Tatsiana Shymanovich

Alkaloid Variation Among Epichloid Endophytes of Sleepygrass (Achnatherum robustum) and Consequences for Resistance to Insect Herbivores

Epichloid endophytes are well known symbionts of many cool-season grasses that may alleviate environmental stresses for their hosts. For example, endophytes produce alkaloid compounds that may be toxic to invertebrate or vertebrate herbivores. Achnatherum robustum, commonly called sleepygrass, was aptly named due to the presence of an endophyte that causes toxic effects to livestock and wildlife. Variation in alkaloid production observed in two A. robustum populations located near Weed and Cloudcroft in the Lincoln National Forest, New Mexico, suggests two different endophyte species are present in these populations. Genetic analyses of endophyte-infected samples revealed major differences in the endophyte alkaloid genetic profiles from the two populations, which were supported with chemical analyses. The endophyte present in the Weed population was shown to produce chanoclavine I, paspaline, and terpendoles, so thus resembles the previously described Epichloë funkii. The endophyte present in the Cloudcroft population produces chanoclavineI, ergonovine, lysergic acid amide, and paspaline, and is an undescribed endophyte species. We observed very low survival rates for aphids feeding on plants infected with the Cloudcroft endophyte, while aphid survival was better on endophyte infected plants in the Weed population. This observation led to the hypothesis that the alkaloid ergonovine is responsible for aphid mortality. Direct testing of aphid survival on oat leaves supplemented with ergonovine provided supporting evidence for this hypothesis. The results of this study suggest that alkaloids produced by the Cloudcroft endophyte, specifically ergonovine, have insecticidal properties.

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

Comparison of electrospray ionization and atmospheric pressure photoionization liquid chromatography mass spectrometry methods for analysis of ergot alkaloids from endophyte-infected sleepygrass (Achnatherum robustum)

100 million annually. In an attempt to shed light on Echinaceas 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.

Interspecific and intraspecific hybrid Epichloë species symbiotic with the North American native grass Poa alsodes

Ergot alkaloids are mycotoxins with an array of biological effects. With this study, we investigated for the first time the application of atmospheric pressure photoionization (APPI) as an ionization method for LC-MS analysis of ergot alkaloids, and compared its performance to that of the more established technique of electrospray ionization (ESI). Samples of the grass Achnatherum robustum infected with the ergot producing Epichloë fungus were extracted using cold methanol and subjected to reserved-phase HPLC-ESI-MS and HPLC-APPI-MS analysis. The ergot alkaloids ergonovine and lysergic acid amide were detected in these samples, and quantified via external calibration. Validation parameters were recorded in accordance with ICH guidelines. A triple quadrupole MS operated in multiple reaction monitoring yielded the lowest detection limits. The performance of APPI and ESI methods was comparable. Both methods were subject to very little matrix interference, with percent recoveries ranging from 82% to 100%. As determined with HPLC-APPI-MS quantification, lysergic acid amide and ergonovine were extracted from an A. robustum sample infected with the Epichloë fungus at concentrations of 1.143±0.051 ppm and 0.2822±0.0071 ppm, respectively. There was no statistically significant difference between these concentrations and those determined using ESI for the same samples.

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

ABSTRACT The endophyte presence and diversity in natural populations of Poa alsodes were evaluated along a latitudinal transect from the southern distribution range in North Carolina to New York. Two distinct Epichloë hybrid taxa were identified from 23 populations. Each taxon could easily be distinguished by polymerase chain reaction (PCR) genotyping with primers designed to mating type genes and alkaloid biosynthesis genes that encode key pathway steps for ergot alkaloids, indole-diterpenes, lolines, and peramine. The most commonly found Epichloë taxon, Poa alsodes Taxonomic Group-1 (PalTG-1), was detected in 22 populations at high infection frequencies (72–100%), with the exception of one population at high elevation (26% infection). The second taxon, PalTG-2, was observed only in five populations in Pennsylvania constituting 12% of infected samples. Phylogenetic analyses placed PalTG-1 as an interspecific hybrid of E. amarillans and E. typhina subsp. poae ancestors, and it is considered a new hybrid species, which the authors name Epichloë alsodes. PalTG-2 is an intraspecific hybrid of two E. typhina subsp. poae ancestors, similar to E. schardlii from the host Cinna arundinacea, which the authors propose as a new variety, Epichloë schardlii var. pennsylvanica. Epichloë alsodes isolates were all mating type MTA MTB and tested positive for dmaW, easC, perA, and some LOL genes, but only the alkaloid N-acetylnorloline was detected in E. alsodes–infected plant material. Epichloë schardlii var. pennsylvanica isolates were all mating type MTB MTB and tested positive for perA, but peramine was not produced. Both E. alsodes and E. schardlii var. pennsylvanica appeared to have complete perA genes, but point mutations were identified in E. alsodes that would render the encoded perA gene nonfunctional.

Anti-insect defenses of Achnatherum robustum (sleepygrass) provided by two Epichloë endophyte species

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.

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

Many pooid grasses (Poaceae) harbor Epichloë species (Hypocreales), endophytic fungi that often produce toxic alkaloids which may provide anti‐insect protection for their hosts. Two natural populations of Achnatherum robustum (Vasey) (sleepygrass), in the Lincoln National Forest, Cloudcroft, and Weed (NM, USA), are infected with the endophyte species Epichloë funkii (KD Craven & Schardl) JF White and Epichloë sp. nov. We tested whether: (1) these endophytes affect survival, growth, and development of the insect herbivore Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae) (fall armyworm), (2) larval diets alter adult fecundity (assessed as number of larvae or eggs produced by females and number of spermatophores that males transfer to females when enclosed in pairs within each feeding group), and (3) infections affect leaf consumption in larval no‐choice and choice experiments. Individual larvae were reared on Epichloë infected vs. uninfected clipped leaves from the Cloudcroft and Weed population plants. Overall, armyworm survival was not affected when fed infected sleepygrass from either population. However, larvae that fed on Weed‐infected plants were smaller and had longer development than larvae that fed on uninfected and Cloudcroft‐infected plants. Males fed on Weed‐infected leaves had reduced mating success. Interestingly, pupal mass increased when larvae fed on either the infected leaf types. However, heavier females from both infected diets did not lay more eggs than lighter females from uninfected diets. In a no‐choice test, larvae on Weed‐infected plants diet consumed more leaf biomass than larvae from three other groups. In choice tests, larvae avoided feeding on leaves infected with either of the endophytes relative to uninfected leaves. Thus, the two Epichloë may provide direct protection to sleepygrass from insect herbivory by deterrence. The Weed population endophyte may provide stronger indirect protection than the Cloudcroft endophyte by reducing insect fitness or increasing risks of predation and parasitism through delayed development, even though larvae may consume more leaf biomass.

Epichloë endophytes of Poa alsodes employ alternative mechanisms for host defense: insecticidal versus deterrence

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.

Plant population and genotype effects override the effects of Epichloë endophyte species on growth and drought stress response of Achnatherum robustum plants in two natural grass populations

Some cool-season pooid grasses partner with symbiotic fungal endophytes in the Epichloë genus for defense against insect herbivores via fungal alkaloids. Poa alsodes, North American woodland grass, independently hosts two species of Epichloë that vary by produced alkaloids. E. alsodes produces insecticidal N-acetylnorloline. E. schardlii var. pennsylvanica (E. schardlii hereafter) has the gene for peramine, an insect-deterring alkaloid, production, but peramine was not detected. We tested the effects of the two endophytes on survival, feeding preference, and plant damage by the generalist herbivore, Spodoptera frugiperda. No larvae survived when feeding on plants harboring E. alsodes. In contrast, survival was only slightly reduced by plants harboring E. schardlii. However, larvae that fed on E. schardlii infected plants experienced delayed development and reduced pupal mass. Uninfected plants and plants infected with E. schardlii were damaged severely when single larvae fed upon them, whereas larvae fed negligibly on plants infected with E. alsodes. Preference did not match performance. Larvae strongly avoided feeding on E. schardlii but not E. alsodes-infected leaves where survival was zero. When E. schardlii was experimentally removed, larval leaf choices suggested that this endophyte is responsible for deterrence. High levels of N-acetylnorloline were detected from E. alsodes infected plants. Peramine was not detected in the experimental plants harboring E. schardlii, so it remains unclear what mechanisms caused avoidance and developmental delays. The two endophytes may protect their common host in different ways: E. alsodes by larval mortality and E. schardlii by deterring feeding and negative effects on development.