Biodegradation and utilization of crop residues contaminated with poisonous pyrrolizidine alkaloids.

Disposal of noxious plant residues is a challenge for farmers and land management dealing with contaminated biomasses. Recent studies confirm the potential threat of transferring toxic plant constituents like pyrrolizidine alkaloids (PAs) from plant residues to non-toxic succeeding agricultural crops via the soil. We studied the degree of biochemical degradation of PAs in the two most important processes, composting and biomethanization. We used lab composting and biogas batches to investigate the potential of PA-degradation of two common PA-containing plants, Lappula squarrosa and Senecio jacobaea. The experiments demonstrated a virtually complete loss of PAs in three months during the composting process and a rapid decomposition of PAs from 3112.6 µg/kg to less than 21.5 µg/kg in L. squarrosa and from 6350.2 µg/kg to less than 539.6 µg/kg in S. jacobaea during biomethanization. The information obtained is a first guide on how to re-utilize PA-contaminated plant matter in a circular bioeconomy.

Arbuscular Mycorrhizal Fungi and Plant Chemical Defence: Effects of Colonisation on Aboveground and Belowground Metabolomes.

Arbuscular mycorrhizal fungal (AMF) colonisation of plant roots is one of the most ancient and widespread interactions in ecology, yet the systemic consequences for plant secondary chemistry remain unclear. We performed the first metabolomic investigation into the impact of AMF colonisation by Rhizophagus irregularis on the chemical defences, spanning above- and below-ground tissues, in its host-plant ragwort (Senecio jacobaea). We used a non-targeted metabolomics approach to profile, and where possible identify, compounds induced by AMF colonisation in both roots and shoots. Metabolomics analyses revealed that 33 compounds were significantly increased in the root tissue of AMF colonised plants, including seven blumenols, plant-derived compounds known to be associated with AMF colonisation. One of these was a novel structure conjugated with a malonyl-sugar and uronic acid moiety, hitherto an unreported combination. Such structural modifications of blumenols could be significant for their previously reported functional roles associated with the establishment and maintenance of AM colonisation. Pyrrolizidine alkaloids (PAs), key anti-herbivore defence compounds in ragwort, dominated the metabolomic profiles of root and shoot extracts. Analyses of the metabolomic profiles revealed an increase in four PAs in roots (but not shoots) of AMF colonised plants, with the potential to protect colonised plants from below-ground organisms.

Metabolite Profiling in the Liver, Plasma and Milk of Dairy Cows Exposed to Tansy Ragwort (Senecio jacobae) Pyrrolizidine Alkaloids.

BACKGROUND: Plant-derived pyrrolizidine alkaloids (PAs) in feed cause metabolic disturbances in farm animals resulting in high economic losses worldwide. The molecular pathways affected by these PAs in cells and tissues are not yet fully understood. The objective of the study was to examine the dose-dependent effects of orally applied PAs derived from tansy ragwort in midlactation dairy cows. METHODS: Twenty Holstein dairy cows were treated with target exposures of 0, 0.47, 0.95 and 1.91 mg of total PA/kg of body weight/d in control, PA1, PA2 and PA3, respectively, for 28 days. Liver tissue biopsy and plasma and milk samples were taken at day 28 of treatment to assess changes in metabolic pathways. A targeted metabolomics approach was performed to detect the metabolite profiles in all compartments. RESULTS: The PA-affected metabolite profiling in liver tissue, plasma and milk revealed changes in three substrate classes: acylcarnitines (ACs), phosphatidylcholines (PCs) and sphingomyelins (SMs). In addition, in the plasma, amino acid concentrations were affected by PA exposure. CONCLUSIONS: PA exposure disturbed liver metabolism at many sites, especially devastating pathways related to energy metabolism and to amino acid utilization, most likely based on mitochondrial oxidative stress. The effects on the milk metabolite profile may have consequences for milk quality.

Grazing Ecology of Sheep and Its Impact on Vegetation and Animal Health on Pastures Dominated by Common Ragwort (Senecio jacobaea L.)-Part 2: Animal Health.

Common ragwort (Senecio jacobaea L.) naturally occurs on species-rich grasslands. Containing pyrrolizidine alkaloids (PA), it endangers livestock health through contaminated feed. Although in vitro studies showed a detoxification capacity of PA in sheep, few field data are available on the ability of grazing sheep to cope with ragwort. During two grazing seasons on a ragwort-rich pasture, we studied: (1) To what extent do sheep voluntarily ingest ragwort and (2) What impact their grazing behavior has on animal health. Ragwort intake was monitored by counting missing plant parts and calculating their weight. From 70 sheep, seven were slaughtered at the beginning and in six-week intervals at the end of each grazing period to monitor blood parameters and liver tissue. Sheep continuously preferred ragwort. The daily intake was above the currently assumed lethal dose, varying between 0.2-4.9 kg per sheep. Clinical, hematologic, and blood biochemistry parameters mostly remained within the reference limits. Initially elevated liver copper content declined over time. The liver of all 70 animals displayed slight to moderate hepatitis, fibrosis, and proliferation of the bile ducts, but no morphological signs of liver cirrhosis. Sheep preferred and tolerated ragwort, making their grazing an option to control ragwort from both an animal health and nature conservation perspective.

Grazing Ecology of Sheep and Its Impact on Vegetation and Animal Health in Pastures Dominated by Common Ragwort (Senecio jacobaea L.)-Part 1: Vegetation.

Species-rich pastures naturally contain potentially toxic plants such as common ragwort (Senecio jacobaea L.), whose pyrrolizidine alkaloids (PA) impose a risk, mainly for cattle and horses. Although in vitro studies showed detoxification capacity of PA in sheep, few field data are available to ascertain whether grazing sheep can both tolerate and reduce ragwort. In a two-year study in a ragwort-rich pasture with a stocking density of 12 sheep/hectare, we documented (1) the extent of voluntarily ingested ragwort, (2) the correlation of nutritional parameters and feeding behavior, and (3) the impact of grazing on the yield proportion and number of flowers of dominant plants. Every six weeks the vegetation underwent a botanical survey and a chemical analysis. Sheep continuously ingested ragwort between 1.2 and 4.9 kg (2020) and 1.0 and 2.2 kg (2021) per individual per day without any impact on animal health. The more biomass ragwort produced, the more it contained sugar (r = 0.59-0.74), and the more sheep ingested it (r = 0.94-0.95). Other herbs increased their yield proportion from 23.3 to 36.5%, while that of ragwort decreased from 26.3 to 18.8% (2020/2021), doubling its flowers. Sheep preferred and tolerated ragwort, making their grazing an option to control ragwort from both an animal health and a nature conservation perspective.

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.

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.

Pyrrolizidine Alkaloids in the Food Chain: Is Horizontal Transfer of Natural Products of Relevance?

Recent studies have raised the question whether there is a potential threat by a horizontal transfer of toxic plant constituents such as pyrrolizidine alkaloids (PAs) between donor-PA-plants and acceptor non-PA-plants. This topic raised concerns about food and feed safety in the recent years. The purpose of the study described here was to investigate and evaluate horizontal transfer of PAs between donor and acceptor-plants by conducting a series of field trials using the PA-plant Lappula squarrosa as model and realistic agricultural conditions. Additionally, the effect of PA-plant residues recycling in the form of composts or press-cakes were investigated. The PA-transfer and the PA-content of soil, plants, and plant waste products was determined in form of a single sum parameter method using high-performance liquid chromatography mass spectroscopy (HPLC-ESI-MS/MS). PA-transfer from PA-donor to acceptor-plants was frequently observed at low rates during the vegetative growing phase especially in cases of close spatial proximity. However, at the time of harvest no PAs were detected in the relevant field products (grains). For all investigated agricultural scenarios, horizontal transfer of PAs is of no concern with regard to food or feed safety.

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.

Transfer of pyrrolizidine alkaloids between living plants: A disregarded source of contaminations.

To elucidate the origin of the wide-spread contaminations of plant derived commodities with various alkaloids, we employed co-cultures of pyrrolizidine alkaloid (PA) containing Senecio jacobaea plants with various alkaloid free acceptor plants. Our analyses revealed that all plants grown in the vicinity of the Senecio donor plants indeed contain significant amounts of the PAs, which previously had been synthesized in the Senecio plants. These findings illustrate that typical secondary metabolites, such as pyrrolizidine alkaloids, are commonly transferred and exchanged between living plants. In contrast to the broad spectrum of alkaloids in Senecio, in the acceptor plants nearly exclusively jacobine is accumulated. This indicates that this alkaloid is exuded specifically by the Senecio roots. Although the path of alkaloid transfer from living donor plants is not yet fully elucidated, these novel insights will extend and change our understanding of plant-plant interactions and reveal a high relevance with respect to the widespread alkaloidal contaminations of plant-derived commodities. Moreover, they could be the basis for the understanding of various so far not fully understood phenomena in cultivation of various crops, e.g. the beneficial effects of crop rotations or the co-cultivation of certain vegetables.

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.

Influence of Storage on the Stability of Toxic Pyrrolizidine Alkaloids and Their N-Oxides in Peppermint Tea, Hay, and Honey.

1,2-Dehydropyrrolizidine alkaloids (PA) and PA- N-oxides (PANO) are phytotoxins, which presumably occur in more than 6,000 plant species worldwide. Plants containing PA/PANO are responsible for various food and feed poisonings recorded for decades. Main reasons of exposition of consumers and livestock are contaminations of food and animal feed with parts, seeds, pollen, or nectar of PA-containing plants. Concerning stability, effects of processing on PA were mainly investigated in the past. The current study examined the behavior of PA/PANO in unprocessed matrices peppermint tea, hay, and honey during storage. Blank samples were fortified with PA/PANO or contaminated with blueweed ( Echium vulgare) and ragwort ( Senecio jacobaea) and stored for 182 d. The time-series analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that all 25 analyzed PA/PANO compounds remained stable in herbal samples. However, the results showed a very fast decrease of PANO in honey samples within hours. These results were discussed with respect to potential consequences for health risk assessment.

Responses of Cynoglossum officinale L. and Senecio jacobaea L. to various degrees of defoliation.

Effects of various degrees of defoliation on Cynoglossum officinale and Senecio jocobaea were studied under favourable growing conditions. Regrowth was found in both species, but did not overcompensate for defoliation. The physiological mechanisms responsible for regrowth after defoliation were found to relate either to an increase in net assimilation rate or to an increase in relative investment in aboveground parts. The morphological mechanism behind regrowth involved an increase in leaf area ratio. In almost completely defoliated plants, a combination of both mechanisms was found. The time needed to re-establish the same amount of biomass as control plants differed between the species and between levels of defoliation. These differences in recovery time after defoliation are discussed in relation to decrees of herbivory found m natural situations.

The effects of host plant defoliation and fertilizer application on larval growth and oviposition behaviour in cinnabar moth.

Defoliated ragwort plants produced regrowth foliage that was higher in alkaloid, but lower in amino acid concentrations than primary foliage. Total N was not affected. 2) Plants fertilized with nitrogen (as ammonium sulphate) had lower amino acid concentrations than unfertilized control plants, slightly increased alkaloid levels but similar total N concentrations. 3) Ovipositing females laid eggs upon plants with equal probability for controls, regrowth and fertilized foliage (one rosette in 5 received an egg batch). However, the probability of receiving eggs was significantly lower on the primary leaves of 'cut-back' plants that had had their lower leaves removed a few days before egg laying (only one rosette in 13 was selected). 4) Egg batch size was higher on fertilized control foliage than on other treatments. 5) Larvae attained greater final weights when fed a diet of regrowth foliage, despite the higher levels of alkaloid they contained. Larval development rate was not affected by experimental treatment of the foliage. 6) Larval growth was lowest on the leaves of fertilized plants. This was associated with significant reductions in the concentrations of three amino acids (methionine down 29%, tyrosine 33% and lysine 25%).

Differential effects of insect herbivory on arbuscular mycorrhizal colonization.

A series of field and laboratory experiments were conducted to examine whether natural levels of insect herbivory affect the arbuscular mycorrhizal (AM) colonization of two plant species. The plant species were the highly mycorrhizal (mycotrophic) Plantago lanceolata, which suffers small amounts of insect damage continuously over a growing season and the weakly mycorrhizal (non-mycotrophic) Senecio jacobaea, which is frequently subject to rapid and total defoliation by moth larvae. Herbivory was found to reduce AM colonization in P. lanceolata, but had no effect in S. jacobaea. Similarly, AM colonization reduced the level of leaf damage in P. lanceolata, but had no such effect in S. jacobaea. AM fungi were found to increase growth of P. lanceolata, but this effect was only clearly seen when insects were absent. AM fungi reduced the growth of S. jacobaea irrespective of whether insects were present. It is concluded that the reduction of AM fungal colonization by herbivory in P. lanceolata is due to the reduced amount of photosynthate available to the symbiont. This may only become apparent at threshold levels of insect damage and, below these, increased photosynthesis elicited by the mycorrhiza is able to compensate for foliage loss to the insects. However, in S. jacobaea, the mycorrhiza appears to be an aggressive parasite and insect attack only exacerbates the reduction in biomass. In mycotrophic plants, insect herbivores may be responsible for poor functioning of the symbiosis in field conditions and there is a symmetrical interaction between insects and fungi. However, in non-mycotrophic plants, the interaction is strongly asymmetrical, being entirely in favour of the mycorrhiza.

Diversity of pyrrolizidine alkaloids in Senecio species does not affect the specialist herbivore Tyria jacobaeae.

The evolution of the diversity of related secondary metabolites in plants is still poorly understood. It is often thought that the evolution of plant secondary metabolites is driven by specialist insect herbivores and under this coevolutionary model it is expected that related compounds differ in their effects on specialist herbivores. Here we focus on the diversity of pyrrolizidine alkaloids (PAs) in Senecio species and their effects on Tyria jacobaeae, a specialist moth on Senecio jacobaea. As a first step to determine the effects of related PAs on T. jacobaeae, we studied larval performance on plants from 11 S. jacobaea populations and eight Senecio species with different PA compositions. Although the populations of S. jacobaea differed in their PA compositions, there was no difference in larval performance among the populations. Larval performance differed among the eight species but we could not show a correlation with PA composition. Oviposition choice experiments showed a strong correlation between oviposition preference and larval performance on the eight species but oviposition preference did not seem to be correlated with PAs. We found no indications that related PAs differ in effects on the specialist T. jacobaeae; therefore it seems unlikely that T. jacobaeae is a driving force behind the evolution of the diversity of PAs. Alternatively, we propose that the evolution of the diversity of PAs is driven by selection pressure from generalist herbivores or that the diversity of PAs may even be selectively neutral.

Cost assessment of the production of pyrrolizidine alkaloids in ragwort (Senecio jacobaea L.).

Costs of pyrrolizidine alkaloid (Pa) production in vegetative ragwort (Senecio jacobaea) were examined under conditions in which plant growth was limited by light, nitrogen and phosphorus. Measurable costs of Pa production were demonstrated under light-limiting conditions. Plants with higher Pa concentrations grew more slowly than those with lower Pa concentration. Under nitrogen- and phosphorus-limited conditions no trade-off between Pa production and growth was observed.

A comparative evaluation of models of cinnabar moth dynamics.

1. A complex model of cinnabar moth dynamics proposed by Dempster and Lakhani (1979) with 23 parameters is reduced to a single equation with five parameters, and the behaviour of the reduced model shown to explain most features of the full model. 2. The efficiency of the full model is compared with the reduced model and with two even simpler models (the two parameter discrete logistic and a four parameter model based on a step-function for mortality) in their abilities to describe time series data of cinnabar moth population densities from Weeting Heath. Models with more parameters were not significantly better than few-parameter models in describing population trajectories. 3. Models that included a driving variable (in this case observed rainfall data) were no better at describing the data than simpler models without driving variables. It appears, therefore, that the routine inclusion of driving variables may be counterproductive, unless there is compelling empirical or theoretical evidence of their importance and the mode of action of the driving variables can be modelled mechanistically. For example, the regression model used to describe the relationship between rainfall and plant biomass in Dempster and Lakhani (1979), breaks down if rainfall is assumed to be constant, because there is no explicit model for the regulation of plant biomass. 4. The parameter values of the cinnabar-ragwort interaction suggest that cinnabar moth dynamics may be chaotic. Whether or not field data exhibit chaos or environmental stochasticity (or a mixture of both) is impossible to determine from inspection of time series data on population density. There is an urgent need for experimental and theoretical protocols to disentangle these two sources of population fluctuation.

Positive and negative effects of herbivory on the population dynamics of Senecio jacobaea L. and Cynoglossum officinale L.

Herbivore effects were studied on populations of the biennial plant species Senecio jacobaea and Cynoglossum officinale. During a three year period (1985-1988) population characteristics (herbivory, number of seedlings, rosettes and flowering plants) were compared in-and outside exclosures, as well as parameters reflecting vegetation cover. In S. jacobaea, a strong negative effect of Tyria jacobaeae was found on seedling establishment, rosette growth and flowering. On the other hand, vertebrate herbivores (mainly rabbits) had an indirect positive effect by limiting the development of the surrounding vegetation (esp. grasses). The increasing vegetation cover in protected populations caused a reduction in germination, seedling- and rosette-growth. Herbivory on C. officinale was low (<10%), no direct effects of herbivores on plant populations were shown. Indirect effects of herbivory through an increasing vegetation were even more pronounced as in S. jacobaea. Therefore, although both plant species may first benefit from herbivore-exclusion, their populations are dependent on rabbits eating other plants (esp. grasses) and reducing competition.

Tritrophic interactions between aphids (Aphis jacobaeae Schrank), ant species, Tyria jacobaeae L., and Senecio jacobaea L. lead to maintenance of genetic variation in pyrrolizidine alkaloid concentration.

We hypothesize that the tritrophic interaction between ants, the aphid Aphis jacobaeae, the moth Tyria jacobaeae, and the plant Senecio jacobaea can explain the genetic variation observed in pyrrolizidine alkaloid concentration in natural populations of S. jacobaea. The ant Lasius niger effectively defends S. jacobaea plants infested with A. jacobaeae against larvae of T. jacobaeae. S. jacobaea plants with A. jacobaeae which are defended by ants escape regular defoliation by T. jacobaeae. Plants with aphids and ants have a lower pyrrolizidine alkaloid concentration than plants without aphids and ants. When these data are fitted to an existing theoretical model for temporal variation in fitness it is shown that varying herbivore pressure by T. jacobaeae in interaction with ants defending aphid-infested plants with a low pyrrolizidine alkaloid concentration can lead to a stable polymorphism in pyrrolizidine alkaloid concentration. Costs of the production and maintenance of pyrrolizidine alkaloids are not accounted for in the model.