Microbial diversity analysis
Endophytes exist in specific tissues of plants and interact with plants through the exchange of nutrients, enzymes (catalase, oxidase, etc.), functional factors (biosurfactants, etc.), and signal transmission (Lu et al., 2021). They colonize in plant tissues for a long time and do not produce negative effects similar to pathogens, such as the destruction of photosynthesis, nutrient transfer, etc. On the contrary, the presence of these endophytes in host plants has beneficial effects on their health and growth. In recent years, high-throughput sequencing technology has been widely used to study the diversity of environmental, food, animal, plant, and microbial communities (Caravaca et al., 2020). Endophytes determination by high-throughput sequencing technology, can obtain detailed information about the microbial structure in plants, analysis of plant internal micro level of ecological and environmental relation. At the same time, the study of endophytes of medicinal plants provide the possibility of screening high-quality strains and fermentation for the production of drug active ingredients, and establish a new mode of genuine identification of medicinal materials, which has gradually become a research focus of microbial resources of medicinal plants (Adeleke and Babalola, 2021).
In this study, the diversity of microorganisms is associated with rhizosphere soil and the different parts of S. sphenanthera. In the case of bacteria, Chao1 indices indicate that the rhizosphere soil samples had the highest diversity among all of the samples from the different parts. Chao1 also revealed that the diversity of root samples is higher than that in the leaf samples (Table 3). On the contrary, fungi (Chao 1) showed an increasing trend of species richness from root to stem and leaf samples. Additionally, the diversity of bacterial microorganisms showed richness than fungal microorganisms (Table 3). PCoA showed that below-ground parts are distinguishable from the above-ground parts (Fig. 7). This result is consistent with the principal component analysis (PCA) results of different parts of S. chamaejasme L, the leaf and stem are distributed in clusters, which are different from the root plots (Jin et al., 2014).
In this study, the results show that Proteobacteria, Cyanobacteria, and Acidobacteria are main bacteria, Ascomycota and Basidiomycota are the main fungi at phylum level (Fig. 3). According to research found that most microbial community have little difference at phylum level, which is basically consistent with the results of this study, which fully demonstrated the similarity of microbes in larger taxonomic units (Rim et al., 2021; Sun et al., 2021). At present, endophytes can be isolated from various parts and organs of studied plants, such as root, stem, leaf, fruit, and seed, and the structure composition and abundance of endophytes will change with different plant varieties, parts, and development periods (Dong et al., 2018; Lv et al., 2021). This study reveals that there are significant differences between the microbial communities screened from rhizosphere soil and endophytic communities screened from different parts of S. sphenanthera (Table S3 and S4). There are also significant differences in the community structure and composition of endophyte in different parts of S. sphenanthera, which may be related to the different physiological structures of different tissues and the diversity of endophyte sources. The microbial communities of rhizosphere soil and root are more similar than those of endophytes from stem, leaf, and fruit, suggesting that endophytes of root invaded from rhizosphere soil and reached root through transport tissue. However, the endophytic communities of stem and fruit are similar to leaf, suggesting that the microorganisms of stem and fruit may enter through the phyllosphere (Figs. 1 and 7).
Bacterial function analysis (generic level)
Most of the bacterial microorganisms identified in this study are involved in microbial degradation, producing a variety of metabolites that affect plant growth and development. They promote plant growth through a variety of direct and indirect mechanisms. Directly mechanisms include dissolution of phosphate, nitrogen fixation, degradation of environmental pollutants, and production of hormones. And indirect mechanisms protect host plants by inhibiting plant-pathogenic bacteria by producing amino acids, polysaccharide, and other metabolites (antibiotics or lyases) (Bhattacharyya et al., 2015). In this study, Achromobacter, Methylobacterium, and Propionibacterium with high abundance have been detected in rhizosphere soil and four parts of S. sphenanthera, among which there are more in above-ground parts (stem, leaf, and fruit) (Table 1). Achromobacter can inoculate into vetiver grass (Chrysopogon zizanioides) which utilizes aromatic compounds as a sole carbon source (Ho et al., 2013). Methylobacterium protects soybean (Glycine max) against pathogens by inducing endophytic community changes (Christian et al., 2021). Propionibacterium is a kind of bacterial microorganism in stem that can synthesize propionic acid using special carboxylic enzymes, and the study shows that Propionibacterium is widely used in the production of vitamin B12, four pyrrole compounds and propionic acid, acid, as well as probiotics and cheese industry (Ames et al., 2012; Guyomarc'h et al., 2020).
In addition, Rhodoplanes, Candidatus Solibacter and Gemmata are also the dominant flora in the below-ground parts (rhizosphere soil and root). Rhodoplanes and Candidatus Solibacter are the exclusive bacteria of the below-ground parts (Table S3). Rhodoplanes are facultative photoorganics and potential nitrate fixation bacteria (Hiraishi and Ueda, 1994). Candidatus Solibacter is associated with food spoilage or foodborne diseases, and Gemmata is pathogenic (the epidemiology of Gemmata bacteremia) (Christen et al., 2018; Muriuki et al., 2021). At the same time, the content of dominant bacteria unique to the above-ground parts is less, and the total content is lower than 4% (3.147% in stem, 2.898% in leaf, and 1.512% in fruit) (Table S3). However, there are still some bacteria (not unique) with the higher contents in the above-ground parts, such as Anoxybacillus, Methyloversatilis, Staphylococcus, Hymenobacter, etc. (Table S3). Bacillus and Anoxybacillus can be used to promote plant growth, they compete to plant roots competitively, and can be used as biological fertilizers and identified root pathogens, such as bacteria, fungi, and nematodes (biological pesticides) (Shaikh and Sayyed, 2015). Methyloversatilis is capable of reducing NO3− or NO2−, and capable of C1-assimilation via the serine cycle and Calvin cycle (Anthony, 1982; Lu et al., 2014). Most of Staphylococcus is non-pathogenic parasitic bacteria, and some can cause infectious diseases in poultry (Othman et al., 2021). Hymenobacter is a radiation-resistant bacterium commonly found in rhizosphere soil and phyllosphere (Park et al., 2022; Stevens et al., 2021). In this experiment, Hymenobacter is only present in leaf (2.210%) (Table S3).
Among the dominant bacteria (genus level) mentioned above, Candidatus Solibacter, Gemmata and Staphylococcus are pathogenic. The remaining bacteria are involved in plant protection, growth promotion, and functional secondary metabolites. The results also confirm that endophytic bacteria and rhizosphere soil microorganisms have a low risk to plants.
Fungal function analysis (generic level)
The total abundance of fungi (genus level) detected in rhizosphere soil and different parts of S. sphenanthera is higher than that of bacteria, especially the fungi in rhizosphere soil is more than four times as abundant as bacteria (Table 4). There are 6 genera of fungi unique to rhizosphere soil (Table S4), and no genus unique to bacteria (Table S3). Among six genera, Hebeloma and Clavulinopsis have higher abundances (Table S4). Hebeloma affects the flow of phosphorus in litter, the transformation of soil organic matter itself, and the retardation of humification by participating in fungal interactions (Mrnka et al., 2020). Clavulinopsis, a member of the Clavariaceae family, assimilates and transfers 15N-depleted N form to the leaves of host plants, and builds fungal cell walls by absorbing 15N-enriched N (Birkebak et al., 2013; Mayor et al., 2009). Moreover, Cortinarius and unclassified_Xylariales are dominant genera in rhizosphere soil, and almost absent from four parts of S. sphenanthera (Table S4). Clavulinopsis and Cortinarius both belong to Agaricales, but most of Cortinarius are edible (Li et al., 2018). On the other hand, Cortinarius can form ectomycorrhiza with some trees and shrubs, and play an important role in plant growth and forest ecosystem (Bödeker et al., 2014). Unclassified_Xylariales, which is within Xylariales, is pathogenicity to plants. And Xylariales has a wide range of metabolites, which can be used as biocontrol agents and biofuel producers (Helaly et al., 2018).
Table 4
The abundance of genus of bacteria and fungi in rhizosphere soil and different parts of S. sphenanthera. Unclassified_Fungi data of fungi are excluded from the statistical data.
Abundance | Bacteria | Fungi |
Rhizosphere soil | 9.378 | 39.006 |
Root | 11.789 | 26.080 |
Stem | 37.004 | 45.683 |
Leaf | 30.749 | 23.212 |
Friut | 18.008 | 45.999 |
In addition to the four dominant fungi mentioned above, unclassified_Helotiales, unclassified_Clavariaceae, unclassified_Thelephoraceae, unclassified_Agaricales, and Tuber are also dominant fungi in rhizosphere soil. These five dominant fungi are also dominant in the root, especially unclassified_Helotiales with high abundance in root, which is belong to Helotiales (Table S4). Helotiales has a high level of species diversity and ecological diversity, which may be reflected in their metabolic diversity (Hosoya, 2020). Unclassified_Clavariaceae belongs to the Clavariaceae family (Agaricales), which is of great importance in studying fungus system. Many of Clavariaceae are ectomycorrhizal fungi, which play an important role in the protection and reconstruction of forest ecosystem, as well as maintaining the material cycle and energy flow of forest ecosystem (Birkebak et al., 2013). And some species of Clavariaceae, such as Ramaria botrytis (Pers.) Ricken and Ramaria botrtoides (Peck) Corner, are not only edible, but also used as medicinal materials in TCM (Dai and Yang, 2008). Unclassified_Thelephoraceae belongs to Thelephoraceae, and unclassified_Agaricales belong to Agaricales. Some of Thelephoraceae can form ectomycorrhizas with a broad range of hosts (Miguel et al., 2016). Some of Tuber has high commercial and gastronomic values (Schneider-Maunoury et al., 2020).
In this study, Botrytis (20.768% in fruit), Stomiopeltis (18.506% in stem), Zygophiala (5.657% in fruit), and Ramularia (3.544% in leaf and 2.815% in fruit) are extremely abundant in the above-ground parts (Table S4). Most of the fungal microorganisms in this study are pathogenic. Although they temporarily lose their pathogenicity, they may recover their pathogenicity under environmental selection, such as Botrytis (Backman and Sikora, 2008; Kan et al., 2014), Stomiopeltis (Ajitomi et al., 2017), and Zygophiala (Batzer et al., 2008). At the same time, pathogens can be used as inducer to enhance the host's resistance to disease, activate the host defense system, and improve the host's defense ability against pathogens (Backman and Sikora, 2008). In addition, some endophytic fungi and their metabolites in above-ground parts can promote the growth and development of host plants, such as Trametes and Eremothecium (Table S4). The secondary metabolites of Eremothecium and Trametes have medicinal value. Eremothecium can synthetize essential oil, which has a composition similar to that of rose oil (Semenova et al., 2022). Trametes versicolor, the most important medicinal mushrooms of Trametes, can produce polysaccharide krestin (PSK) and polysaccharide peptide (PSP), which can reduce cancer metastases (Zmitrovich et al., 2012). Moreover, the abundances of Cercospora and Alternaria (especially in stems) in common genus of fungal in four parts are very high (Table 2). The extracts of Alternaria can be used as biological attractants to increase the content of secondary metabolites in Catharanthus roseus (Birat et al., 2021). The secondary metabolites of Cercospora can inhibit the growth of Candida albicans (Bashir et al., 2022).
The abundance (genus level) of fungi detected in this experiment is higher than that of bacteria (except leaf) (Table 4), and the dominant fungi are far more than that of bacteria. Endophytic fungi have multiple functions. Unclassified_Xylariales, Botrytis, Stomiopeltis, and Zygophiala are pathogenic, but can be used as inducers to enhance the host's resistance to disease. There are several kinds of fungi belonging to Agaricales, mainly distributed in the below-ground part, which can be eaten and some can also be used as TCM. In addition, some fungi belong to growth-promoting fungi, whose metabolites can promote the growth and development of plants. And these growth-promoting fungi mainly distributed in four parts of S. sphenanthera (Table 2 and S4).
In view of the roles played by endophytes in plant growth, our findings contribute to the expansion of endophyte use in the production of S. sphenanthera and its important metabolites. The information on the differences of endophytes in the above-ground and below-ground parts can serve as basis for the selection of functional microorganisms.