Influence of sampling location and processing on the assembly and network of Polygoni Multiflori Radix surface microbiome.

The raw and processed roots of Polygonum multiflorum Thunb is a popular traditional Chinese medicine. However, Polygoni Multiflori Radix is easily contaminated by toxigenic fungi and mycotoxins during harvesting, processing, and transportation, thereby posing a health risk for consumers. This study aims to investigate the presence of fungi on the surface of raw and processed Polygoni Multiflori Radix collected from four producing areas using high-throughput sequencing. Results showed that the phyla Ascomycota and Basidiomycota, the genera Xeromyces, Cystofilobasidium, Eurotium, and Aspergillus were the dominant fungus, and significant differences are presented in four areas and two processed products. Three potential mycotoxin-producing fungi were detected, namely Trichosporon cutaneum, Aspergillus restrictus, and Fusarium oxysporum. The α-diversity and network complexity showed significant differences in four areas. Chao 1 and Shannon were highest in Yunnan (YN), then incrementally decreased from SC (Sichuan) to AH (Anhui) and GD (Guangdong) areas. Meanwhile, α-diversity was also strongly influenced by processing. Chao 1 and Shannon indices were higher in the raw group, however, the network complexity and connectivity were higher in the processed group. In conclusion, the assembly and network of the surface microbiome on Polygoni Multiflori Radix were influenced by sampling location and processing. This work provides details on the surface microbiome of Polygoni Multiflori Radix samples, which could ensure the drug and consumers' safety.

Sampling locations and processing methods shape fungi microbiome on the surface of edible and medicinal Arecae semen.

Introduction: Arecae semen, which is derived from the dried ripe seed of Areca catechu L., has been commonly used as one of the major traditional Chinese medicines (TCMs). Three types of crude herbal preparations, namely, raw Arecae semen (AS), Arecae semen tostum (SAS), and Arecae semen carbonisata (FAS), are available for different clinical applications in TCMs. Although aflatoxin contamination in Arecae semen has been reported preliminarily, only a few studies have been conducted on fungal contamination. Methods: In this study, the presence of fungi on the surface of three Arecae semen (AS, SAS, and FAS) that collected from four provinces were investigated using high-throughput sequencing and internal transcribed spacer 2. Results: Results showed that the phyla Ascomycota (75.45%) and Basidiomycota (14.29%) and the genera Wallemia (7.56%), Botryosphaeria (6.91%), Davidiella (5.14%), and Symbiotaphrina (4.87%) were the dominant fungi, and they presented significant differences in four areas and three processed products (p < 0.05). The α-diversity and network complexity exhibited significant differences in the four sampling locations (p < 0.05), with higher in Yunnan (Chao 1, 213.45; Shannon, 4.61; average degree, 19.96) and Hainan (Chao 1, 198.27; Shannon, 4.21; average degree, 22.46) provinces. Significant differences were noted in the three processed samples; and SAS group had highest α-diversity (Chao 1, 167.80; Shannon, 4.54) and network complexity (average degree, 18.32). Conclusions: In conclusion, the diversity and composition of microbiome on the surface of Arecae semen were shaped by sampling location and processing methods. This work provides details on the surface microbiome of Arecae semen samples and highlights the importance of roles of origin and processing methods in microbiomes, ensuring drug efficacy and food safety.

Occurrence of fungi and mycotoxins in herbal medicines and rapid detection of toxin-producing fungi.

Contamination from external hazardous materials may greatly influence the safety and efficacy of herbal medicines. This paper aimed to evaluate the levels of contamination by mycotoxins and toxigenic fungi in herbal medicines and establish a rapid method for detecting toxin-producing fungi. Herein, 62.92%, 36.25%, and 64.17% of herbal medicines were contaminated by aflatoxins (AFs), ochratoxins, and fumonisins, respectively. Aspergillus (43.77%), Fusarium (5.17%), and Cladosporium (4.46%) were the three predominant genera. Spearman's correlation results showed that Aspergillus and Fusarium were significantly and positively correlated with mycotoxin content (R > 0.5, P < 0.05). In addition, 323 fungal strains were isolated from herbal medicines, and 20 species were identified, mainly belonging to Aspergillus and Penicillium. Analysis of potential mycotoxin-producing fungi showed that Aspergillus flavus can produce AFs, and Aspergillus ochraceus and Aspergillus niger can produce ochratoxin A (OTA). Multiplex real-time polymerase chain reaction showed that A. flavus harbored AF synthesis genes (aflR), and A. ochraceus and A. niger harbored OTA synthesis genes (aoksl). With these synthesis genes, 67.07% and 37.20% of 164 herbal medicines were positive for toxigenic genes. Furthermore, an excellent correlation was found between the above gene copies and mycotoxin content (R2 = 0.99). Our results confirmed the high detection rate of mycotoxins in herbal medicines and identified pivotal AF- and OTA-producing fungi. In conclusion, this paper provided the contamination status of fungi and mycotoxins in herbal medicines and established a rapid method for detecting toxigenic fungi.

[Research summary of chemical constituents and pharmacological effects of Panax notoginseng and predictive analysis on its Q-markers].

Panax notoginseng contains triterpene saponins, flavonoids, amino acids, polysaccharides, volatile oil and other active components, which have the effects of promoting blood circulation, stopping bleeding, removing blood stasis, etc. This study summarized the herbal research, chemical constituents and main pharmacological activities of P. notoginseng, and based on the theory of Q-markers of traditional Chinese medicine, predicted and analyzed the Q-markers of P. notoginseng from the aspects of plant kinship, efficacy, drug properties, measurability of chemical components, etc. It was found that ginsenosides Rg_1, Re, and Rb_1 with specific content ratio, ginsenosides Rb_2, Rb_3, Rc, Rd, Rh_2, and Rg_3, notoginseng R_1, dencichine and quercetin could be used as potential Q-markers of P. notoginseng, which facilitated the formulation of quality standards reflecting the efficacy of P. notoginseng.

[Traditional Chinese medicine microbiomics and its research strategies].

The tight relationships between microbiome and traditional Chinese medicine(TCM)have been widely recognized. New technologies, results, and theories are emerging in the field of microbiomics in recent years with the advances in high-throughput sequencing and multi-omics technologies. Based on the previous research, the present study has proposed the concept of TCM microbiomics(TCMM), which is an interdisciplinary subject aiming at elucidating the functions and applications of microbiome in the areas of herb resources, herb processing, herb storage, and clinical effects by using modern technology of biology, ecology, and informatics. This subject essentially contains the structures, functions, interactions, molecular mechanisms, and application strategies of the microbiome associated with the quality, safety, and efficacy of TCM. Firstly, the development of the TCMM concept was summarized, with the profound understanding of TCMM on the complexity and entirety of microbiome being emphasized. Then, the research contents and applications of TCMM in promoting the sustainable development of herb resources, improving the standardization and diversification of herb fermentation, strengthening the safety of herb storage, and resolving the scientific connotation of theories and clinical efficacy of TCM are reviewed. Finally, the research strategies and methods of TCM microbiomics were elaborated from basic research, application research, and system research. TCMM is expected to promote the integrative development of TCM with frontier science and technology, thereby expanding the depth and scope of TCM study and facilitating TCM modernization.

Core rhizosphere microbiome of Panax notoginseng and its associations with belowground biomass and saponin contents.

The core rhizosphere microbiome is critical for plant fitness. However, its contribution to the belowground biomass and saponin contents of Panax notoginseng remains unclear. High-throughput sequencing of amplicon and metagenome was performed to obtain the microbiome profiles and functional traits in P. notoginseng rhizosphere across a large spatial scale. We obtained 639 bacterial and 310 fungal core OTUs, which were mainly affected by soil pH and organic matter (OM). The core taxa were grouped into four ecological clusters (i.e. high pH, low pH, high OM and low OM) for sharing similar habitat preferences. Furthermore, structural equation modelling (SEM) and correlation analyses revealed that the diversity and composition of core microbiomes, as well as the metagenome-derived microbial functions, were related to belowground biomass and saponin contents. Key microbial genera related to the two plant indicators were also identified. In short, this study explored the main driving environmental factors of core microbiomes in the P. notoginseng rhizosphere and revealed that the core microbiomes and microbial functions potentially contributed to the belowground biomass and saponin contents of the plant. This work may enhance our understanding of interactions between microbes and perennial plants and improve our ability to manage root microbiota for the sustainable production of herbal medicine.

Temporal Dynamics of Rhizosphere Communities Across the Life Cycle of Panax notoginseng.

Rhizosphere microbiome promotes plant growth; however, the succession of rhizosphere microbial community during the growth stages of perennial medicinal plant Panax notoginseng (P. notoginseng) is still unclear. Here, amplicon sequencing was performed to assess the succession characteristics of rhizosphere microbiomes during developmental stages. Results showed that bacterial and fungal communities were mainly shaped by the development stages. The microbial α-diversities first increased and then decreased with plant growth and the variation in microbial composition was active at the 3-year root growth (3YR) stage. The variation trend of cross-domain co-occurrence network complexity was similar to that of α-diversities. Cross-domain nodes decreased at the 3YR stage and fungal nodes increased at the 3YR stage. This study provided a detailed and systematic survey of rhizosphere microbiomes during the growth stages of P. notoginseng. The findings revealed that the development stages of P. notoginseng drove the temporal dynamics of rhizosphere communities. This study helps in harnessing the power of microbiomes to evaluate herbal medicine growth and provides valuable information to guide the microbial breeding of medical plants.

Evidence for saponin diversity-mycobiome links and conservatism of plant-fungi interaction patterns across Holarctic disjunct Panax species.

Although interplays between plant and coevolved microorganisms are believed to drive landscape formation and ecosystem services, the relationships between the mycobiome and phytochemical evolution and the evolutionary characteristics of plant-mycobiome interaction patterns are still unclear. The present study explored fungal communities from 405 multiniche samples of three Holarctic disjunct Panax species. The overall mycobiomes showed compartment-dominated variations and dynamic universality. Neutral models were fitted for each compartment at the Panax genus (I) and species (II) levels to infer the community assembly mechanism and identify fungal subgroups potentially representing different plant-fungi interaction results, i.e., the potentially selected, opposed, and neutral taxa. Selection contributed more to the endosphere than to external compartments. The nonneutral taxa showed significant phylogenetic clustering. In Model I, the opposed subgroups could best reflect Panax saponin diversities (r = 0.69), and genera with highly positive correlations to specific saponins were identified using machine learning. Although mycobiomes in the three species differed significantly, subgroups in Model II were phylogenetically clustered based on potential interaction type rather than plant species, indicating potentially conservative plant-fungi interactions. In summary, the finding of strong links between invaders and saponin diversity can help explore the underlying mechanisms of saponin biosynthesis evolution from microbial insights, which is important to understanding the formation of the current landscape. The potential conservatism of plant-fungi interaction patterns suggests that the related genetic modules and selection pressures were convergent across Panax species, advancing our understanding of plant interplay with biotic environments.

Contents of lobetyolin, syringin, and atractylolide III in Codonopsis pilosula are related to dynamic changes of endophytes under drought stress.

BACKGROUND: Codonopsis pilosula, an important medicinal plant, can accumulate certain metabolites under moderate drought stress. Endophytes are involved in the metabolite accumulations within medicinal plants. It is still unknown that the endophytes of C. pilosula are associated with the accumulations of metabolites. This study aims to investigate the promoting effect of endophytes on the accumulations of active substances in C. pilosula under drought stress. METHODS: High-performance liquid chromatography and high-throughput sequencing technology were performed to investigate changes in the contents of secondary metabolite and endophyte abundances of C. pilosula under drought stress, respectively. Spearman's correlation analysis was further conducted to identify the endophytic biomarkers related to accumulations of pharmacodynamic compounds. Culture-dependent experiments were performed to confirm the functions of endophytes in metabolite accumulations. RESULTS: The distribution of pharmacological components and diversity and composition of endophytes showed tissue specificity within C. pilosula. The contents of lobetyolin, syringin, and atractylolide III in C. pilosula under drought stress were increased by 8.47%‒86.47%, 28.78%‒230.98%, and 32.17%‒177.86%, respectively, in comparison with those in untreated groups. The Chao 1 and Shannon indices in different parts of drought-stressed C. pilosula increased compared with those in untreated parts. The composition of endophytic communities in drought treatment parts of C. pilosula was different from that in control parts. A total of 226 microbial taxa were identified as potential biomarkers, of which the abundances of 42 taxa were significantly and positively correlated to the pharmacodynamic contents. Culture-dependent experiments confirmed that the contents of lobetyolin and atractylolide III were increased by the application of Epicoccum thailandicum, Filobasidium magnum, and Paraphoma rhaphiolepidis at the rates of 11.12%‒46.02%, and that the content of syringin was increased by Pseudomonas nitroreducens at the rates of 118.61%‒119.36%. CONCLUSIONS: Certain endophytes participated in the accumulations of bioactive metabolites, which provided a scientific evidence for the development and application of microorganisms to improve the quality of traditional Chinese medicine.

Dispersal Limitation Plays Stronger Role in the Community Assembly of Fungi Relative to Bacteria in Rhizosphere Across the Arable Area of Medicinal Plant.

Understanding the ecological patterns of rhizosphere microbial communities is critical for propelling sustainable agriculture and managing ecosystem functions by exploiting microorganisms. However, this knowledge is still unclear, especially under host-associated large-scale and regarding the comparison between bacteria and fungi. We examined community assembly processes and community characters including environmental thresholds and co-occurrence patterns across the cultivatable area of Panax notoginseng for bacteria and fungi. Both are vital members of the rhizosphere but differ considerably in their life history and dispersal potentiality. Edaphic factors drove the parallel variations of bacterial and fungal communities. Although bacterial and fungal communities exhibited similar biogeographic patterns, the assembly of fungi was more driven by dispersal limitation than selection compared with bacteria. This finding supported the 'size-dispersal' hypothesis. pH and total nitrogen respectively mediated the relative importance of deterministic and stochastic processes in shaping bacterial and fungal communities. In addition, fungal communities exhibited potentially broader environmental thresholds and more modular co-occurrence patterns than bacteria (bacteria: 0.67; fungi: 0.78). These results emphasized the importance of dispersal limitation in structuring rhizosphere microbiota and shaping community features of ecologically distinct microorganisms. This study provides insights into the improved prediction and management of the key functions of rhizosphere microbiota.

Endophytes isolated from Panax notoginseng converted ginsenosides.

Endophytes may participate in the conversion of metabolites within medicinal plants, influencing the efficacy of host. However, the distribution of endophytes within medicinal plants P. notoginseng and how it contributes to the conversion of saponins are not well understood. Here, we determined the distribution of saponins and endophytes within P. notoginseng compartments and further confirm the saponin conversion by endophytes. We found metabolites showed compartment specificity within P. notoginseng. Potential saponin biomarkers, such as Rb1, Rg1, Re, Rc and Rd, were obtained. Endophytic diversity, composition and co-occurrence networks also showed compartment specificity, and bacterial alpha diversity values were highest in root compartment, consistently decreased in the stem and leaf compartments, whereas those of fungi showed the opposite trend. Potential bacterial biomarkers, such as Rhizobium, Bacillus, Pseudomonas, Enterobacter, Klebsiella, Pantoea and fungal biomarkers Phoma, Epicoccum, Xylariales, were also obtained. Endophytes related to saponin contents were found by Spearman correlation analysis, and further verification experiments showed that Enterobacter chengduensis could convert ginsenoside Rg1 to F1 at a rate of 13.24%; Trichoderma koningii could convert ginsenoside Rb1 to Rd at a rate of 40.00% and to Rg3 at a rate of 32.31%; Penicillium chermesinum could convert ginsenoside Rb1 to Rd at a rate of 74.24%.

Composition and function of rhizosphere microbiome of Panax notoginseng with discrepant yields.

BACKGROUND: Panax notoginseng is a highly valuable medicinal plant. Reduced P. notoginseng yield is a common and serious problem that arises in a continuous cropping system. Variation in the composition and function of soil microbial community is considered the primary cause of yield reduction. METHODS: This study used shotgun metagenomic sequencing approaches to describe the taxonomic and functional features of P. notoginseng rhizosphere microbiome and screen microbial taxa and functional traits related to yields. RESULTS: At the family and genus level, a total of 43 families and 45 genera (relative abundance > 0.1%) were obtained, and the correlation with the yield of P. notoginseng was further analyzed. Nitrosomonadaceae, Xanthomonadaceae, Mycobacterium and Arthrobacter that were enriched in soils with higher yields were positively correlated with P. notoginseng yields, thereby suggesting that they might increase yields. Negative correlation coefficients indicated that Xanthobacteraceae, Caulobacteraceae, Oxalobacteraceae, Chitinophagaceae, Sphingomonas, Hyphomicrobium, Variovorax and Phenylobacterium might be detrimental to P. notoginseng growth. A total of 85 functional traits were significantly (P < 0.05) correlated with P. notoginseng yields. Functional traits, likely steroid biosynthesis and MAPK signaling pathway were positively correlated with P. notoginseng yields. In contrast, functional traits, such as bacterial secretion system, ABC transporters, metabolism of xenobiotics by cytochrome P450 and drug metabolism-cytochrome P450, were negatively associated with yields. CONCLUSIONS: This study describes an overview of the rhizosphere microbiome of P. notoginseng with discrepant yields and identifies the taxa and functional traits related to yields. Our results provide valuable information to guide the isolation and culture of potentially beneficial microorganisms and to utilize the power of the microbiome to increase plant yields in a continuous cropping system.