Impact of Phylogenetically Diverse Bacterial Endophytes of Bergenia pacumbis on Bergenin Production in the Plant Cell Suspension Cultures.

Plant in vitro cultures are potential sources for secondary metabolites. However, low productivity is often a major drawback for industrial application. Elicitation is an important strategy to improve product formation in vitro. In this context, endophytes are of special interest as biotic elicitors due to their possible interaction with the metabolism of the host plant. A total of 128 bacterial endophytes were isolated from the medicinal plant Bergenia pacumbis and taxonomically classified using 16S rRNA gene sequencing. Five strains belonging to different genera were grown in lysogeny broth and tryptic soy broth medium and cells as well as spent media were used as elicitors in cell suspension cultures of B. pacumbis. Production of the main bioactive compound bergenin was enhanced 3-fold (964 µg/g) after treatment with cells of Moraxella sp. or spent tryptic soy broth medium of Micrococcus sp. These results indicate that elicitation of plant cell suspension cultures with endophytic bacteria is a promising strategy for enhancing the production of desired plant metabolites.

Biosynthetic Potential of the Endophytic Fungus Helotiales sp. BL73 Revealed via Compound Identification and Genome Mining.

Endophytic fungi have been recognized as prolific producers of chemically diverse secondary metabolites. In this work, we describe a new representative of the order Helotiales isolated from the medicinal plant Bergenia pacumbis. Several bioactive secondary metabolites were produced by this Helotiales sp. BL 73 isolate grown on rice medium, including cochlioquinones and isofusidienols. Sequencing and analysis of the approximately 59-Mb genome revealed at least 77 secondary metabolite biosynthesis gene clusters, of which several could be associated with detected compounds or linked to previously reported molecules. Four terpene synthase genes identified in the BL73 genome were codon optimized and expressed, together with farnesyl-, geranyl-, and geranylgeranyl-pyrophosphate synthases, in Streptomyces spp. An analysis of recombinant strains revealed the production of linalool and its oxidized form, terpenoids typically associated with plants, as well as a yet unidentified terpenoid. This study demonstrates the importance of a complex approach to the investigation of the biosynthetic potential of endophytic fungi using both conventional methods and genome mining. IMPORTANCE Endophytic fungi represent an as yet underexplored source of secondary metabolites, of which some may have industrial and medical applications. We isolated a slow-growing fungus belonging to the order Helotiales from the traditional medicinal plant Bergenia pacumbis and characterized its potential to biosynthesize secondary metabolites. We used cultivation of the isolate with a subsequent analysis of compounds produced, bioinformatics-based mining of the genome, and heterologous expression of several terpene synthase genes. Our study revealed that this Helotiales isolate has enormous potential to produce structurally diverse natural products, including polyketides, nonribosomally synthesized peptides, terpenoids, and ribosomally synthesized and posttranslationally modified peptides (RiPPs). Identification of meroterpenoids and xanthones, along with establishing a link between these molecules and their putative biosynthetic genes, sets the stage for investigation of the respective biosynthetic pathways. The heterologous production of terpenoids suggests that this approach can be used for the discovery of new compounds belonging to this chemical class using Streptomyces bacteria as hosts.

Endophytic Akanthomyces sp. LN303 from Edelweiss Produces Emestrin and Two New 2-Hydroxy-4 Pyridone Alkaloids.

In the search for new antibiotics, several fungal endophytes were isolated from the medicinal plant Leontopodium nivale subsp. alpinum (Edelweiss). The extract from one of these fungi classified as Akanthomyces sp. displayed broad-spectrum antibiotic activity against gram-negative bacteria and fungi. Further investigation into the composition of this extract using bioactivity-guided fractionation, HRMS, and nuclear magnetic resonance revealed two new 4-hydroxy-2-pyridone alkaloids (1, 2) and emestrin (3), an epidithiodioxopiperazine not previously known to be produced by a member of Cordycipitaceae. Further testing of purified compounds 1 and 2 proved that they are devoid of antibiotic activity, and all the activities observed in the crude extract could be assigned to emestrin (3), whose configuration was confirmed by crystallographic data. This study demonstrates, for the first time, that endophytic fungi from Edelweiss can produce new compounds, prompting further investigation into them for drug discovery.

Piperazic acid containing peptides produced by an endophytic Streptomyces sp. isolated from the medicinal plant Atropa belladonna.

The culture broth of endophytic Streptomyces sp. AB100, isolated from the shoots of medicinal plant Atropa belladonna (L.) was investigated for the presence of antibacterial compounds. After initial testing followed by bioactivity-guided fractionation, six new piperazic acid (PA)-containing congeners of two known peptides, JBIR-39 and JBIR-40, were identified by HR-MS/MS and NMR analyses. Only the dehydroxylated hexapeptidic derivatives with unusual incorporation of four PA moieties exhibited weak antibacterial activity against Gram-positive test organism Bacillus subtilis. A 16S rDNA-based phylogenetic tree of known Streptomyces spp. producing PA-containing hexapeptides isolated from different habitats and endophyte Streptomyces AB100 showed considerable diversity, suggesting that these metabolites may play an important environmental role beyond their antibacterial activity.

Boron application increases growth of Brazilian Cerrado grasses.

Nutrients are known to limit productivity of plant communities around the world. In the Brazilian Cerrado, indirect evidences point to phosphorus as the main limiting nutrient, but some fertilization experiments suggest that one or more micronutrients might play this role. Boron is one of the essential micronutrients for plants. Agronomically, it received some attention, but it has mostly been neglected in ecological studies assessing the effects of nutrients on plant growth. Through field fertilization and mesocosm experiments in a degraded area in the Cerrado, we show that boron addition increased biomass production of herbaceous vegetation. This could be related to a lower aluminum uptake in the boron fertilized plants. Even considering that plant growth was promoted by boron addition due to aluminum toxicity alleviation, this is the first study reporting boron limitation in natural, noncultivated plant communities and also the first report of this kind in vegetative grasses. These results contribute to disentangling patterns of nutrient limitation among plant species of the species-rich, aluminum-rich, and nutrient-poor Cerrado biome and highlight the potential role of micronutrients, such as boron, for growth of noncrop plants. Understanding how nutrient limitation differs among functional groups in the highly biodiverse areas founded on ancient tropical soils may help managing these plant communities in a changing world.

Exploring Actinobacteria Associated With Rhizosphere and Endosphere of the Native Alpine Medicinal Plant Leontopodium nivale Subspecies alpinum.

The rhizosphere of plants is enriched in nutrients facilitating growth of microorganisms, some of which are recruited as endophytes. Endophytes, especially Actinobacteria, are known to produce a plethora of bioactive compounds. We hypothesized that Leontopodium nivale subsp. alpinum (Edelweiss), a rare alpine medicinal plant, may serve as yet untapped source for uncommon Actinobacteria associated with this plant. Rhizosphere soil of native Alpine plants was used, after physical and chemical pre-treatments, for isolating Actinobacteria. Isolates were selected based on morphology and identified by 16S rRNA gene-based barcoding. Resulting 77 Actinobacteria isolates represented the genera Actinokineospora, Kitasatospora, Asanoa, Microbacterium, Micromonospora, Micrococcus, Mycobacterium, Nocardia, and Streptomyces. In parallel, Edelweiss plants from the same location were surface-sterilized, separated into leaves, roots, rhizomes, and inflorescence and pooled within tissues before genomic DNA extraction. Metagenomic 16S rRNA gene amplicons confirmed large numbers of actinobacterial operational taxonomic units (OTUs) descending in diversity from roots to rhizomes, leaves and inflorescences. These metagenomic data, when queried with isolate sequences, revealed an overlap between the two datasets, suggesting recruitment of soil bacteria by the plant. Moreover, this study uncovered a profound diversity of uncultured Actinobacteria from Rubrobacteridae, Thermoleophilales, Acidimicrobiales and unclassified Actinobacteria specifically in belowground tissues, which may be exploited by a targeted isolation approach in the future.

Does hybridization of endophytic symbionts in a native grass increase fitness in resource-limited environments?

Hybridization is common among plants, animals and microbes. However, the ecological consequences of hybridization for microbes are far less understood than for plants and animals. For symbiotic Epichloë fungi, hybridization is widespread and may augment the well-known benefits of the endophytes to their grass hosts, especially in stressful environments. We tested the hybrid fitness hypothesis (HFH) that hybrid endophytes enhance fitness in stressful environments relative to non-hybrid endophytes. In a long-term field experiment, we monitored growth and reproduction of hybrid-infected (H+), non-hybrid infected (NH+), naturally endophyte free (E-) plants and those plants from which the endophyte had been experimentally removed (H- and NH-) in resource-rich and resource-poor environments. Infection by both endophyte species enhanced growth and reproduction. H+ plants outperformed NH+ plants in terms of growth by the end of the experiment, supporting HFH. However, H+ plants only outperformed NH+ plants in the resource-rich treatment, contrary to HFH. Plant genotypes associated with each endophyte species had strong effects on growth and reproduction. Our results provide some support the HFH hypothesis but not based upon adaptation to stressful environments. Our results reinforce the notion of a complex interplay between endophyte and plant genotype and environmental factors that determine fitness of the symbiotum.

Effects of Hybrid and Non-hybrid Epichloë Endophytes and Their Associated Host Genotypes on the Response of a Native Grass to Varying Environments.

Asexual Epichloë endophytes are prevalent in cool season grasses, and many are of hybrid origin. Hybridization of asexual endophytes is thought to provide a rapid influx of genetic variation that may be adaptive to endophyte-host grass symbiota in stressful environments. For Arizona fescue (Festuca arizonica), hybrid symbiota are commonly found in resource-poor environments, whereas non-hybrid symbiota are more common in resource-rich environments. There have been very few experimental tests where infection, hybrid and non-hybrid status, and plant genotype have been controlled to tease apart their effects on host phenotype and fitness in different environments. We conducted a greenhouse experiment where hybrid (H) and non-hybrid (NH) endophytes were inoculated into plant genotypes that were originally uninfected (E-) or once infected with either the H or NH endophytes. Nine endophyte and plant genotypic group combinations were grown under low and high water and nutrient treatments. Inoculation with the resident H endophyte enhanced growth and altered allocation to roots and shoots, but these effects were greatest in resource-rich environments, contrary to expectations. We found no evidence of co-adaptation between endophyte species and their associated host genotypes. However, naturally E- plants performed better when inoculated with the hybrid endophyte, suggesting these plants were derived from H infected lineages. Our results show complex interactions between endophyte species of hybrid and non-hybrid origin with their host plant genotypes and environmental factors.

Secondary Metabolites from Fungal Endophytes of Echinacea purpurea Suppress Cytokine Secretion by Macrophage-Type Cells.

Botanical extracts of Echinacea purpurea have been widely used for the treatment of upper respiratory infections. We sought to chemically examine fungal endophytes inhabiting E. purpurea, and to identify compounds produced by these endophytes with in vitro cytokine-suppressive activity. Twelve isolates from surface sterilized seeds of E. purpurea were subjected to fractionation and major components were isolated. Sixteen secondary metabolites belonging to different structural classes were identified from these isolates based on NMR and mass spectrometry data. The compounds were tested for their influence on cytokine secretion by murine macrophage-type cells. Alternariol (1), O-prenylporriolide (4), porritoxin (10) ß-zearalenol (13), and (S)-zearalenone (14) inhibited production of TNF-α from RAW 264.7 macrophages stimulated with LPS in the absence of any significant cytotoxicity. This is the first report of a cytokine-suppressive effect for 4. The results of this study are particularly interesting given that they show the presence of compounds with cytokine-suppressive activity in endophytes from a botanical used to treat inflammation. Future investigations into the role of fungal endophytes in the biological activity of E. purpurea dietary supplements may be warranted.

Ethanolic Echinacea purpurea Extracts Contain a Mixture of Cytokine-Suppressive and Cytokine-Inducing Compounds, Including Some That Originate from Endophytic Bacteria.

Echinacea preparations, which are used for the prevention and treatment of upper respiratory infections, account for 10% of the dietary supplement market in the U.S., with sales totaling more than $100 million annually. In an attempt to shed light on Echinacea's mechanism of action, we evaluated the effects of a 75% ethanolic root extract of Echinacea purpurea, prepared in accord with industry methods, on cytokine and chemokine production from RAW 264.7 macrophage-like cells. We found that the extract displayed dual activities; the extract could itself stimulate production of the cytokine TNF-α, and also suppress production of TNF-α in response to stimulation with exogenous LPS. Liquid:liquid partitioning followed by normal-phase flash chromatography resulted in separation of the stimulatory and inhibitory activities into different fractions, confirming the complex nature of this extract. We also studied the role of alkylamides in the suppressive activity of this E. purpurea extract. Our fractionation method concentrated the alkylamides into a single fraction, which suppressed production of TNF-α, CCL3, and CCL5; however fractions that did not contain detectable alkylamides also displayed similar suppressive effects. Alkylamides, therefore, likely contribute to the suppressive activity of the extract but are not solely responsible for that activity. From the fractions without detectable alkylamides, we purified xanthienopyran, a compound not previously known to be a constituent of the Echinacea genus. Xanthienopyran suppressed production of TNF-α suggesting that it may contribute to the suppressive activity of the crude ethanolic extract. Finally, we show that ethanolic extracts prepared from E. purpurea plants grown under sterile conditions and from sterilized seeds, do not contain LPS and do not stimulate macrophage production of TNF-α, supporting the hypothesis that the macrophage-stimulating activity in E. purpurea extracts can originate from endophytic bacteria. Together, our findings indicate that ethanolic E. purpurea extracts contain multiple constituents that differentially regulate cytokine production by macrophages.

Effects of natural hybrid and non-hybrid Epichloë endophytes on the response of Hordelymus europaeus to drought stress.

Interspecific hybrid endophytes of the genus Epichloë (Ascomycota, Clavicipitaceae) are prevalent in wild grass populations, possibly because of their larger gene variation, resulting in increased fitness benefits for host plants; however, the reasons are not yet known. We tested hypotheses regarding niche expansion mediated by hybrid endophytes, population-dependent interactions and local co-adaptation in the woodland grass Hordelymus europaeus, which naturally hosts both hybrid and non-hybrid endophyte taxa. Seedlings derived from seeds of four grass populations made endophyte free were re-inoculated with hybrid or non-hybrid endophyte strains, or left endophyte free. Plants were grown in the glasshouse with or without drought treatment. Endophyte infection increased plant biomass and tiller production by 10-15% in both treatments. Endophyte types had similar effects on growth, but opposite effects on reproduction: non-hybrid endophytes increased seed production, whereas hybrid endophytes reduced or prevented it completely. The results are consistent with the observation that non-hybrid endophytes in H. europaeus prevail at dry sites, but cannot explain the prevalence of hybrid endophytes. Thus, our results do not support the hypothesis of niche expansion of hybrid-infected plants. Moreover, plants inoculated with native relative to foreign endophytes yielded higher infections, but both showed similar growth and survival, suggesting weak co-adaptation.

The Epichloe endophytes associated with the woodland grass Hordelymus europaeus including four new taxa.

Epichloë endophytes (Clavicipitaceae, Ascomycota), including asexual forms placed in Neotyphodium, are common in cool-season grasses. Here we characterize the endophytes of the European woodland grass Hordelymus europaeus based on growth characteristics, morphology of conidiophores and conidia and phylogenetic relationships. Of the six different taxa found on H. europaeus, four are new, for which we propose the species names E. hordelymi, E. disjuncta, E. danica and subspecies E. sylvatica subsp. pollinensis. The other two are assigned to previously described E. bromicola and E. sylvatica. E. hordelymi, E. disjuncta and E. danica are asexual interspecific hybrids, while the other taxa are haploid. Only E. sylvatica subsp. pollinensis was found to reproduce sexually on H. europaeus. The high diversity of endophytes may be explained by repeated host jumps to H. europaeus with and without subsequent interspecific hybridizations.

Genetic diversity in epichloid endophytes of Hordelymus europaeus suggests repeated host jumps and interspecific hybridizations.

Epichloid fungal endophytes (Epichloë and Neotyphodium spp.) are excellent model systems for studying speciation processes because of their variable life history traits that are linked to host grass fitness. Presumed jumps to new hosts and subsequent somatic hybridizations appear to be common among epichloid endophytes resulting in increased genetic variation upon which selection can act and speciation be initiated. In this study, we explored the endophyte diversity of a rare European native woodland grass species, Hordelymus europaeus, along a latitudinal transect covering the entire distribution range of H. europaeus. From 28 populations in six countries, isolates were sampled and molecularly characterized. Based on the sequences of tubB and tefA, six distinct epichloid taxa (interspecific hybrid or cryptic haploid species) were found, of which four were novel and two have been previously reported from this host. Of the novel endophytes, two were presumed to be interspecific hybrids and two of nonhybrid origin. While previously known endophytes of H. europaeus are seed-born and strictly asexual, one of the novel nonhybrid endophytes found in the glacial refugium of the Apennine peninsula reproduced sexually in cultured plants. This is the first case of a seed-borne, but sexually reproducing endophyte of this host. We discuss the origin, and possible ancestral species, of the six epichloid taxa using phylogenetic analyses. Repeated host jumps and somatic hybridizations characterize the diversity of the endophytes. To date, no other grass species is known to host a larger diversity of endophytes than H. europaeus.