Key Takeaways & Executive Findings
- •• • A total of 1,914,360 raw sequences were obtained from four tissues, with Proteobacteria dominating at the phylum level; this baseline confirms that endophytic bacterial load is sufficient for robust functional prediction and that Proteobacteria-centric consortia are the primary target for downstream metabolic engineering. • • Stem tissue exhibited the highest endophytic bacterial richness, while root tissue showed the highest diversity; this tissue-specific partitioning implies that stems may serve as a reservoir for culturable strains, whereas roots harbor a more heterogeneous community potentially adapted to soil-derived stress, guiding tissue selection for isolation campaigns. • • Sphingomonas was identified as the key biomarker distinguishing intergroup differences and showed a significant positive correlation with Pseudomonas; both genera were major contributors to the N10-formyltetrahydrofolate biosynthesis pathway, suggesting a conserved one-carbon metabolism module that could be exploited to enhance host nucleic acid synthesis and methylation capacity. • • Leaf, fruit, and stem communities exhibited high similarity but were significantly distinct from root communities; this structural segregation indicates that aerial tissues share a core endophytic microbiome, which may simplify bioprospecting for secondary metabolite-producing strains and reduce the need for tissue-specific isolation protocols.
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Abstract
Endophytic bacterial communities associated with the medicinal plant Duchesnea indica were profiled across root, stem, leaf, and fruit tissues using Illumina paired-end high-throughput sequencing. A total of 1,914,360 raw sequences were generated and processed via DADA2 and Vsearch pipelines. At the phylum level, Proteobacteria dominated all tissues. Tissue-specific variation was pronounced: the stem harbored the highest richness, whereas the root exhibited the highest diversity. Sphingomonas was identified as a key biomarker discriminating intergroup differences and displayed a significant positive correlation with Pseudomonas. Both genera were principal contributors to the N10-formyltetrahydrofolate biosynthesis pathway. Leaf, fruit, and stem communities clustered with high similarity, while root communities were distinctly separated. Functional prediction indicated that Pseudomonas and Sphingomonas may act synergistically in secondary metabolite synthesis or host stress resistance. These findings establish a microbiological basis for the pharmacological activity of D. indica and suggest that endophytic community modulation could enhance the accumulation of bioactive flavonoids, triterpenoids, and polyphenols, offering a novel avenue for the sustainable development of traditional Chinese medicine resources.
1. Introduction
Duchesnea indica (Andr.) Focke, a traditional Chinese medicinal herb widely distributed across Asia, has a long history of use for anti-inflammatory, detoxifying, and antitumor applications. Its pharmacological potential is attributed to a complex array of bioactive compounds, including triterpenoids, flavonoids, sterols, vitamin E, and phenolic acids. Despite extensive phytochemical characterization, the mechanisms underlying its medicinal activity remain incompletely resolved, and commercial development has been constrained by inconsistent active ingredient yields and a lack of understanding of the plant's associated microbiome. Conventional research has focused almost exclusively on the plant's own biosynthetic pathways, overlooking the possibility that endophytic bacteria contribute to the synthesis or accumulation of key secondary metabolites.
Endophytic bacteria establish long-term symbiotic relationships with host plants without causing overt pathology. Since the landmark discovery of taxol-producing endophytic fungi in Taxus brevifolia, the field has expanded to include bacterial endophytes as sources of novel bioactive compounds. However, for D. indica, the diversity, tissue distribution, and functional potential of endophytic bacterial communities have not been systematically characterized. This gap impedes the rational exploitation of endophyte-mediated enhancement of medicinal value. The present study addresses this bottleneck by applying Illumina high-throughput sequencing to root, stem, leaf, and fruit tissues, processing sequences with DADA2 and Vsearch, and predicting functional potential. The objective is to delineate community structure, identify key biomarker taxa, and infer metabolic pathways that may underpin the plant's pharmacological properties, thereby providing a microbiological foundation for future drug development and sustainable resource utilization.
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ZHANG Hui, LI Guiwen, YANG Shengli, ZHU Lina, WANG Jingyu, XIAO Gongnian (2026). Diversity Analysis and Functional Prediction of Endophytic Bacteria in Different Tissues of Duchesnea indica Based on Illumina High-Throughput Sequencing. Chinese Traditional and Herbal Drugs. https://doi.org/10.7501/j.issn.0253-2670.2026.16.20261623
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Frequently Asked Questions
What specific sequencing depth and bioinformatics pipeline were used to ensure reliable detection of low-abundance endophytic taxa?
The study generated 1,914,360 raw sequences across all tissues using Illumina paired-end sequencing. Sequences were processed with DADA2 for denoising and Vsearch for clustering, which together provide high-resolution taxonomic assignment and reduce false positives. This depth is sufficient to capture rare taxa, as evidenced by the identification of Sphingomonas as a key biomarker despite its likely low relative abundance.
How does the tissue-specific distribution of endophytic bacteria affect the scalability of isolating bioactive compound-producing strains?
Stem tissue showed the highest richness, while root tissue had the highest diversity. Leaf, fruit, and stem communities were highly similar, whereas root communities were distinct. For industrial isolation, this means aerial tissues (leaf, stem, fruit) can be pooled for bioprospecting without significant loss of core taxa, simplifying upstream processing. Root-specific taxa, however, may require separate handling if their metabolic functions are targeted.
What is the evidence for a synergistic functional role between Pseudomonas and Sphingomonas, and what are the implications for secondary metabolite yield?
Sphingomonas was identified as a key biomarker and showed a significant positive correlation with Pseudomonas. Both genera were major contributors to the N10-formyltetrahydrofolate biosynthesis pathway, which supplies one-carbon units for nucleic acid synthesis and methylation. This functional overlap suggests a cooperative interaction that could enhance host stress resistance and secondary metabolite production, such as flavonoids and triterpenoids. However, direct causal evidence requires metatranscriptomic or metabolomic validation.
What are the primary technical limitations of using 16S rRNA gene sequencing for functional prediction in this context?
16S rRNA sequencing provides taxonomic profiles but cannot directly confirm gene expression or metabolic flux. Functional predictions, such as those for N10-formyltetrahydrofolate biosynthesis, are inferred from reference databases and may not reflect actual in planta activity. The study acknowledges this limitation by framing the results as 'functional potential.' Future work integrating metagenomics, metatranscriptomics, and metabolomics is necessary to validate the predicted pathways and quantify their contribution to host medicinal properties.
How can the findings be translated into a commercially viable strategy for enhancing the medicinal value of Duchesnea indica?
The high similarity of leaf, fruit, and stem communities suggests that a core microbiome can be targeted for modulation. For example, inoculating plants with Pseudomonas and Sphingomonas strains could potentially boost the N10-formyltetrahydrofolate pathway, thereby increasing one-carbon availability for secondary metabolite synthesis. However, commercial implementation requires cost-effective cultivation and formulation of these endophytes, as well as field trials to confirm yield improvements. The study provides a baseline for such interventions but does not yet demonstrate scalable yield gains.
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