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.

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.

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.

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.

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.