Echinacea purpurea microbiota: bacterial-fungal interactions and the interplay with host and non-host plant species in vitro dual culture.

Important evidence is reported on the antimicrobial and antagonistic properties of bacterial endophytes in Echinacea purpurea and their role in the modulation of plant synthesis of bioactive compounds. Here, endophytic fungi were isolated from E. purpurea, and the dual culture approach was applied to deepen insights into the complex plant-microbiome interaction network. In vitro experiments were carried out to evaluate the species specificity of the interaction between host (E. purpurea) and non-host (E. angustifolia and Nicotiana tabacum) plant tissues and bacterial or fungal endophytes isolated from living E. purpurea plants to test interactions between fungal and bacterial endophytes. A higher tropism towards plant tissue and growth was observed for both fungal and bacterial isolates compared to controls without plant tissue. The growth of all fungi was significantly inhibited by several bacterial strains that, in turn, were scarcely affected by the presence of fungi. Finally, E. purpurea endophytic bacteria were able to inhibit mycelial growth of the phytopathogen Botrytis cinerea. Bacteria and fungi living in symbiosis with wild Echinacea plants interact with each other and could represent a potential source of bioactive compounds and a biocontrol tool.

Volatile profile of Echinacea purpurea plants after in vitro endophyte infection.

The differences in volatile profile of Echinacea purpurea plants not-inoculated (EpC) and inoculated with their endophytes from roots (EpR) and stem/leaves (EpS/L) were analysed and compared by GC-FID/GC-MS in an in vitro model. Non-terpenes and sesquiterpene hydrocarbons were the most abundant classes with an opposite behaviour of EpS/L showing a decreased emission of sesquiterpenes and an increase of non-terpene derivatives. The main compounds obtained from EpS/L were (Z)-8-dodecen-1-ol and 1-pentadecene, while germacrene D and ß-caryophyllene were the key compounds in EpC and EpR. For the first time, this work indicates that bacterial endophytes modify the aroma profiles of infected and non-infected in vitro plants of the important medicinal plant E. purpurea. Therefore, our model of infection could permit to select endophytic strains to use as biotechnological tool in the production of medicinal plants enriched in volatile bioactive compounds.

The influence of Echinacea purpurea leaf microbiota on chicoric acid level.

The controversial anti-proliferative effects of Echinacea purpurea (L.) Moench (Asteraceae) might be related to different plant metabolites contained in plant samples, extracts and products. The influence of bacterial endophytes on the synthesis of bioactive compounds in the medicinal plants has been previously demonstrated but there are only few studies addressing anticancer effects and mechanisms of E. purpurea extracts following endophytic colonization. The present study aimed to test and compare the lactate dehydrogenase (LDH) inhibition potential of n-hexane and methanol extracts from in vitro endophyte non-inoculated and inoculated E. purpurea plants. An in vitro model was previously set up to perform the infection of axenic E. purpurea plants with bacterial endophytic strains isolated from E. purpurea aerial part. Only methanol extracts showed LDH5 inhibition, in particular the richest in chicoric acid and most strongly inhibiting extract was obtained from inoculated stem and leaves of E. purpurea (IC50 = 0.9 mg/ml). Chicoric acid showed an IC50 value (66.7 µM) in enzymatic assays better than that of the reference compound galloflavin. Modeling studies were carried out to suggest the putative interaction mode of chicoric acid in the enzyme active site. This in vitro model on plant-bacterial interaction may lead to obtain extracts from plants enriched in bioactive compounds and it is a new approach for the discovery of novel anticancer compounds.

Tissue specificity and differential effects on in vitro plant growth of single bacterial endophytes isolated from the roots, leaves and rhizospheric soil of Echinacea purpurea.

BACKGROUND: Echinacea-endophyte interaction might affect plant secondary metabolites content and influence bacterial colonization specificity and plant growth, but the underlying mechanisms need deepening. An in vitro model, in which E. purpurea axenic plants as host species and E. angustifolia and Nicotiana tabacum as non-host species inoculated with single endophytes isolated from stem/leaf, root and rhizospheric soil, were used to investigate bacterial colonization. RESULTS: Colonization analysis showed that bacteria tended to reach tissues from which they were originally isolated (tissue-specificity) in host plants but not in non-host ones (species-specificity). Primary root elongation inhibition as well as the promotion of the growth of E. purpurea and E. angustifolia plants were observed and related to endophyte-produced indole-3-Acetic Acid. Bacteria-secreted substances affected plant physiology probably interacting with plant regulators. Plant metabolites played an important role in controlling the endophyte growth. CONCLUSIONS: The proposed in vitro infection model could be, generally used to identify novel bioactive compounds and/or to select specific endophytes contributing to the host metabolism properties.

Synergy and antagonism in natural product extracts: when 1 + 1 does not equal 2.

Covering: 2000 to 2019 According to a 2012 survey from the Centers for Disease Control and Prevention, approximately 18% of the U.S. population uses natural products (including plant-based or botanical preparations) for treatment or prevention of disease. The use of plant-based medicines is even more prevalent in developing countries, where for many they constitute the primary health care modality. Proponents of the medicinal use of natural product mixtures often claim that they are more effective than purified compounds due to beneficial "synergistic" interactions. A less-discussed phenomenon, antagonism, in which effects of active constituents are masked by other compounds in a complex mixture, also occurs in natural product mixtures. Synergy and antagonism are notoriously difficult to study in a rigorous fashion, particularly given that natural products chemistry research methodology is typically devoted to reducing complexity and identifying single active constituents for drug development. This report represents a critical review with commentary about the current state of the scientific literature as it relates to studying combination effects (including both synergy and antagonism) in natural product extracts. We provide particular emphasis on analytical and Big Data approaches for identifying synergistic or antagonistic combinations and elucidating the mechanisms that underlie their interactions. Specific case studies of botanicals in which synergistic interactions have been documented are also discussed. The topic of synergy is important given that consumer use of botanical natural products and associated safety concerns continue to garner attention by the public and the media. Guidance by the natural products community is needed to provide strategies for effective evaluation of safety and toxicity of botanical mixtures and to drive discovery in botanical natural product research.

Plant microbiome-dependent immune enhancing action of Echinacea purpurea is enhanced by soil organic matter content.

We previously demonstrated that extracts from Echinacea purpurea material varied substantially in their ability to activate macrophages in vitro and that this variation was due to differences in their content of bacterial components. The purpose of the current study was to identify soil conditions (organic matter, nitrogen, and moisture content) that alter the macrophage activation potential of E. purpurea and determine whether these changes in activity correspond to shifts in the plant-associated microbiome. Increased levels of soil organic matter significantly enhanced macrophage activation exhibited by the root extracts of E. purpurea (p < 0.0001). A change in soil organic matter content from 5.6% to 67.4% led to a 4.2-fold increase in the macrophage activation potential of extracts from E. purpurea. Bacterial communities also differed significantly between root materials cultivated in soils with different levels of organic matter (p < 0.001). These results indicate that the level of soil organic matter is an agricultural factor that can alter the bacterial microbiome, and thereby the activity, of E. purpurea roots. Since ingestion of bacterial preparation (e.g., probiotics) is reported to impact human health, it is likely that the medicinal value of Echinacea is influenced by cultivation conditions that alter its associated bacterial community.

Antagonism and antibiotic resistance drive a species-specific plant microbiota differentiation in Echinacea spp.

A key factor in the study of plant-microbes interactions is the composition of plant microbiota, but little is known about the factors determining its functional and taxonomic organization. Here we investigated the possible forces driving the assemblage of bacterial endophytic and rhizospheric communities, isolated from two congeneric medicinal plants, Echinacea purpurea (L.) Moench and Echinacea angustifolia (DC) Heller, grown in the same soil, by analysing bacterial strains (isolated from three different compartments, i.e. rhizospheric soil, roots and stem/leaves) for phenotypic features such as antibiotic resistance, extracellular enzymatic activity, siderophore and indole 3-acetic acid production, as well as cross-antagonistic activities. Data obtained highlighted that bacteria from different plant compartments were characterized by specific antibiotic resistance phenotypes and antibiotic production, suggesting that the bacterial communities themselves could be responsible for structuring their own communities by the production of antimicrobial molecules selecting bacterial-adaptive phenotypes for plant tissue colonization.

Preliminary data on antibacterial activity of Echinacea purpurea-associated bacterial communities against Burkholderia cepacia complex strains, opportunistic pathogens of Cystic Fibrosis patients.

Burkholderia cepacia complex bacteria (Bcc) represent a serious threat for immune-compromised patient affected by Cystic Fibrosis (CF) since they are resistant to many substances and to most antibiotics. For this reason, the research of new natural compounds able to inhibit the growth of Bcc strains has raised new interest during the last years. A source of such natural compounds is represented by medicinal plants and, in particular, by bacterial communities associated with these plants able to produce molecules with antimicrobial activity. In this work, a panel of 151 (endophytic) bacteria isolated from three different compartments (rhizospheric soil, roots, and stem/leaves) of the medicinal plant Echinacea purpurea were tested (using the cross-streak method) for their ability to inhibit the growth of 10 Bcc strains. Data obtained revealed that bacteria isolated from the roots of E. purpurea are the most active in the inhibition of Bcc strains, followed by bacteria isolated from the rhizospheric soil, and endophytes from stem/leaf compartment. At the same time, Bcc strains of environmental origin showed a higher resistance toward inhibition than the Bcc strains with clinical (i.e. CF patients) origin. Differences in the inhibition activity of E. purpurea-associated bacteria are mainly linked to the environment -the plant compartment- rather than to their taxonomical position.

Phenotypic and genomic characterization of the antimicrobial producer Rheinheimera sp. EpRS3 isolated from the medicinal plant Echinacea purpurea: insights into its biotechnological relevance.

In recent years, there has been increasing interest in plant microbiota; however, despite medicinal plant relevance, very little is known about their highly complex endophytic communities. In this work, we report on the genomic and phenotypic characterization of the antimicrobial compound producer Rheinheimera sp. EpRS3, a bacterial strain isolated from the rhizospheric soil of the medicinal plant Echinacea purpurea. In particular, EpRS3 is able to inhibit growth of different bacterial pathogens (Bcc, Acinetobacter baumannii, and Klebsiella pneumoniae) which might be related to the presence of gene clusters involved in the biosynthesis of different types of secondary metabolites. The outcomes presented in this work highlight the fact that the strain possesses huge biotechnological potential; indeed, it also shows antimicrobial effects upon well-described multidrug-resistant (MDR) human pathogens, and it affects plant root elongation and morphology, mimicking indole acetic acid (IAA) action.

Activities and Prevalence of Proteobacteria Members Colonizing Echinacea purpurea Fully Account for Macrophage Activation Exhibited by Extracts of This Botanical.

Evidence supports the theory that bacterial communities colonizing Echinacea purpurea contribute to the innate immune enhancing activity of this botanical. Previously, we reported that only about half of the variation in in vitro monocyte stimulating activity exhibited by E. purpurea extracts could be accounted for by total bacterial load within the plant material. In the current study, we test the hypothesis that the type of bacteria, in addition to bacterial load, is necessary to fully account for extract activity. Bacterial community composition within commercial and freshly harvested (wild and cultivated) E. purpurea aerial samples was determined using high-throughput 16S rRNA gene pyrosequencing. Bacterial isolates representing 38 different taxa identified to be present within E. purpurea were acquired, and the activity exhibited by the extracts of these isolates varied by over 8000-fold. Members of the Proteobacteria exhibited the highest potency for in vitro macrophage activation and were the most predominant taxa. Furthermore, the mean activity exhibited by the Echinacea extracts could be solely accounted for by the activities and prevalence of Proteobacteria members comprising the plant-associated bacterial community. The efficacy of E. purpurea material for use against respiratory infections may be determined by the Proteobacterial community composition of this plant, since ingestion of bacteria (probiotics) is reported to have a protective effect against this health condition.

Antagonistic interactions between endophytic cultivable bacterial communities isolated from the medicinal plant Echinacea purpurea.

In this work we have studied the antagonistic interactions existing among cultivable bacteria isolated from three ecological niches (rhizospheric soil, roots and stem/leaves) of the traditional natural medicinal plant Echinacea purpurea. The three compartments harboured different taxonomic assemblages of strains, which were previously reported to display different antibiotic resistance patterns, suggesting the presence of differential selective pressure due to antagonistic molecules in the three compartments. Antagonistic interactions were assayed by the cross-streak method and interpreted using a network-based analysis. In particular 'within-niche inhibition' and 'cross-niche inhibition' were evaluated among isolates associated with each compartment as well as between isolates retrieved from the three different compartments respectively. Data obtained indicated that bacteria isolated from the stem/leaves compartment were much more sensitive to the antagonistic activity than bacteria from roots and rhizospheric soil. Moreover, both the taxonomical position and the ecological niche might influence the antagonistic ability/sensitivity of different strains. Antagonism could play a significant role in contributing to the differentiation and structuring of plant-associated bacterial communities.

Antibiotic resistance differentiates Echinacea purpurea endophytic bacterial communities with respect to plant organs.

Recent findings have shown that antibiotic resistance is widespread in multiple environments and multicellular organisms, as plants, harboring rich and complex bacterial communities, could be hot spot for emergence of antibiotic resistances as a response to bioactive molecules production by members of the same community. Here, we investigated a panel of 137 bacterial isolates present in different organs of the medicinal plant Echinacea purpurea, aiming to evaluate if different plant organs harbor strains with different antibiotic resistance profiles, implying then the presence of different biological interactions in the communities inhabiting different plant organs. Data obtained showed a large antibiotic resistance variability among strains, which was strongly related to the different plant organs (26% of total variance, P < 0.0001). Interestingly this uneven antibiotic resistance pattern was present also when a single genus (Pseudomonas), ubiquitous in all organs, was analyzed and no correlation of antibiotic resistance pattern with genomic relatedness among strains was found. In conclusion, we speculate that antibiotic resistance patterns are tightly linked to the type of plant organ under investigation, suggesting the presence of differential forms of biological interaction in stem/leaves, roots and rhizosphere.

Endophytic and rhizospheric bacterial communities isolated from the medicinal plants Echinacea purpurea and Echinacea angustifolia

In this work we analyzed the composition and structure of cultivable bacterial communities isolated from the stem/leaf and root compartments of two medicinal plants, Echinacea purpurea (L.) Moench and Echinacea angustifolia (DC.) Hell, grown in the same soil, as well as the bacterial community from their rhizospheric soils. Molecular PCR-based techniques were applied to cultivable bacteria isolated from the three compartments of the two plants. The results showed that the two plants and their respective compartments were characterized by different communities, indicating a low degree of strain sharing and a strong selective pressure within plant tissues. Pseudomonas was the most highly represented genus, together with Actinobacteria and Bacillus spp. The presence of distinct bacterial communities in different plant species and among compartments of the same plant species could account for the differences in the medicinal properties of the two plants (AU)

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Endophytic and rhizospheric bacterial communities isolated from the medicinal plants Echinacea purpurea and Echinacea angustifolia.

In this work we analyzed the composition and structure of cultivable bacterial communities isolated from the stem/leaf and root compartments of two medicinal plants, Echinacea purpurea (L.) Moench and Echinacea angustifolia (DC.) Hell, grown in the same soil, as well as the bacterial community from their rhizospheric soils. Molecular PCR-based techniques were applied to cultivable bacteria isolated from the three compartments of the two plants. The results showed that the two plants and their respective compartments were characterized by different communities, indicating a low degree of strain sharing and a strong selective pressure within plant tissues. Pseudomonas was the most highly represented genus, together with Actinobacteria and Bacillus spp. The presence of distinct bacterial communities in different plant species and among compartments of the same plant species could account for the differences in the medicinal properties of the two plants.

Total bacterial load within Echinacea purpurea, determined using a new PCR-based quantification method, is correlated with LPS levels and in vitro macrophage activity.

Our previous studies indicate that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea depends on bacterial components. In the present study, total bacterial load was determined within E. purpurea samples and ranged from 6.4 × 10(6) to 3.3 × 10(8) bacteria/g of dry plant material. To estimate total bacterial load, we developed a PCR-based quantification method that circumvents the problems associated with nonviable/nonculturable cells (which precludes using plate counts) or the coamplification of mitochondrial or chloroplast DNA with the use of universal bacterial primers (which precludes the use of qPCR). Differences in total bacterial load within Echinacea samples were strongly correlated with the activity (NF-κB activation in THP-1 cells) and content of bacterial lipopolysaccharides within extracts of this plant material. These results add to the growing body of evidence that bacteria within Echinacea are the main source of components responsible for enhancing innate immune function.

Chromatographic methods for metabolite profiling of virus- and phytoplasma-infected plants of Echinacea purpurea.

This study was focused on the effects of virus and phytoplasma infections on the production of Echinacea purpurea (L.) Moench secondary metabolites, such as caffeic acid derivatives, alkamides, and essential oil. The identification of caffeic acid derivatives and alkamides was carried out by means of high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-electrospray ionization-mass spectrometry (ESI-MS), and MS(2). Quantitative analysis of these compounds was carried out using HPLC-DAD. The results indicated that the presence of the two pathogens significantly decreases (P < 0.05) the content of cichoric acid, the main caffeic acid derivative. Regarding the main alkamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, a significant decrease (P < 0.05) in the content of this secondary metabolite was observed in virus-infected plants in comparison with healthy plants, while in the phytoplasma-infected sample the variation of this secondary metabolite was not appreciable. The % relative area of the E/Z isomers of this alkamide was also found to change in infected samples. The gas chromatography (GC) and GC-MS analysis of E. purpurea essential oil enabled the identification of 30 compounds. The main significant differences (P < 0.05) in the semiquantitative composition were observed for three components: limonene, cis-verbenol, and verbenone. The results indicate that the presence of virus and phytoplasma has an appreciable influence on the content of E. purpurea secondary metabolites, which is an important issue in defining the commercial quality, market value, and therapeutic efficacy of this herbal drug.

Hairy root cultures for secondary metabolites production.

Hairy roots (HRs) are differentiated cultures of transformed roots generated by the infection of wounded higher plants with Agrobacterium rhizogenes. This pathogen causes the HR disease leading to the neoplastic growth of roots that are characterized by high growth rate in hormone free media and genetic stability. HRs produce the same phytochemicals pattern of the corresponding wild type organ. High stability and productivity features allow the exploitation of HRs as valuable biotechnological tool for the production of plant secondary metabolites. In addition, several elicitation methods can be used to further enhance their accumulation in both small and large scale production. However, in the latter case, cultivation in bioreactors should be still optimized. HRs can be also utilised as biological farm for the production of recombinant proteins, hence holding additional potential for industrial use. HR technology has been strongly improved by increased knowledge of molecular mechanisms underlying their development. The present review summarizes updated aspects of the hairy root induction, genetics and metabolite production.

Effect of high pressure pasteurization on bacterial load and bioactivity of Echinacea purpurea.

UNLABELLED: High hydrostatic pressure (HHP) technology was applied to organic Echinacea purpurea (E. purpurea) roots and flowers to determine the feasibility of using this technology for cold herb pasteurization, to produce microbiologically safe and shelf-stable products for the natural health products (NHPs) industry. HHP significantly (P < 0.01) reduced microbial contamination in both roots and flowers without affecting the phytochemical retention of chicoric and chlorogenic acids, and total alkamide contents. The antioxidant activity of E. purpurea methanol-derived extracts, evaluated in both chemical (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) [ABTS] and oxygen radical absorption capacity [ORAC] assay) and in cell culture models (RAW264, 7 macrophage, H(2)O(2)-induced intracellular oxidation, and lipopolysaccharide [LPS]-induced nitric oxide production), was not adversely affected by the application of HHP at both 2 and 5 min at 600 mPa. Furthermore, HHP did not affect the capacity of E. purpurea extracts to suppress nitric oxide production in LPS-activated macrophage cells. Therefore, our results show that HHP is an effective pasteurization process treatment to reduce microbial-contamination load while not adversely altering chemical and bioactive function of active constituents present in organic E. purpurea. PRACTICAL APPLICATION: Our study reports for the first time, the effectiveness of using high hydrostatic pressure (HHP) technology pressure to pasteurize E. purpurea root and flower, and the comparative retention of bioactive phytochemicals. Therefore, this technique can be used in food and natural health product industries to produce high-quality, microbiologically safe, and shelf-stable products.

Root colonization by an arbuscular mycorrhizal (AM) fungus increases growth and secondary metabolism of purple coneflower, Echinacea purpurea (L.) Moench.

Purple coneflower, Echinacea purpurea (L.) Moench, is an important phytomedicinal species that contains phenolics and alkamides with antipathogenic properties. This study aimed to examine the effect of arbuscular mycorrhizal (AM) colonization on the physiology and biochemistry of E. purpurea. It was hypothesized that AM colonization enhances the growth and secondary metabolism in E. purpurea. In this regard, a 13-week factorial greenhouse experiment was performed with E. purpurea, inoculated (or not) with the AM fungus Glomus intraradices Schenck & Smith. Overall, the results indicated that AM colonization significantly increased the mass of shoots and roots and the concentrations of proteins and most of the phenolics in the roots. Hence, the selected trait of mycorrhiza could play an important role in optimizing the growth of E. purpurea by inducing the production of secondary phytomedicinal metabolites.

Genetic transformation of Echinacea purpurea with Agrobacterium rhizogenes and bioactive ingredient analysis in transformed cultures.

A method of the transformed hairy roots cultures of Echinacea purpurea was established by infecting different types of explants with three type strains of Agrobacterium rhizogenes (A4, R1601 and R1000) in this paper. We obtained that the transformed percentage of E. purpurea leaves with A4, R1601 and R1000 were 80%, 60%, 40%, respectively and that of E. purpurea leafstalks were 10%, 30%, 45%, respectively. The contents of polysaccharides and phenolic compounds were measured in transformed hairy roots and non-transformed roots after 2 months in culture. For transformed hairy roots, the contents of polysaccharides and phenolic compounds were 236.0 and 18.9 mg g(-1) DW, respectively. While the contents of polysaccharides and phenolic compounds in non-transformed roots were 161.5 and 33.3 mg g(-1) DW, respectively.