Dose-response study on the transfer of pyrrolizidine alkaloids from a tansy ragwort extract (Jacobaea vulgaris Gaertn.) to bovine milk.

Ragworts like tansy ragwort (J. vulgaris Gaertn., syn. Senecio jacobaea L.) contain hepatotoxic and cancerogenic pyrrolizidine alkaloids (PA) and their corresponding pyrrolizidine alkaloid N-oxides (PANO). Due to increasing spread of ragworts (Jacobaea spp.) PA/PANO may pose a health risk to animals and humans consuming contaminated feed and food. Therefore, the aim of the present study was to investigate the transfer of individual PA/PANO originating from a well-defined PA/PANO extract into the milk of dairy cows. For this objective, 16 German Holstein cows were assigned to four treatment groups (n = 4) in a 28-day dose-response study. Administration into the reticulorumen was performed daily by gavage after the morning milking. Three groups received different amounts of the J. vulgaris extract resulting in a PA/PANO exposure of 0.47, 0.95, or 1.91 mg PA/PANO/kg body weight/day, respectively. Furthermore, a control group received molasses to account for the sugar content of the used PA/PANO extract. While the composition of the PA/PANO extract was more diverse, the PA/PANO pattern in milk was dominated by the PA in their free base form. It was shown that mainly PA considered stable in the rumen environment were transferred into the milk. The main compounds in milk were jacoline (74.3 ± 2.4% of the PA/PANO sum), jaconine (11.2 ± 1.3%), and jacobine (7.2 ± 0.6%) with concentrations up to 29.7, 4.65 µg/l, or in the highest exposed group, 3.44 µg/l. There was no dose-dependent effect on the total PA/PANO transfer rate into the milk. The average transfer rate was 0.064 ± 0.005% of the administered content.

Dihydrostilbenes and flavonoids from whole plants of Jacobaea vulgaris.

The isolation of previously undescribed 12 compounds from the MeOH extract of Jacobaea vulgaris whole plants is disclosed, comprising 11 dihydrostilbenes (1-11) and one flavanone (12), and eight known compounds (six flavonoids, one dihydrostilbene, and one caffeoylquinic acid). Structural elucidation employed spectroscopic methods, including 1D and 2D NMR spectroscopy, HRESIMS, and ECD calculations. Evaluation of the compounds' effects on PCSK9 and LDLR mRNA expression revealed that compounds 1 and 3 downregulated PCSK9 mRNA while increasing LDLR mRNA expression, suggesting potential cholesterol-lowering properties.

No wonder, it is a hybrid. Natural hybridization between Jacobaea vulgaris and J. erucifolia revealed by molecular marker systems and its potential ecological impact.

Progressive changes in the environment are related to modifications of the habitat. Introducing exotic species, and interbreeding between species can lead to processes that in the case of rare species or small populations threatens their integrity. Given the declining trends of many populations due to increased hybridization, early recognition of hybrids becomes important in conservation management. Natural hybridization is prevalent in Jacobaea. There are many naturally occurring interspecific hybrids in this genus, including those between Jacobaea vulgaris and its relatives. Although Jacobaea erucifolia and J. vulgaris often co-occur and are considered closely related, apart from the few reports of German botanists on the existence of such hybrids, there is no information on research confirming hybridization between them. Morphologically intermediate individuals, found in the sympatric distributions of J. vulgaris and J. erucifolia, were hypothesized to be their hybrids. Two molecular marker systems (nuclear and chloroplast DNA markers) were employed to test this hypothesis and characterize putative hybrids. Nuclear and chloroplast DNA sequencing results and taxon-specific amplified fragment length polymorphism (AFLP) fragment distribution analysis confirmed the hybrid nature of all 25 putative hybrids. The AFLP patterns of most hybrids demonstrated a closer relationship to J. erucifolia, suggesting frequent backcrossing. Moreover, they showed that several individuals previously described as pure were probably also of hybrid origin, backcrosses to J. erucifolia and J. vulgaris. This study provides the first molecular confirmation that natural hybrids between J. vulgaris and J. erucifolia occur in Poland. Hybridization appeared to be bidirectional but asymmetrical with J. vulgaris as the usual maternal parent.

Rumen Metabolism of Senecio Pyrrolizidine Alkaloids May Explain Why Cattle Tolerate Higher Doses Than Monogastric Species.

Rumen metabolism of Senecio pyrrolizidine alkaloids (PAs) and their N-oxide forms was studied by mass spectrometry in in vitro batch culture incubates and confirmed in in vivo samples. Most N-oxides were found to undergo rapid conversion to their corresponding free bases, followed by biotransformation to metabolites hydrogenated at both the necine base and the necic acid moiety. Therefore, rumen metabolism can be considered a detoxification step, as saturated necine base structures are known as the platyphylline type, which is regarded as less or nontoxic. Individual Senecio PAs, such as jacoline, are metabolized slowly during rumen fermentation. PAs that showed limited biotransformation in the rumen in this study also showed limited transformation and CYP-mediated bioactivation in the liver in other studies. This could not only explain why PAs that are comparatively metabolically stable can pass into milk but also suggest that such PAs might be considered compounds of lesser concern.

Utilization of Biomasses from Landscape Conservation Growths Dominated by Common Ragwort (Jacobaea vulgaris Gaertn.) for Biomethanization.

The highly toxic species common ragwort (Jacobaea vulgaris Gaertn.) prefers to migrate into protected dry grassland biotopes and limits the use of the resulting biomass as animal feed. There is an urgent need for a safe alternative use of the contaminated biomass apart from landfill disposal. We investigated the optional utilization of biomethanization of fresh and ensiled common ragwort biomasses and evaluated their energetic potentials by estimation models based on biochemical characteristics and by standardized batch experiments. The fresh and ensiled substrates yielded 174 LN∙kg-1 oDM methane and 185 LN∙kg-1 oDM, respectively. Ensiling reduced the toxic pyrrolizidine alkaloid content by 76.6%; a subsequent wet fermentation for an additional reduction is recommended. In comparison with other biomasses from landscape cultivation, ragwort biomass can be ensiled readily but has a limited energy potential if harvested at its peak flowering stage. Considering these properties and limitations, the energetic utilization is a promising option for a sustainable handling of Senecio-contaminated biomasses in landscape conservation practice and represents a safe alternative for reducing pyrrolizidine alkaloid entry into the agri-food sector.

Pyrrolizidine alkaloids and unusual millingtojanine A-B from Jacobaea vulgaris (syn. Senecio jacobaea L.).

Four undescribed pyrrolizidine alkaloids (seneciojanine A-D), two enantiomeric pairs of unusual alkaloids (millingtojanine A-B) with a unique tricyclic core, and nine known pyrrolizidine alkaloids were isolated from whole plant extracts of Jacobaea vulgaris Gaertn. The structures of the undescribed compounds were established by extensive spectroscopic and spectrometric analyses and comparison of theoretical and experimental ECD data. Several of the structures were also confirmed by X-ray diffraction analysis. Seneciojanine A-D possess a rare natural necine moiety with an α,ß-unsaturated carbonyl group located at C-3 and a hydroxyl substituent at C-8.

Evolutionary changes in an invasive plant support the defensive role of plant volatiles.

It is increasingly evident that plants interact with their outside world through the production of volatile organic compounds,1-5 but whether the volatiles have evolved to serve in plant defense is still a topic of considerable debate.3,6-8 Unharmed leaves constitutively release small amounts of volatiles, but when the leaves are damaged by herbivorous arthropods, they emit substantially more volatiles. These herbivore-induced plant volatiles (HIPVs) attract parasitoids and predators that kill insect herbivores,9-12 and this can benefit the plants.13,14 As yet, however, there is no tangible evolutionary evidence that this tritrophic interplay contributes to the selection forces that have shaped the volatile emissions of plants.2,3,5-8,15 With this in mind, we investigated the evolutionary changes in volatile emissions in invasive common ragwort and the respective defensive roles of its constitutive and inducible volatiles. This Eurasian plant has invaded other continents, where it evolved for many generations in the absence of specialized herbivores and their natural enemies. We found that, compared to native ragworts, invasive plants release higher levels of constitutive volatiles but considerably lower levels of herbivore-induced volatiles. As a consequence, invasive ragwort is more attractive to a specialist moth but avoided by an unadapted generalist moth. Importantly, conforming to the indirect defense hypothesis, a specialist parasitoid was much more attracted to caterpillar-damaged native ragwort, which was reflected in higher parasitism rates in a field trial. The evolution of foliar volatile emissions appears to be indeed driven by their direct and indirect roles in defenses against insects.

Occurrence of pyrrolizidine alkaloids in ragwort plants, soils and surface waters at the field scale in grassland.

Pyrrolizidine alkaloids (PA)s are natural toxins produced by a variety of plants including ragwort. The PAs present a serious health risk to human and livestock. Although these compounds have been extensively studied in food and feed, little is known regarding their environmental fate. To fill this data gap, we investigated the occurrence of PAs in ragwort plants, soils and surface waters at three locations where ragwort was the dominant plant species to better understand their environmental distribution. The concentrations of PAs were quantified during the full growing season (April-November) and assessed in relation to rain events. PA concentrations ranged from 3.2-6.6 g/kg dry weight (dw) in plants, 0.8-4.0 mg/kg dw in soils, and 6.0-529 µg/L in surface waters. Maximum PA concentrations in the soil (4 mg/kg) and water (529 µg/L) were in mid-May just before flowering. The average distribution of PAs in water was approximately 5 g/10,000 L, compared to the average amounts present in ragwort (506 kg/ha), and soil (1.7 kg/ha). In general, concentrations of PAs increase in the soil and surface water following rain events.

Spread of Jacobaea vulgaris and Occurrence of Pyrrolizidine Alkaloids in Regionally Produced Honeys from Northern Germany: Inter- and Intra-Site Variations and Risk Assessment for Special Consumer Groups.

Pyrrolizidine alkaloids (PA) and PA N-oxides (PANO) are secondary plant metabolites exhibiting genotoxic and carcinogenic properties. Apart from the roots and leaves, PA/PANO are particularly present in pollen and nectar. Therefore, the spread of Jacobaea vulgaris in certain regions of northern Germany has an impact on the safety of honey produced in that region. In this study, raw honey samples (n = 437) were collected from usually three individual beehives per site (n = 73) in the district of Ostholstein and analyzed for 25 PA/PANO. The results reveal mean levels of 8.4, 1.5, and 72.6 µg/kg and maximum levels of 111, 59.4, and 3313 µg/kg, depending on the season (summer 2015 and spring/summer 2016, respectively). As far as individual data are concerned, sites near areas with J. vulgaris growth did not necessarily result in high PA/PANO values. Furthermore, intra-site investigations revealed remarkable differences in PA/PANO levels of raw honey collected by different bee colonies at the same site. Consumption of these regionally produced honeys entails an increased exposure to PA/PANO, especially in children and high consumers. Margin of exposure values of <10,000 and an exceedance of the health-based guidance value highlight that regionally produced and marketed honey must be considered with care for a proper risk assessment and risk management.

Soil Inoculation Alters Leaf Metabolic Profiles in Genetically Identical Plants.

Abiotic and biotic properties of soil can influence growth and chemical composition of plants. Although it is well-known that soil microbial composition can vary greatly spatially, how this variation affects plant chemical composition is poorly understood. We grew genetically identical Jacobaea vulgaris in sterilized soil inoculated with live soil collected from four natural grasslands and in 100% sterilized soil. Within each grassland we sampled eight plots, totalling 32 different inocula. Two samples per plot were collected, leading to three levels of spatial variation: within plot, between and within grasslands. The leaf metabolome was analysed with 1H Nuclear magnetic resonance spectroscopy (NMR) to investigate if inoculation altered the metabolome of plants and how this varied between and within grasslands. Inoculation led to changes in metabolomics profiles of J. vulgaris in two out of four sites. Plants grown in sterilized and inoculated soils differed in concentrations of malic acid, tyrosine, trehalose and two pyrrolizidine alkaloids (PA). Metabolomes of plants grown in inoculated soils from different sites varied in glucose, malic acid, trehalose, tyrosine and in one PA. The metabolome of plants grown in soils with inocula from the same site was more similar than with inocula from distant sites. We show that soil influences leaf metabolomes. Performance of aboveground insects often depends on chemical composition of plants. Hence our results imply that soil microbial communities, via affecting aboveground plant metabolomes, can impact aboveground plant-insect food chains but that it is difficult to make general predictions due to spatial variation in soil microbiomes.

Genetic and chemical diversity of the toxic herb Jacobaea vulgaris Gaertn. (syn. Senecio jacobaea L.) in Northern Germany.

Tansy ragwort, Jacobaea vulgaris Gaertn. (syn. Senecio jacobaea L.), is a common Asteraceae in Europe and Asia and known to be an invasive pest in several regions in the world. Recently it is also spreading immensely in native regions like Northern Germany. Pyrrolizidine alkaloids (PAs), which are found in high amounts in Jacobaea vulgaris, are toxic for humans and potentially lethal for grazing animals. In this study we investigated 27 populations of tansy ragwort in Northern Germany for their PA concentration and composition using liquid chromatography coupled to high resolution mass spectrometry. Furthermore, we investigated the genetic structure of selected populations using amplified length polymorphism markers. We detected 98 different PAs in the samples and considerable differences of PA composition between populations. In contrast, PA content of populations did not differ significantly. Genetic (4%) differentiation among populations was low while average genetic diversity was high (0.35). There was no correlation between genetic and geographic distance. Neither genetic markers nor chemical composition revealed any connection to the geographic pattern. As we could not detect any pattern in genetic or chemical diversity, we suggest that the existence of this diversity is a result of a broad interaction with the environment rather than that of evolutionary constraints in the current selection process driving PA composition in J. vulgaris in certain chemotypes.

Microbiomes of a specialist caterpillar are consistent across different habitats but also resemble the local soil microbial communities.

BACKGROUND: Insect-associated microorganisms can provide a wide range of benefits to their host, but insect dependency on these microbes varies greatly. The origin and functionality of insect microbiomes is not well understood. Many caterpillars can harbor symbionts in their gut that impact host metabolism, nutrient uptake and pathogen protection. Despite our lack of knowledge on the ecological factors driving microbiome assemblages of wild caterpillars, they seem to be highly variable and influenced by diet and environment. Several recent studies have shown that shoot-feeding caterpillars acquire part of their microbiome from the soil. Here, we examine microbiomes of a monophagous caterpillar (Tyria jacobaeae) collected from their natural host plant (Jacobaea vulgaris) growing in three different environments: coastal dunes, natural inland grasslands and riverine grasslands, and compare the bacterial communities of the wild caterpillars to those of soil samples collected from underneath each of the host plants from which the caterpillars were collected. RESULTS: The microbiomes of the caterpillars were dominated by Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Only 5% of the total bacterial diversity represented 86.2% of the total caterpillar's microbiome. Interestingly, we found a high consistency of dominant bacteria within the family Burkholderiaceae in all caterpillar samples across the three habitats. There was one amplicon sequence variant belonging to the genus Ralstonia that represented on average 53% of total community composition across all caterpillars. On average, one quarter of the caterpillar microbiome was shared with the soil. CONCLUSIONS: We found that the monophagous caterpillars collected from fields located more than 100 km apart were all dominated by a single Ralstonia. The remainder of the bacterial communities that were present resembled the local microbial communities in the soil in which the host plant was growing. Our findings provide an example of a caterpillar that has just a few key associated bacteria, but that also contains a community of low abundant bacteria characteristic of soil communities.

Removal of soil biota alters soil feedback effects on plant growth and defense chemistry.

We examined how the removal of soil biota affects plant-soil feedback (PSF) and defense chemistry of Jacobaea vulgaris, an outbreak plant species in Europe containing the defense compounds pyrrolizidine alkaloids (PAs). Macrofauna and mesofauna, as well as fungi and bacteria, were removed size selectively from unplanted soil or soil planted with J. vulgaris exposed or not to above- or belowground insect herbivores. Wet-sieved fractions, using 1000-, 20-, 5- and 0.2-µm mesh sizes, were added to sterilized soil and new plants were grown. Sieving treatments were verified by molecular analysis of the inocula. In the feedback phase, plant biomass was lowest in soils with 1000- and 20-µm inocula, and soils conditioned with plants gave more negative feedback than without plants. Remarkably, part of this negative PSF effect remained present in the 0.2-µm inoculum where no bacteria were present. PA concentration and composition of plants with 1000- or 20-µm inocula differed from those with 5- or 0.2-µm inocula, but only if soils had been conditioned by undamaged plants or plants damaged by aboveground herbivores. These effects correlated with leaf hyperspectral reflectance. We conclude that size-selective removal of soil biota altered PSFs, but that these PSFs were also influenced by herbivory during the conditioning phase.

Uncertainties in the determination of pyrrolizidine alkaloid levels in naturally contaminated honeys and comparison of results obtained by different analytical approaches.

The contamination of honey with hepatotoxic pyrrolizidine alkaloids (PAs) is a well-known hazard for food safety. While management strategies and controls of the honey industry aim to reduce the PA levels, uncertainties remain with regard to the safety of regionally produced and marketed honey. In addition, a previous study showed large differences of results obtained after various periods of storage and apparent differences between the analytical results of different laboratories. Therefore, this study aimed at examining these uncertainties by monitoring the impact of storage on the PA and PA N-oxide (PANO) content of two freshly harvested honeys and on possible demixing effects caused by pollen settling. Additionally, three analytical approaches - target analysis with matrix-matched calibration or standard addition and a sum parameter method - were applied for a comparative analysis of 20 honeys harvested in summer 2016. All samples originated from Schleswig-Holstein in Northern Germany where the PA plant Jacobaea vulgaris is currently observed on a massive scale. The results of the time series analyses showed that PANO levels markedly decreased within a few weeks and practically reached the LOD 16 weeks after harvest. Tertiary PAs, by contrast, remained stable and did not increase as a consequence of PANO decrease. The experiments on a putative demixing, which may result in a heterogeneous distribution of PAs/PANOs, revealed that there was no such effect during storage of up to 12 weeks. A comparison of the PA/PANO levels obtained by different analytical approaches showed that in some cases the sum parameter method yielded much higher levels than the target approaches, whereas in other cases, the target analysis with standard addition found higher levels than the other two methods. In summary, the results of this study highlight uncertainties regarding the validity and comparability of analytical results and consequently regarding health risk assessment.

The effect of structurally related metabolites on insect herbivores: A case study on pyrrolizidine alkaloids and western flower thrips.

Plant specialised metabolites (SMs) are very diverse in terms of both their number and chemical structures with more than 200,000 estimated compounds. This chemical diversity occurs not only among different groups of compounds but also within the groups themselves. In the context of plant-insect interactions, the chemical diversity within a class of structurally related metabolites is generally also related to their bioactivity. In this study, we tested firstly whether individual SMs within the group of pyrrolizidine alkaloids (PAs) differ in their effects on insect herbivores (western flower thrips, Frankliniella occidentalis). Secondly, we tested combinations of PA N-oxides to determine whether they are more active than their individual components. We also evaluated the bioactivity of six PA free bases and their corresponding N-oxides. At concentrations similar to that in plants, several PAs reduced thrip's survival but the effect also differed strongly among PAs. In general, PA free bases caused a lower survival than their corresponding N-oxides. Among the tested PA free bases, we found jacobine and retrorsine to be the most active against second instar larvae of thrips, followed by erucifoline and seneciphylline, while senecionine and monocrotaline did not exhibit significant dose-dependent effects on thrip's survival. In the case of PA N-oxides, we found that only senecionine N-oxide and jacobine N-oxide reduced thrip's survival, although the effect of senecionine N-oxide was weak. Combinations of PA N-oxides showed no synergistic effects. These findings indicate the differences observed in the effect of structurally related SMs on insect herbivores. It is of limited value to study the bioactivity of combined groups, such as PAs, without taking their composition into account.