ASV Full Form
After quality control and chimera sequences removal, a total of 1,003,042 effective sequences and 8928 ASVs were obtained of bacterial 16S rRNA genes. The average number of the obtained bacterial sequence was 34,449, and the average length was about 376bp. Similarly, a total of 8,06,544 effective sequences and 303 ASVs were obtained of fungal ITS regions after quality control and chimera sequences removal. The average length of the obtained fungal sequence was 243 bp. Amusingly, for both root and leaf samples, the amount of ASVs in 16S rRNA gene sequencing analysis was > 28 times more than the ASVs in ITS analysis (Supplementary Table 1). When searched in the SILVA database, these bacterial ASVs corresponded to 3458 ASVs in roots, 2142 ASVs in stems, and 2952 ASVs in leaves. These fungal ASVs corresponded to 82 ASVs from roots, 35 ASVs from stems, and 70 ASVs from leaves were identified using the UNITE database (Fig.1).
Alpha diversity of bacterial and fungal communities
Calculate alpha diversity to analyze the richness and diversity of ginseng endophytes. The diversity of endophytes in three different organs were statistically analyzed by the Kruskal-Wallis test and Dunn post hoc tests. The alpha diversity indexes of different ginseng organs are different (Fig. 2). The coverage rate of the sample is greater than 84.5%, indicating that the possibility of not detecting the sequence is small, and there are enough sequences for endophyte diversity analysis. We found that roots had the highest Shannon diversity index, which indicated that the root has the highest endophytic bacterial community diversity. The index of Chao1 indicates that roots have the highest endophytic bacterial community richness. Further analysis revealed that the Shannon index of endophytic bacteria of the root was significantly higher than the stem (p<0.05) (Fig. 2b). The endophytic fungal community diversity in the ginseng roots, stems, and leaves was less affected by the organ and showed no marked change in the diversity.
Beta diversity of bacterial and fungal communities
Use Bray-Curtis distance matrix to perform Principal coordinate (PCoA) analysis and Non-Metric multidimensional scaling (NMDS) analysis on each sample, combined with Anosim analysis, to show the overall relationship of endophytic bacterial and fungal communities structure between the samples. PCoA was achieved to determine the overall resemblance of endophytic microbial community structure among organs; it showed a considerably different community compositions of endophytic bacteria (R = 0.6459, p =0.001) and endophytic fungi (R = 0.6803, p =0.001) in different organs (Fig. 3a, b). PCoA analysis showed that the abscissa was the main coordinate component that caused differences in the composition of the endophytic microbial community in different organ samples. In terms of ASV, PC1 contributed 36.1% and 28.6% to the differences in community composition of endophytic bacteria and fungi, respectively (Fig. 3a, b). The NMDS map showed that the microbial community structure of the three organs was different (bacteria: R = 0.6459, p =0.001, fungus: R = 0.6803, p =0.001) (Fig. 3c, d).
Relative Microbial Abundances
The bacterial phyla detected in different organs of ginseng were Proteobacteria, Bacteroidetes,, and Actinobacteria (Fig. 4a). In the root, the main phyla were Proteobacteria (63.74%), Bacteroidetes (8.58%), Actinobacteria (17.77%). The main phylum in stems and leaves were Proteobacteria (81.80% and 66.57%, respectively). Analyze the top 10 genera of the three organs (Fig. 4b). The main genera observed in roots were Ochrobactrum (7.07%), Cupriavidus (6.14%), and Mycobacterium (7.77%). Ochrobactrum was the main genus of stems and leaves (15.91% and 13.12%, respectively). Both phylum and genus account for more than 1%.
Fungal ASVs primarily consisted of phyla Basidiomycota, Ascomycota, and Mortierellomycota. Ascomycota was the most abundant in ginseng roots, stems, and leaves (Fig. 5a). In the root, the main phyla were Ascomycota (86.89%), Mortierellomycota (2.44%), Basidiomycota (10.18%). The main phyla in the stem were Ascomycota (80.42%) and Basidiomycota (17.27%). The main phyla in leaves were Ascomycota (80.95%), Basidiomycota (18.99%), Mortierellomycota did not exist in the leaf. Analyze the top 10 genera of the three organs (Fig. 5b). The main genera observed in roots were Aspergillus (15.48%), Cadophora (13.40%), Tetracladium (7.2%). The main genera of stems were Umbilicaria (10.56%), Simplicillium (7.46%), Monilinia (6.80%). Monilinia (33.52%) was the main genus of leaves.
LEfSe analysis of the endogenous microbiome
To further clarify the possible interactions between the identified endophytic microbes dependencies in ginseng organ samples, linear discriminant effect size (LEfSe) was used to quantitatively analyze the biomarkers of different organs. We detected significant differences in biomarker abundance of endophytic bacteria from different groups, and identified a total of 9 biomarkers from all organ samples, as shown in the branch diagram (Fig. 6b). In the root, the significantly abundant taxa were the family Mycobacteriaceae, genus Mycobacterium and Devosia. In the stem, the significant taxa belonged to the class Gammaproteobacteria, orders Betaproteobacteriales, Sphingomonadales, family Burkholderiaceae, Sphingomonadaceae, and genus Sphingomonas, which were all abundant.
Similarly, a total of 6 biomarkers have been identified for endophytic fungi in different organs, as shown in the branch diagram (Fig. 6d). The significant taxa in the leaves were affiliated with different phylogenetic groups, including the order Capnodiales, family Sclerotiniaceae, and genus Monilinia. In the stem, the significant taxa belonged to the class Sordariomycetes, class Saccharomycetes, and order Saccharomycetales, which were all abundant.
Functional Characteristics of the endophytic microbiome
According to the function prediction results based on 16S rRNA analysis, we found that the microbiota of three organ-associated endophytic bacteria had relatively similar functions in 60 MetaCyc functions, and was mainly enriched in the amino acid biosynthesis; carbohydrate biosynthesis; Cofactor, Prosthetic Group, Electron Carrier, and Vitamin Biosynthesis, and so on. According to the function prediction results based on ITS analysis, we found that the microbiota of three organ-associated endophytic fungal also had relatively similar functions in 29 MetaCyc functions, and was mainly enriched in the nucleoside and nucleotide biosynthesis; fatty acid and lipid biosynthesis; etc.