other element or there may be an intervening element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or there may be an intervening element. The terms "vertical," "horizontal," "left," "right," and similar expressions used herein are for illustrative purposes only and do not represent the only possible implementation.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] This application provides a method for promoting the growth of *Anoectochilus roxburghii* seedlings. To better understand this method, the following further explanation is provided:

[0028] One method for promoting the growth of Anoectochilus roxburghii seedlings involves watering the seedlings with a culture solution of mycorrhizal fungi from the orange-yellow jade phoenix flower, so that the mycorrhizal fungi culture solution soaks into the stems and leaves of the seedlings.

[0029] The above-mentioned method for promoting the growth of *Anoectochilus roxburghii* seedlings involves irrigating the stems and leaves of the seedlings with a culture solution of mycorrhizal fungi from *Anoectochilus roxburghii*. This allows the mycorrhizal fungi culture solution to permeate the stems and leaves of the seedlings, enabling the mycorrhizal fungi culture solution to act directly on the stems and leaves of the seedlings. This promotes the entry of the endophytic fungi of *Anoectochilus roxburghii* into the interior of the stems and leaves of the seedlings, effectively enhancing the stress resistance of the seedlings. In particular, it has a good antagonistic effect on the pathogens causing stem rot, thereby significantly improving the survival rate of the seedlings.

[0030] In one embodiment, the orange-yellow jade phoenix flower was collected from the Xiangtoushan Nature Reserve. More specifically, the orange-yellow jade phoenix flower was collected from a location at 114° east longitude, 23° north latitude, and an altitude of 460m.

[0031] In one embodiment, the seedlings of *Anoectochilus roxburghii* are irrigated with a culture solution of mycorrhizal fungi of *Anoectochilus roxburghii*, including the following steps:

[0032] S110. Obtain *Anoectochilus roxburghii* seedlings. Furthermore, the *Anoectochilus roxburghii* seedlings came from the *Anoectochilus roxburghii* planting and breeding base of Hujinyuan Ecological Agriculture Development Co., Ltd., Longmen County, Huizhou City, Guangdong Province.

[0033] S120. Spray the seedlings of *Anoectochilus roxburghii* with mycorrhizal fungal culture solution so that the mycorrhizal fungal culture solution adheres to the stems and leaves of *Anoectochilus roxburghii*.

[0034] It is understandable that spraying the mycorrhizal fungal culture solution directly onto the stems and leaves of the *Anoectochilus roxburghii* seedlings allows for effective contact and immersion, facilitating the entry of the endophytic fungi of *Anoectochilus roxburghii* into the seedlings. Furthermore, during this direct spraying, some of the mycorrhizal fungal culture solution flows down to the roots of the seedlings, promoting a symbiotic relationship between the endophytic fungi and the root system, thus enhancing the overall resilience of the seedlings.

[0035] In one embodiment, after the obtained *Anoectochilus roxburghii* seedlings are planted in a planting substrate, the seedlings are then sprayed with mycorrhizal fungal culture solution.

[0036] In one embodiment, the planting substrate is peat moss, which is beneficial to the growth of *Anoectochilus roxburghii* seedlings. In another embodiment, after obtaining the *Anoectochilus roxburghii* seedlings and before spraying them with mycorrhizal fungal culture solution, the method for promoting the growth of *Anoectochilus roxburghii* seedlings further includes the following step: treating the mycorrhizal fungal culture solution or the *Anoectochilus roxburghii* seedlings to promote the adhesion and penetration of the mycorrhizal fungal culture solution onto the surface of the stems and leaves of the *Anoectochilus roxburghii* seedlings. Further, the treatment of the mycorrhizal fungal culture solution to promote penetration specifically involves adding tetrahydropiperine to the mycorrhizal fungal culture solution. Further, the mass percentage of tetrahydropiperine in the mycorrhizal fungal culture solution is 0.02% to 0.1%. It is understandable that the use of tetrahydropiperine effectively promotes the invasion of endophytic fungi of *Anoectochilus roxburghii* into the stems and leaves of *Anoectochilus roxburghii* seedlings, thus better promoting the entry of endophytic fungi of *Anoectochilus roxburghii* into the stems and leaves of *Anoectochilus roxburghii* seedlings, better ensuring the enhanced stress resistance of *Anoectochilus roxburghii* seedlings, and thus better ensuring the survival rate of *Anoectochilus roxburghii* seedlings.

[0037] In one embodiment, the *Anoectochilus roxburghii* seedlings are subjected to a penetration-enhancing treatment. Specifically, the surface of the seedlings is locally rubbed to create wounds on the cortical layer of the stems and leaves. Further, no more than 0.1% of the seedlings have wounds on the cortical layer of their stems and leaves. It is understood that controlling the number of wounds to no more than 0.1% of the seedlings' stems and leaves will not affect their growth. Furthermore, even 0.1% of the seedlings having wounds effectively promotes the invasion of *Anoectochilus roxburghii* endophytic fungi into the stems and leaves of the *Anoectochilus roxburghii* seedlings, thus better facilitating the entry of these fungi into the interior of the seedlings' stems and leaves, better ensuring the enhanced stress resistance of the seedlings, and consequently, better ensuring their survival rate.

[0038] In one embodiment, the amount of mycorrhizal fungal culture solution used is 5ml to 15ml. It is understood that when the stems and leaves of *Anoectochilus roxburghii* seedlings are fully immersed in the mycorrhizal fungal culture solution via spraying, the antagonistic effect against the pathogens causing stem rot in *Anoectochilus roxburghii* seedlings is better when the amount of mycorrhizal fungal culture solution used is 5ml to 15ml. However, if the amount used is less, the dominance of the endophytic fungi of *Anoectochilus roxburghii* is weaker, making it less effective in enhancing the resistance of *Anoectochilus roxburghii* seedlings. Conversely, if the amount used is more, the dominance of the endophytic fungi of *Anoectochilus roxburghii* is stronger, which may affect the dominance of the native fungi in *Anoectochilus roxburghii* seedlings, thus negatively impacting the growth of *Anoectochilus roxburghii* seedlings.

[0039] In one embodiment, the mycorrhizal fungal culture medium is obtained through the following steps:

[0040] S210, Picking wild orange-yellow jade phoenix flowers;

[0041] S220. Treat the rhizomes of the orange-yellow jade phoenix flower to clean the surface of the rhizomes and obtain new nutrient roots that form mycorrhizae.

[0042] S230 performs a scraping operation on the newly grown nutrient roots to extract the fungal clusters on the new nutrient roots, thus obtaining the orange-yellow jade phoenix flower fungal community.

[0043] S240. The mycorrhizal fungi culture solution was obtained by propagating and culturing the orange-yellow jade phoenix fungus.

[0044] It is understood that the endophytic fungi of *Anoectochilus roxburghii* are cultured together to form a mycorrhizal fungi culture solution. The presence of at least one endophytic fungus of *Anoectochilus roxburghii* can effectively enhance the stress resistance of *Anoectochilus roxburghii* seedlings. This application does not purify or isolate the specific species of endophytic fungi, but only protects the endophytic fungi of *Anoectochilus roxburghii* by culturing them together to form a mycorrhizal fungi culture solution. When used on *Anoectochilus roxburghii* seedlings, it plays a good role in enhancing the stress resistance. It is worth mentioning that although there are multiple endophytic fungi in the mycorrhizal fungi culture solution, it will not affect the growth of *Anoectochilus roxburghii* seedlings when the amount used is controlled.

[0045] In one embodiment, the rhizome treatment of the orange-yellow jade phoenix flower specifically includes the following steps:

[0046] S310. The rhizomes of the orange-yellow jade phoenix flower are rinsed and cut to remove particulate matter from the surface of the rhizomes, resulting in multiple rhizome segments.

[0047] S320. Deeply clean the root and stem segments of the orange-yellow jade phoenix flower to remove bacteria and harmful substances from the surface of the root and stem segments.

[0048] S330. The root and stem segments of the orange-yellow jade phoenix flower after deep cleaning are cleaned to remove the liquid on the surface of the root and stem segments and obtain new nutrient roots.

[0049] It is understandable that by cleaning the surface of the roots and stems of the Orange-Yellow Jade Phoenix Flower separately to remove particulate matter, bacteria, and harmful substances, it is easier for the newly formed nutrient roots to obtain a high purity of the Orange-Yellow Jade Phoenix Flower's endophytic fungal community. This helps to reduce the impact of miscellaneous bacteria or other substances on the growth of the fungal community at the newly formed nutrient roots during the propagation and culture process.

[0050] In one embodiment, the rhizomes of the orange-yellow jade phoenix flower are rinsed and cut. Specifically, the rhizomes of the orange-yellow jade phoenix flower are rinsed under tap water, and then cut into segments to obtain rhizome segments. This effectively ensures the removal of particulate matter on the surface of the rhizomes of the orange-yellow jade phoenix flower, and the formation of rhizome segments is beneficial for scraping the mycelial masses of newly formed nutrient roots later.

[0051] In one embodiment, the length of the rootstock segment of the orange-yellow jade phoenix flower is 1cm to 2cm, which is beneficial for scraping out the mycelial mass of newly formed nutrient roots in the later stage.

[0052] In one embodiment, the root and stem segments of the orange-yellow jade phoenix flower were subjected to deep cleaning treatment. Specifically, the root and stem segments of the orange-yellow jade phoenix flower were soaked in sodium hypochlorite solution, and then taken out and soaked in ethanol solution, which effectively removed bacteria and harmful substances from the surface of the root and stem segments of the orange-yellow jade phoenix flower.

[0053] In one embodiment, the sodium hypochlorite solution has a mass percentage of 2.5%. Furthermore, soaking the rhizome segments of *Echeveria elegans* in the sodium hypochlorite solution for 0.5 to 1 minute further effectively removes bacteria and harmful substances from the surface of the rhizome segments.

[0054] In one embodiment, the ethanol solution has a mass percentage of 70%. Furthermore, immersing the rhizome segments of *Echeveria elegans* in the ethanol solution for 0.8 min to 1 min further effectively removes bacteria and harmful substances from the surface of the rhizome segments.

[0055] In one embodiment, the root and stem segments of the orange-yellow jade phoenix flower after deep cleaning are further cleaned by rinsing the root and stem segments of the orange-yellow jade phoenix flower with sterile water at least three times, and then using sterile absorbent paper to dry the liquid on the surface of the root and stem segments of the orange-yellow jade phoenix flower, which further reduces the residue of various reagents or bacteria on the surface of the orange-yellow jade phoenix flower.

[0056] In one embodiment, a mycelial clump scraping operation is performed on the newly formed nutrient roots. Specifically, the newly formed nutrient roots are placed in a culture dish with sterile water, and a single mycelial clump of the newly formed nutrient roots is scraped off using a sterilized inoculation needle, which effectively achieves the acquisition of endophytic fungi on the newly formed nutrient roots.

[0057] In one embodiment, before the step of propagating and culturing the *Pterocarya stenoptera* mycorrhizal fungi and after the step of scraping the mycorrhizal fungi from the newly formed vegetative roots, the mycorrhizal fungi culture solution further includes the following step: selectively culturing the *Pterocarya stenoptera* mycorrhizal fungi to inhibit the excessive growth of other fungi in the *Pterocarya stenoptera* mycorrhizal fungi.

[0058] In one embodiment, selective culture of the *Pterocarya stenoptera* fungal community was performed by adding fluconazole to the PDB medium. Further, the amount of fluconazole used was 0.1 g / L to 0.5 g / L.

[0059] It is understandable that, since there are many types of endophytic fungi in *Anoectochilus roxburghii*, and at least one of these endophytic fungi enhances the stress resistance of *Anoectochilus roxburghii* seedlings, fluconazole was used for selective proliferation culture of the endophytic fungi in order to better achieve the effective growth of the endophytic fungi that enhance the stress resistance of *Anoectochilus roxburghii* seedlings. In this way, the resulting mycorrhizal fungal culture solution has a better enhancing effect on the stress resistance of *Anoectochilus roxburghii* seedlings.

[0060] In one embodiment, the preparation of PDB culture medium is specifically carried out as follows: 200g of peeled potatoes are boiled in boiling water for 30 minutes, filtered through gauze, 20g of glucose is added, water is added to make up to 1L, and sterilized at 121℃ for 30 minutes.

[0061] In one embodiment, the *Anoectochilus roxburghii* fungal community was proliferated and cultured. Specifically, the fungal community was inoculated into PDB medium, and then the PDB medium was placed in a shaker at a temperature of 25°C–28°C for cultivation. Further, the shaker speed was 110–120 rpm. Further, the PDB medium volume was 100–150 ml. Further, the PDB medium was cultured in a shaker at 25°C–28°C for at least 3 days. This effectively achieved rapid proliferation of the *Anoectochilus roxburghii* fungal community, reduced mutual interference among the fungal communities, and thus ensured the enhanced stress resistance effect of the mycorrhizal fungal culture solution formed after the proliferation and cultivation of the *Anoectochilus roxburghii* fungal community on the *Anoectochilus roxburghii* seedlings. It is understandable that if the *Anoectochilus roxburghii* fungal community overgrows, it will affect the mutual interference among the fungal communities, thus affecting the effect of the mycorrhizal fungal culture solution on enhancing the stress resistance of *Anoectochilus roxburghii* seedlings. If the *Anoectochilus roxburghii* fungal community does not grow sufficiently, the concentration of endophytic fungi of *Anoectochilus roxburghii* in the mycorrhizal fungal culture solution will be low, making it difficult to meet the needs of enhancing the stress resistance of *Anoectochilus roxburghii* seedlings.

[0062] In one embodiment, the PDB medium is placed in a shaker at a temperature of 25°C to 28°C for cultivation, and then the PDB medium is broken up and mixed so that the clumps of mycelia in the PDB medium are evenly dispersed in the PDB medium to obtain mycorrhizal fungi culture medium.

[0063] Compared with the prior art, the present invention has at least the following advantages:

[0064] The method for promoting the growth of *Anoectochilus roxburghii* seedlings of this invention involves irrigating the stems and leaves of *Anoectochilus roxburghii* seedlings with a culture solution of mycorrhizal fungi from *Anoectochilus roxburghii*. This allows the mycorrhizal fungi culture solution to directly act on the stems and leaves of the *Anoectochilus roxburghii* seedlings, promoting the entry of endophytic fungi from *Anoectochilus roxburghii* into the interior of the seedlings. This effectively enhances the stress resistance of the *Anoectochilus roxburghii* seedlings, especially showing a good antagonistic effect against pathogens causing stem rot, thereby significantly improving the survival rate of the *Anoectochilus roxburghii* seedlings.

[0065] The following are some specific examples. Where %, it refers to a percentage by weight. It should be noted that the following examples do not exhaustively cover all possible scenarios, and unless otherwise specified, the materials used in the following examples are commercially available.

[0066] Example 1

[0067] Sampling: Orange-yellow jade phoenix flower, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m altitude);

[0068] Preparation of PDB medium: Boil 200g of potatoes (peeled) in boiling water for 30 minutes, filter with gauze, add 20g of glucose, add water to make up to 1L, put it in an electric pressure steam sterilizer at 121℃ for 30 minutes, divide into Erlenmeyer flasks and set aside. The capacity of the Erlenmeyer flasks is 100ml.

[0069] Rinse the collected roots of the orange-yellow jade phoenix flower with running water to remove sand and other particles from the surface of the rootstock. Select the rootstock of the orange-yellow jade phoenix flower, cut it with scissors, place it in a fixed cup, and transfer it to an ultra-clean workbench. Cut the rootstock of the orange-yellow jade phoenix flower into small sections of 1.5±0.5cm each. Then soak them in 2.5% sodium hypochlorite solution for 0.5min. Then soak each sample plant in 70% ethanol solution for 0.8min. Wash with sterile water (rinse 3 times). Gently scrape the surface of the rootstock with a sterile inoculation needle. Rinse with sterile water 5 times. Place it on sterile absorbent paper to dry, and obtain the new nutrient roots.

[0070] Place the newly formed nutrient roots in a sterile water petri dish and scrape off a single mycelial mass from the newly formed nutrient roots using a sterilized inoculation needle.

[0071] On a clean bench, single mycelial clusters of newly formed vegetative roots are picked up with sterilized pipettes and bamboo sticks and placed into PDB medium. They are then cultured for 3 days on a constant temperature shaker at 25°C and 110 r / min. The mycelial balls in the PDB medium are then cut into pieces with sterilized forceps and scissors, and the mycelium and mycelium solution are evenly mixed with the liquid using a vortex shaker to prepare mycorrhizal fungi culture solution.

[0072] Example 2

[0073] Sampling: Orange-yellow jade phoenix flower, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m altitude);

[0074] Preparation of PDB medium: Boil 200g of potatoes (peeled) in boiling water for 30 minutes, filter with gauze, add 20g of glucose, add water to make up to 1L, put it in an electric pressure steam sterilizer at 121℃ for 30 minutes, divide into Erlenmeyer flasks and set aside. The capacity of the Erlenmeyer flasks is 150ml.

[0075] Rinse the collected roots of the orange-yellow jade phoenix flower with running water to remove sand and other particles from the surface of the rootstock. Select the rootstocks of the orange-yellow jade phoenix flower, cut them with scissors, place them in a fixed cup, and transfer them to a clean bench. Cut the rootstocks of the orange-yellow jade phoenix flower into small sections of 1.5±0.5cm each. Then soak them in 2.5% sodium hypochlorite solution for 0.8min. Then soak each sample plant in 70% ethanol solution for 0.9min. Wash them with sterile water (rinse three times). Gently scrape the surface of the rootstock with a sterile inoculation needle. Rinse them with sterile water 6 times. Place them on sterile absorbent paper to dry, and obtain new nutrient roots.

[0076] Place the newly formed nutrient roots in a sterile water petri dish and scrape off a single mycelial mass from the newly formed nutrient roots using a sterilized inoculation needle.

[0077] On a clean bench, single mycelial clusters of newly formed vegetative roots are picked up into PDB medium using sterilized pipettes and bamboo sticks and cultured on a constant temperature shaker at 26°C and 116 r / min for 4 days. The mycelial balls in the PDB medium are then cut into pieces with sterilized forceps and scissors, and the mycelium and mycelium solution are evenly mixed using a vortex shaker to prepare mycorrhizal fungi culture solution.

[0078] Example 3

[0079] Sampling: Orange-yellow jade phoenix flower, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m altitude);

[0080] Preparation of PDB medium: Boil 200g of potatoes (peeled) in boiling water for 30 minutes, filter with gauze, add 20g of glucose, add water to make up to 1L, put it in an electric pressure steam sterilizer at 121℃ for 30 minutes, divide into Erlenmeyer flasks and set aside. The capacity of the Erlenmeyer flasks is 150ml.

[0081] Rinse the collected roots of the orange-yellow jade phoenix flower with running water to remove sand and other particles from the surface of the rootstock. Select the rootstock of the orange-yellow jade phoenix flower, cut it with scissors, place it in a fixed cup, and transfer it to an ultra-clean workbench. Cut the rootstock of the orange-yellow jade phoenix flower into small sections of 1.5±0.5cm each. Then soak them in 2.5% sodium hypochlorite solution for 1 minute. Then soak each sample plant in 70% ethanol solution for 1 minute. Wash with sterile water (rinse three times). Gently scrape the surface of the rootstock with a sterile inoculation needle. Rinse with sterile water 6 times. Place it on sterile absorbent paper to dry, and obtain the new nutrient roots.

[0082] Place the newly formed nutrient roots in a sterile water petri dish and scrape off a single mycelial mass from the newly formed nutrient roots using a sterilized inoculation needle.

[0083] On a clean bench, single mycelial clusters of newly formed vegetative roots are picked up with sterilized pipettes and bamboo sticks and placed into PDB medium. They are then cultured for 5 days on a constant temperature shaker at 28°C and 120 r / min. The mycelial balls in the PDB medium are then cut into pieces with sterilized forceps and scissors, and the mycelium and mycelium solution are evenly mixed using a vortex shaker to prepare mycorrhizal fungi culture solution.

[0084] Example 4

[0085] Sampling: Orange-yellow jade phoenix flower, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m altitude);

[0086] Preparation of PDB medium: Boil 200g of potatoes (peeled) in boiling water for 30 minutes, filter with gauze, add 20g of glucose and 0.1g / L of fluconazole, add water to make up to 1L, put it in an electric pressure steam sterilizer at 121℃ for 30 minutes, divide into Erlenmeyer flasks and set aside. The capacity of the Erlenmeyer flasks is 150ml.

[0087] Rinse the collected roots of the orange-yellow jade phoenix flower with running water to remove sand and other particles from the surface of the rootstock. Select the rootstock of the orange-yellow jade phoenix flower, cut it with scissors, place it in a fixed cup, and transfer it to an ultra-clean workbench. Cut the rootstock of the orange-yellow jade phoenix flower into small sections of 1.5±0.5cm each. Then soak them in 2.5% sodium hypochlorite solution for 1 minute. Then soak each sample plant in 70% ethanol solution for 1 minute. Wash with sterile water (rinse three times). Gently scrape the surface of the rootstock with a sterile inoculation needle. Rinse with sterile water 6 times. Place it on sterile absorbent paper to dry, and obtain the new nutrient roots.

[0088] Place the newly formed nutrient roots in a sterile water petri dish and scrape off a single mycelial mass from the newly formed nutrient roots using a sterilized inoculation needle.

[0089] On a clean bench, single mycelial clusters of newly formed vegetative roots are picked up with sterilized pipettes and bamboo sticks and placed into PDB medium. They are then cultured for 5 days on a constant temperature shaker at 28°C and 120 r / min. The mycelial balls in the PDB medium are then cut into pieces with sterilized forceps and scissors, and the mycelium and mycelium solution are evenly mixed using a vortex shaker to prepare mycorrhizal fungi culture solution.

[0090] Example 5

[0091] Sampling: Orange-yellow jade phoenix flower, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m altitude);

[0092] Preparation of PDB medium: Boil 200g of potatoes (peeled) in boiling water for 30 minutes, filter with gauze, add 20g of glucose and 0.2g / L of fluconazole, add water to make up to 1L, put it in an electric pressure steam sterilizer at 121℃ for 30 minutes, divide into Erlenmeyer flasks and set aside. The capacity of the Erlenmeyer flasks is 150ml.

[0093] Rinse the collected roots of the orange-yellow jade phoenix flower with running water to remove sand and other particles from the surface of the rootstock. Select the rootstock of the orange-yellow jade phoenix flower, cut it with scissors, place it in a fixed cup, and transfer it to an ultra-clean workbench. Cut the rootstock of the orange-yellow jade phoenix flower into small sections of 1.5±0.5cm each. Then soak them in 2.5% sodium hypochlorite solution for 1 minute. Then soak each sample plant in 70% ethanol solution for 1 minute. Wash with sterile water (rinse three times). Gently scrape the surface of the rootstock with a sterile inoculation needle. Rinse with sterile water 6 times. Place it on sterile absorbent paper to dry, and obtain the new nutrient roots.

[0094] Place the newly formed nutrient roots in a sterile water petri dish and scrape off a single mycelial mass from the newly formed nutrient roots using a sterilized inoculation needle.

[0095] On a clean bench, single mycelial clusters of newly formed vegetative roots are picked up with sterilized pipettes and bamboo sticks and placed into PDB medium. They are then cultured for 5 days on a constant temperature shaker at 28°C and 120 r / min. The mycelial balls in the PDB medium are then cut into pieces with sterilized forceps and scissors, and the mycelium and mycelium solution are evenly mixed using a vortex shaker to prepare mycorrhizal fungi culture solution.

[0096] Example 6

[0097] Sampling: Orange-yellow jade phoenix flower, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m altitude);

[0098] Preparation of PDB medium: 200g of potatoes (peeled) were boiled in boiling water for 30 minutes, filtered through gauze, 20g of glucose and 0.3g / L of fluconazole were added, and water was added to make up to 1L. The mixture was then placed in an electric pressure steam sterilizer at 121℃ for 30 minutes and sterilized. The mixture was then divided into 150ml Erlenmeyer flasks for later use.

[0099] Rinse the collected roots of the orange-yellow jade phoenix flower with running water to remove sand and other particles from the surface of the rootstock. Select the rootstock of the orange-yellow jade phoenix flower, cut it with scissors, place it in a fixed cup, and transfer it to an ultra-clean workbench. Cut the rootstock of the orange-yellow jade phoenix flower into small sections of 1.5±0.5cm each. Then soak them in 2.5% sodium hypochlorite solution for 1 minute. Then soak each sample plant in 70% ethanol solution for 1 minute. Wash with sterile water (rinse three times). Gently scrape the surface of the rootstock with a sterile inoculation needle. Rinse with sterile water 6 times. Place it on sterile absorbent paper to dry, and obtain the new nutrient roots.

[0100] Place the newly formed nutrient roots in a sterile water petri dish and scrape off a single mycelial mass from the newly formed nutrient roots using a sterilized inoculation needle.

[0101] On a clean bench, single mycelial clusters of newly formed vegetative roots are picked up with sterilized pipettes and bamboo sticks and placed into PDB medium. They are then cultured for 5 days on a constant temperature shaker at 28°C and 120 r / min. The mycelial balls in the PDB medium are then cut into pieces with sterilized forceps and scissors, and the mycelium and mycelium solution are evenly mixed using a vortex shaker to prepare mycorrhizal fungi culture solution.

[0102] Example 7

[0103] Sampling: Orange-yellow jade phoenix flower, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m altitude);

[0104] Preparation of PDB medium: Boil 200g of potatoes (peeled) in boiling water for 30 minutes, filter with gauze, add 20g of glucose and 0.5g / L of fluconazole, add water to make up to 1L, put it in an electric pressure steam sterilizer at 121℃ for 30 minutes, divide into Erlenmeyer flasks and set aside. The capacity of the Erlenmeyer flasks is 150ml.

[0105] Rinse the collected roots of the orange-yellow jade phoenix flower with running water to remove sand and other particles from the surface of the rootstock. Select the rootstock of the orange-yellow jade phoenix flower, cut it with scissors, place it in a fixed cup, and transfer it to an ultra-clean workbench. Cut the rootstock of the orange-yellow jade phoenix flower into small sections of 1.5±0.5cm each. Then soak them in 2.5% sodium hypochlorite solution for 1 minute. Then soak each sample plant in 70% ethanol solution for 1 minute. Wash with sterile water (rinse three times). Gently scrape the surface of the rootstock with a sterile inoculation needle. Rinse with sterile water 6 times. Place it on sterile absorbent paper to dry, and obtain the new nutrient roots.

[0106] Place the newly formed nutrient roots in a sterile water petri dish and scrape off a single mycelial mass from the newly formed nutrient roots using a sterilized inoculation needle.

[0107] On a clean bench, single mycelial clusters of newly formed vegetative roots are picked up with sterilized pipettes and bamboo sticks and placed into PDB medium. They are then cultured for 5 days on a constant temperature shaker at 28°C and 120 r / min. The mycelial balls in the PDB medium are then cut into pieces with sterilized forceps and scissors, and the mycelium and mycelium solution are evenly mixed using a vortex shaker to prepare mycorrhizal fungi culture solution.

[0108] The mycorrhizal fungal culture solutions obtained in Examples 1 to 7 were used for the cultivation of Anoectochilus roxburghii seedlings.

[0109] 1. Materials:

[0110] 1.1 The Anoectochilus roxburghii seedlings with the same growth rate were sourced from the Anoectochilus roxburghii planting and breeding base of Hujinyuan Ecological Agriculture Development Co., Ltd. in Longmen County, Huizhou City, Guangdong Province;

[0111] 1.2 Cultivation substrate: peat moss;

[0112] 2. Cultivation conditions:

[0113] The cultivation experiment of Anoectochilus roxburghii was conducted at the School of Life Sciences, Huizhou University, Huizhou City, Guangdong Province. The geographical coordinates are 114.42°E, 23.03°N. The area is located in a subtropical marine monsoon climate zone with an average annual temperature of 22℃ and an annual precipitation of 2200mm, which is concentrated between April and September.

[0114] 3. Test methods:

[0115] 3.1 A completely randomized design was adopted, with 10 seedlings of Anoectochilus roxburghii forming one group and 3 groups of seedlings forming one large group, which were numbered in total, with the 8th large group serving as the blank control group.

[0116] 3.2 The amount of mycorrhizal fungal culture medium used is as follows:

[0117] The seedlings of *Anoectochilus roxburghii* in groups 1 to 7 were respectively treated with the mycorrhizal fungi culture solutions in Examples 1 to 7;

[0118] For each of the seven groups: Group 1 used 5 ml of mycorrhizal fungal culture solution for Anoectochilus roxburghii seedlings; Group 2 used 10 ml of mycorrhizal fungal culture solution for Anoectochilus roxburghii seedlings; and Group 3 used 15 ml of mycorrhizal fungal culture solution for Anoectochilus roxburghii seedlings.

[0119] Group 8: The seedlings of Anoectochilus roxburghii in each group are given the same amount of sterile water as the seedlings of Anoectochilus roxburghii in each group of the other groups.

[0120] 3.3 Spray the corresponding amount of mycorrhizal fungal culture solution for each Anoectochilus roxburghii seedling onto the stems and leaves of the corresponding Anoectochilus roxburghii seedling, and collect the solution after the seedlings have grown to 150 cm.

[0121] 4. Measurement method:

[0122] The survival rate and plant height of each group of *Anoectochilus roxburghii* seedlings were recorded; and the biomass of each group of *Anoectochilus roxburghii* seedlings was measured and recorded: the drying method was used, and the seedlings were dried at 105℃ for 2 hours until constant weight. After drying, the dry weight of the seedlings was weighed, and the biomass and water content of the seedlings were calculated using the formula.

[0123] 5. Measurement Results:

[0124] 5.1 Table 1: Plant height growth of *Anoectochilus roxburghii* seedlings (unit: cm)

[0125]

[0126]

[0127] 5.2 Table 2: Survival rate of Anoectochilus roxburghii seedlings (unit: %)

[0128]

[0129]

[0130] 5.3 Table 3: Biomass of Anoectochilus roxburghii seedlings (total chlorophyll and flavonoids in mg / g; reducing sugars and polyphenols in μg / g)

[0131]

[0132]

[0133] Table 1 shows that the height growth of *Anoectochilus roxburghii* seedlings treated with mycorrhizal fungi culture solution was significantly faster than that of seedlings not treated with the solution, indicating that the mycorrhizal fungi culture solution has a positive promoting effect on the stem and leaf growth of *Anoectochilus roxburghii* seedlings. Table 2 shows that the survival rate of *Anoectochilus roxburghii* seedlings treated with mycorrhizal fungi culture solution was significantly higher than that of seedlings not treated with the solution, indicating that the mycorrhizal fungi culture solution has a positive promoting effect on the survival of *Anoectochilus roxburghii* seedlings, enabling them to effectively... Antagonizing the pathogens of stem rot; As shown in Table 3, the total chlorophyll content, reducing sugar, flavonoids and polyphenols of *Anoectochilus roxburghii* seedlings treated with mycorrhizal fungi culture solution were significantly increased compared with those not treated with mycorrhizal fungi culture solution. The increase in the total chlorophyll content, reducing sugar, flavonoids and polyphenols of *Anoectochilus roxburghii* seedlings will promote the improvement of the stress resistance of *Anoectochilus roxburghii* seedlings. In other words, mycorrhizal fungi culture solution has a significant effect on improving the stress resistance of *Anoectochilus roxburghii* seedlings.

[0134] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for promoting the growth of *Anoectochilus roxburghii* seedlings, characterized in that, The mycorrhizal fungi culture solution of *Anoectochilus roxburghii* was used to irrigate the seedlings of *Anoectochilus roxburghii*, so that the mycorrhizal fungi culture solution could soak the stems and leaves of the seedlings. The mycorrhizal fungi culture medium is obtained through the following steps: Picking wild orange-yellow jade phoenix flowers; The rootstock of the orange-yellow jade phoenix flower is treated to clean the surface of the rootstock and obtain new nutrient roots that form mycorrhizae. The newly formed nutrient roots are scraped to extract the fungal colonies from them, thus obtaining the orange-yellow jade phoenix flower fungal community. The orange-yellow jade phoenix flower fungus community was proliferated and cultured to obtain the mycorrhizal fungus culture medium; Before the step of propagating and culturing the mycorrhizal fungi in *Gymnocalycium chinense*, and after the step of scraping the mycorrhizal fungi from the newly formed vegetative roots, the mycorrhizal fungi culture solution also includes the following steps: selectively culturing the *Gymnocalycium chinense* fungi to inhibit the excessive growth of miscellaneous bacteria in the *Gymnocalycium chinense* fungi. Selective culture of the orange-yellow jade flower fungus was carried out by adding fluconazole to PDB medium at a concentration of 0.1 g / L to 0.5 g / L.

2. The method for promoting the growth of *Anoectochilus roxburghii* seedlings according to claim 1, characterized in that, The seedlings of *Anoectochilus roxburghii* were irrigated with a culture solution of mycorrhizal fungi from *Anoectochilus roxburghii*, including the following steps: Obtain the aforementioned *Anoectochilus roxburghii* seedlings; The seedlings of *Anoectochilus roxburghii* were sprayed with the mycorrhizal fungal culture solution so that the culture solution adhered to the stems and leaves of the seedlings.

3. The method for promoting the growth of *Anoectochilus roxburghii* seedlings according to claim 1 or 2, characterized in that, The amount of mycorrhizal fungi culture medium used is 5ml to 15ml.

4. The method for promoting the growth of *Anoectochilus roxburghii* seedlings according to claim 1, characterized in that, The newly formed nutrient roots are scraped to remove mycelial aggregates. The specific procedure is as follows: The newly formed nutrient roots were placed in a sterile water petri dish, and a single mycelial mass of the newly formed nutrient roots was scraped off using a sterilized inoculation needle.

5. The method for promoting the growth of *Anoectochilus roxburghii* seedlings according to claim 1, characterized in that, The orange-yellow jade phoenix flower microbial community was proliferated and cultured, specifically as follows: The orange-yellow jade phoenix fungus was inoculated into PDB medium, and then the PDB medium was placed in a shaker at a temperature of 25℃~28℃ for culture.

6. The method for promoting the growth of *Anoectochilus roxburghii* seedlings according to claim 5, characterized in that, The rotational speed of the shaking table is 110 r / min to 120 r / min.

7. The method for promoting the growth of *Anoectochilus roxburghii* seedlings according to claim 5, characterized in that, The PDB culture medium is available in 100ml to 150ml containers.

8. The method for promoting the growth of *Anoectochilus roxburghii* seedlings according to claim 7, characterized in that, The PDB culture medium is placed in a shaker at 25℃~28℃ and cultured for at least 3 days.

9. The method for promoting the growth of *Anoectochilus roxburghii* seedlings according to claim 5, characterized in that, The PDB culture medium is placed in a shaker at a temperature of 25℃~28℃ and then the PDB culture medium is broken up and mixed to evenly disperse the clumps of mycelia in the PDB culture medium, thus obtaining the mycorrhizal fungi culture solution.

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