Comparison and correlation study on chemical composition and endophytic bacteria of two epiphytic methods of Dendrobium nobile under simulated cultivation
The medicinal use of Dendrobium officinale was first recorded in the “Shennong Bencao Jing” and is classified as a top grade with a long history. The name “Dendrobium nobile Lindl.” originated from the Song Dynasty’s “Compendium of Materia Medica”. Dendrobium nobile Lindl. is an epiphytic herbaceous plant belonging to the Orchidaceae family and is one of the medicinal varieties of Dendrobium included in the Chinese Pharmacopoeia. The “Compendium of Materia Medica” records it as “clustered on rocks. Its stems and leaves are all green, while its stems and leaves are yellow, and it blooms with red flowers. It is found everywhere, and the one from Sichuan is the best. The Commentary on the Compendium of Materia Medica states: “Dendrobium grows on stones, fine and solid… On oak trees, it is called Dendrobium. It grows to the point of deficiency and does not contain pills or powder, but can be used for making soup with wine stains. The “Compendium of Materia Medica” records: “Only those that grow on stones are superior. There are also those that grow on oak trees, called dendrobium, which are useless. The Compendium of Materia Medica also records: “It is difficult to find effective Dendrobium, but it is useless to obtain Dendrobium.” It can be seen that throughout history, there have been records of Dendrobium being superior to Dendrobium.
Modern research has shown that the active ingredients of Dendrobium nobile include alkaloids, flavonoids, polysaccharides, benzyl compounds and their derivatives, which have protective effects on gastric mucosa and neurons, anti-tumor, antioxidant and immune regulatory activities. Due to its low reproduction rate, slow growth, and strict habitat requirements in its natural state, as well as human over excavation and habitat destruction, the application of artificial cultivation techniques such as greenhouse cultivation and simulated environment cultivation under forest has greatly improved the problem of resource scarcity in Dendrobium officinale. Among them, the simulated environment cultivation technique that simulates the wild habitat of medicinal plants has been applied to Dendrobium officinale, reflecting the coordinated development of medicinal production and ecological environment, with attached stones and trees as the main cultivation modes. Previous studies have comprehensively evaluated the quality of Dendrobium chrysotoxum from the aspects of active ingredient content and yield, and found that it is superior to Dendrobium chrysotoxum cultivation. Moreover, our research group has previously reported that the relative content of alkaloids and flavonoids secondary metabolites in Dendrobium officinale is higher than that in Dendrobium officinale, and the antioxidant activity of Dendrobium officinale is higher than that of Dendrobium officinale.
Endophytic fungi are closely related to the quality of medicinal herbs and plant growth patterns, among which mycorrhizal fungi are a typical mycorrhizal structure of orchids, and mycorrhizal symbiosis accompanies the entire life cycle of plants. The cultivation method is an important factor affecting the root microbiota and the quality of medicinal materials. For example, the composition and distribution of endophytic bacteria in Dendrobium officinale are affected by different cultivation methods and tissue parts. Endophytic bacteria can also significantly improve the stress resistance of Dendrobium plants and promote plant growth and accumulation of secondary metabolites. The research team conducted preliminary studies on the rhizosphere microorganisms of Dendrobium officinale using two types of epiphytic substrates: stone and wood. It was found that the relative abundance of fungal species in the rhizosphere of Dendrobium officinale was higher, and the dominant bacterial genera were more obvious. This suggests that epiphytic substrates are a key factor affecting the rhizosphere microorganisms of Dendrobium officinale. However, there is currently no research reporting the differences in different epiphytic substrates of Dendrobium nobile from the perspective of endophytic bacteria in the roots.
Therefore, in this study, 16S and ITS technologies were used to sequence the endophytic bacteria in the roots of Dendrobium officinale under simulated cultivation of stone epidermal culture (SE) and tree epidermal culture (TE), and the correlation between endophytic bacteria and dendrobine, total alkaloids, total flavonoids, and total polysaccharides was preliminarily analyzed. The diversity and differences of endophytic bacteria in the roots of Dendrobium officinale under two different attachment modes were studied, and the accumulation patterns of endophytic bacteria and secondary metabolites enriched in Dendrobium officinale under different attachment modes were explored to investigate the correlation between endophytic bacteria and secondary metabolites in Dendrobium officinale roots. Lay the foundation and provide scientific basis for the saying ‘the one who grows on the stone wins’ in Dendrobium officinale.

This article analyzes for the first time the diversity and differences of endophytic bacteria in the roots of Dendrobium chrysotoxum under simulated environment cultivation, and for the first time correlates them with the content of major chemical components. The research results show that there are differences in the enrichment of endophytic bacteria in the roots of Dendrobium chrysotoxum under the two epiphytic methods, and the relative abundance of endophytic bacteria is correlated with the content of chemical components.
The root microbiota of medicinal plants plays an important role in obtaining nutrients, resisting environmental stress, and resisting diseases and pests. The dominant fungi in the results of this study are mainly Ascomycota and Basidiomycota, while the dominant bacteria are mainly Actinobacteria, Proteobacteria, Cyanobacteria, and Acidobacteria. In addition, SE also includes Firmicutes, Myxomycetes, and Chloroflexites, and TE also includes Bacteroidetes. Previous studies have shown that the Bacillus genus under the Firmicutes phylum, the Rhizobium genus and Chronic Rhizobium genus under the Proteobacteria phylum, and the Actinobacteria genus under the Actinobacteria phylum are common nitrogen fixing bacteria that can promote growth by fixing nitrogen elements in the environment. They are key factors affecting the absorption and transformation of nutrients by Dendrobium plants. SE advantage: Fungi of the genus Trichoderma and bacteria of the genus Bacillus can promote the production of metabolites that are beneficial for the growth of orchids. Fusarium fungi, as pathogenic bacteria, have a higher relative abundance in SE than in TE, which is one of the reasons for the disease in SE Dendrobium officinale plants. In addition, the enrichment of Fusarium fungi and Ralstonia bacteria in TE can easily lead to root rot in Dendrobium officinale plants, which is related to the characteristics of wood matrix such as easy absorption of water, lack of air permeability, and easy mold growth.
The formation of endophytic bacteria in medicinal plants is closely related to the quality of medicinal materials. Currently, there are literature reports on the significant correlation between active ingredients and endophytic bacteria, which is of great significance for revealing the influence of plant environmental factors on the quality of medicinal materials and the mechanism of authentic medicinal material formation. There is currently no research reporting on the correlation between endophytic bacteria and active ingredients in the roots of Dendrobium nobile. Dendrobine is a characteristic component of Dendrobium officinale. The 2020 edition of the Chinese Pharmacopoeia stipulates that the content of dendrobine in Dendrobium officinale should not be less than 0.4%. The research results show that the content of dendrobine in both epiphytic methods of Dendrobium officinale meets the requirements, and SE is higher than TE. The content of total alkaloids and flavonoids is higher than TE, while the content of total polysaccharides is lower than TE. Correlation analysis was conducted between the selected advantageous bacterial groups and key species and the content of active ingredients, and for the first time, it was found that the relative abundance of Pseudomonas and Rhizobium bacteria enriched in SE was significantly positively correlated with the content of dendrobine; The enrichment of thermophilic acid bacteria, heterotrophic algae, and chain acidophilic bacteria in TE showed a significant negative correlation with the content of dendrobine. Among them, rhizobia were the dominant beneficial bacteria in SE, suggesting that SE matrix is easy to enrich and promote the dominant community of dendrobine production and accumulation. The study also found that the enriched communities in SE were significantly positively correlated with total alkaloid and total flavonoid content, and significantly negatively correlated with polysaccharides; TE is significantly negatively correlated with the content of total alkaloids and flavonoids, and significantly positively correlated with polysaccharides. Endophytic bacteria can directly participate in the synthesis of medicinal plant metabolites and induce the production of secondary metabolites. Therefore, the enriched endophytic bacterial community in SE is conducive to the accumulation of dendrobine, alkaloids, and flavonoids, while TE is conducive to the accumulation of polysaccharides, thereby promoting the growth of Dendrobium officinale and improving the quality of medicinal materials.
Simulated cultivation techniques can enhance the immune system and resistance to environmental stress of medicinal plants. The endophytic bacterial community in medicinal herbs is closely related to the growth and development of Dendrobium plants. Dendrobium officinale is a special epiphytic mycorrhizal plant, and the epiphytic substrate is an important medium for plants to absorb nutrients and water. The growth and development of plant root microorganisms are affected by the type of substrate. Stone substrates are more nutrient poor and water poor than woody substrates. Therefore, the dominant bacterial community in the roots of Dendrobium officinale is more abundant, and the stone substrate is easier to enrich the endophytic bacterial community that promotes nitrogen fixation, growth, and the accumulation of metabolites, which improves the quality of medicinal herbs and lays the foundation for the study of the correlation between “substrate root endophytic bacteria plant growth” in Dendrobium officinale. This provides a scientific basis for the “victory of those who grow on stones” in Dendrobium officinale. The sentence is:. At the same time, previous studies have found that endophytic bacteria in the roots of Dendrobium plants can promote the absorption of elements in the matrix, and there is a certain correlation between the content of matrix mineral elements and the content of medicinal components. Further research is needed on the relationship between the content of SE and TE matrix mineral elements and the growth and development, accumulation of secondary metabolites, and endophytic bacteria in Dendrobium plants.