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Lolium temulentum, typically known as darnel, poison darnel, darnel ryegrass or cockle, is an annual plant of the genus Lolium within the family Poaceae. The plant stem can grow up to one meter tall, with inflorescence in the ears and purple grain. It has a cosmopolitan distribution. Darnel is toxic and competes with wheat plants. People have taken advantage of its hallucinogenic properties by using it to bake bread and brew beer.

Growth


Darnel usually grows in the same production zones as wheat and was a serious weed of cultivation until modern sorting machinery enabled darnel seeds to be separated efficiently from seed wheat. The similarity between these two plants is so great that in some regions, darnel is referred to as "false wheat". It bears a close resemblance to wheat until the ear appears. The spikes of L. temulentum are more slender than those of wheat. The spikelets are oriented edgeways to the rachis and have only a single glume, while those of wheat are oriented with the flat side to the rachis and have two glumes. Wheat will appear brown when ripe, whereas darnel is black.

Darnel can be infected by an endophytic fungus of the genus Neotyphodium and the endophyte-produced, insecticidal loline alkaloids were first isolated from this plant.

The French word for darnel is ivraie (from Latin ebriacus, intoxicated), which expresses the drunken nausea from eating the infected plant, which can be fatal. The French name echoes the scientific name, Latin temulentus "drunk."

Toxicity
Darnel is an example of a poisonous grass. Its toxicity, combined with its difficulty of separation from staple crops like wheat, have made it problematic for farmers since the time of the New Testament. The symptoms of darnel poisoning include "dizziness, headache, mental confusion with a sense of apprehension and difficulty in thinking, visual and speech difficulties (even loss of speech), decrease in salivary secretion, vomiting, inability to walk, griping, rarely diarrhea, tremor, general weakness and finally coma." Tests have also shown that darnel extracts are fatal to rodents.

Despite much interest in the chemical mechanism behind its toxicity, the exact cause has been difficult to pin down. It is well known that darnel is frequently infected by the fungus Neotyphodium occultans, which produces loline alkaloids in the plant. While it was initially assumed that the toxicity was due to these alkaloids, recent tests have shown that they have no effect on humans or livestock except in exceedingly large concentrations.

The symptoms of darnel poisoning are consistent with those caused by ergot contamination, which has led to a theory that the two are related. Notably, the N. occultans fungus is in the same family, Clavicipitaceae, as the ergot organism. In fact, Lolium endophytes even possess the genes to synthesize ergot toxins. It has thus been hypothesized that under certain conditions, especially in the presence of high concentrations of potassium sulfate and ammonium chloride, ergot alkaloids could be responsible for darnel's toxicity.

Beyond alkaloids, darnel contains a variety of other toxic compounds. Lolium endophytes contain indolediterpene neurotoxins that are highly potent in mammals and have been linked to perennial ryegrass staggers in livestock. Darnel is also susceptible to infection from more than fungus. A bacterium called Clavibacter toxicus has been shown to infect Lolium species. This bacterium produces corynetoxin, a class of toxic antibiotics, and is implicated in annual ryegrass toxicity in livestock.

Historical Uses
Despite its classification as a weed, darnel has been used for a variety of purposes throughout history. The Ancient Greeks knew it as "the plant of frenzy" and took advantage of its hallucinogenic properties. For example, some historians believe that darnel was an ingredient in a hallucinogenic brew used by the priestesses of Demeter and Persephone for the Eleusinian Mysteries. . Similarly, darnel is one of the many hallucinogenic plants that have been proposed to be used by the cult of Apollo at Delphi to induce visions in the oracle.

While the presence of darnel was generally undesirable in wheat harvests or flour because of its strong taste and toxic properties, the seeds were also sometimes used as a flavoring for beer. In these cases, the hallucinogenic nature of the darnel was an added bonus to increase the intoxicating nature of the beer. While many civilizations actively tried to avoid consuming darnel in their bread, some were unable to do so. In early modern Europe, for example, darnel was included in grain mixtures for making bread out of desperation in times of famine. The bread produced with the grain mixture was noted to daze the people who ate it and induce drunken behavior. Using darnel to make brad was only done in desperate times and it was not adopted as a true crop, as cities like Tuscany reported sorting their crops to avoid darnel contamination. It has also been suggested that darnel bread was deliberately used by some governments to keep their populations in a low-level daze to avoid mass discontent during difficult times. Despite its possible uses, finding ways to alleviate darnel's toxicity was of high interest. One proposed method was to ferment the darnel in water and then cook it before combining it with other ingredients for consumption.

Scholars and physicians have also explored the medicinal properties of darnel. The physician Rembert Dodoens recorded its anasthetic properties in his herbal. It was also traditionally used in the Middle East as an anasthetic. The naturalist Ulisse Aldrovandi reported that exposing chickens to darnel was an effective fumigation treatment against diseases.

Literary references



 * The ancient Greek botanist Theophrastus stated in his De causis plantarum (8:7 §1) that wheat can transform (metaballein) into darnel (aira), since fields sown to wheat are often darnel when reaped.


 * Darnel is mentioned in Horace's Satire 2.6 (eaten by the Country mouse while he serves his guest fancier foods)


 * Darnel may have been the plant in the Parable of the Tares in the Gospel of Matthew:
 * Darnel and tares have frequently been used synonymously, and the weeds have since been used as a metaphor for heresy and evil in religious contexts.


 * In ordering the St. Brice's Day massacre of all the Danes in England, Æthelred the Unready observed that "all the Danes who had sprung up in this island, sprouting like cockle amongst the wheat, were to be destroyed by a most just extermination."
 * Darnel is also mentioned as a weed in Shakespeare's King Lear.
 * The plant is used as a symbol of betrayal and the onset of madness. King Lear's crown of darnel would have indicated his mental deterioration given its association with the enemies of Christ.
 * Darnel is one of the many ingredients in mithridate, which Mithridates, the king of ancient Pontus, is supposed to have used every day to render him immune to poisoning.
 * Darnel is mentioned in the Mishnah in Kilayim (1:1) as זונין (zunin), similar to the Arabic زؤان (zuʾān).

Discovery
A member of the loline alkaloids was first isolated from the grass Lolium temulentum and its elemental composition determined in 1892. It was initially named temuline and later renamed norloline. (Reviewed by Schardl et al. (2007).) Studies in the 1950s and 1960s by Russian researchers established the name loline and identified the characteristic 2,7 ether bridge in its molecular structure. Since then the analytical methods for purification and analysis of the lolines have been refined and several different loline species have been identified in many Lolium and related grasses infected by the Epichloë/Neotyphodium (epichloae) endophytes. Lolines are absent in grass plants that do not harbor the epichloae endophytes, and not all epichloae produce the lolines. Because of the very intimate association of plant and endophyte and difficulties to reproduce the symbiotic conditions in vitro, it was long unknown if the fungus was the producer of the lolines, or if they were synthesized by the plant in response to endophyte infection. In 2001, it was demonstrated that the endophyte Neotyphodium uncinatum produces lolines in some chemically defined growth media, which suggests that the endophyte is also the producer of the lolines in the grass plant. The lolines have also been reported from some plants in several plant families, suggesting a more widespread occurrence of these compounds in nature.

Chemical Synthesis
Loline alkaloids have proven to be challenging synthetic targets. They are deceptively complex, with a strained heterotricyclic molecular skeleton containing a bridgehead ether. The polar functional groups in relatively close proximity only add to the challenge. Due to their toxicity to insects and inactivity in mammalian systems, loline alkaloids have received attention as a natural insecticide and several attempts were made to synthesize them before being abandoned due to the unexpected difficulty. Despite the fact that loline alkaloids have been known for more than a century, the first successful synthesis was only completed in 1986 by Joseph Tufariello at the State University of New York at Buffalo. The 1986 synthesis produced a racemic mixture of loline products starting from a nitrone substrate, and it was not until 14 years later that the first asymmetric synthesis, which required twenty steps starting from (-)-malic acid, was achieved in the Blakemore lab. Since that synthesis, additional processes have been developed with the aim of increasing efficiency or accessing a wider range of alkaloids.

Mechanism of action
Lolines are insecticidal and deterrent to a broad range of insects, including species in the Hemiptera, Coleoptera, Hymenoptera, Lepidoptera, and Blattodea, such as the bird cherry-oat aphid (genus Rhopalosiphum), large milkweed bug (Oncopeltus fasciatus), and American cockroach (Periplaneta americana). LC50 values of N-formylloline or N-acetylloline from grass seed extracts are 1-20 μg/ml for aphids and milkweed bugs and impair insect development and fecundity and cause avoidance of loline-containing grass tissues. However, results of feeding tests with grass extracts are occasionally difficult to interpret due to the presence of other endophyte alkaloids in these extracts, and the exact mechanisms of the insecticidal actions of the lolines are unknown. The lolines may be neurotoxic to the insects, and differences in the chemical groups at the C-1-amine result in different levels of insect toxicity; for example, N-formylloline (see Fig. 2), which occurs in higher concentrations in endophyte-infected grass plants, has greater insect toxicity than some other lolines, which occur at lower concentrations in the grass plant.



Loline bioactivities show some unexpected variability with variation in their concentration in grass tissues. For example, the tall fescue endophyte, N. coenophialum, has been associated with enhanced resistance to the migratory root-endoparasitic nematode, Pratylenchus scribneri. At low concentrations, N-formylloline serves as a chemoattractant for P. scribneri, but acts as a repellant at higher concentrations. However, ergot alkaloids also have repellent and immobilizing effects on P. scribneri, and an endophyte of perennial ryegrass lacking lolines, and genetically engineered to produce no ergot alkaloids, exhibits resistance to this nematode. Therefore, the relative importance of the loline and ergot alkaloids to nematode resistance remains unclear.

Many epichloae endophytes—including N. coenophialum symbiotic with Lolium arundinaceum (syn. Festuca arundinacea, tall fescue)—also produce ergot alkaloids that are toxic to mammalian herbivores. The ergot alkaloids occur at relatively low concentrations in the plant and are often difficult to detect analytically. By contrast, the lolines frequently accumulate to very high levels in grass tissues, and were, therefore, initially associated also with toxicity to mammalian herbivores. Specifically, the lolines were thought to be responsible for toxic symptoms called fescue toxicosis displayed by livestock grazing on grasses infected by N. coenophialum. However, subsequently it was demonstrated that only the endophyte-produced ergot alkaloids are responsible for the symptoms of fescue toxicosis (or summer syndrome), and not the lolines which, even at high doses, have only very small physiological effects on mammalians feeders. Another group of alkaloids, the senecio-type alkaloids, are produced by various plants and like the lolines, the senecio alkaloids possess a pyrrolizidine ring structure. Unlike the lolines, however, the senecio alkaloids exhibit strong hepatotoxicity, owing to a double bond between C-1 and C-2 in their ring structure. This double bond is absent in the lolines, explaining the lack of hepatotoxicity of this group of compounds. The lolines have been suggested to inhibit seed germination or growth of other plants (allelopathy), and to increase resistance of infected grasses against drought, but such effects have not been substantiated under more natural conditions of cultivation or in habitats.

Production and distribution in the grass plant
Lolines are produced by several grass–endophyte symbioses involving epichloae species, often along with other bioactive metabolites including ergot alkaloids and indole diterpenoids, and the unusual pyrrolopyrazine alkaloid, peramine, which is not found in other biological communities or organisms. The lolines are produced at levels, however, that can exceed 10 mg/g grass tissue (ranging from 2–20,000 μg/g ), exceeding the concentrations of the other endophyte alkaloids by >1000-fold. Lolines produced in the grasses Lolium pratense (syn. Festuca pratensis, meadow fescue) and tall fescue infected by N. uncinatum and N. coenophialum (see Fig. 3), respectively, exhibit variable concentrations in grass tissues. Higher loline concentrations (100–1000 μg/g) are present in the seeds and in younger leaf tissues, and the lolines display seasonal changes in concentration levels throughout the plant. The periodical appearance of tissues with high loline concentrations, such as flowering stems and seeds, contributes to this seasonal variation. Loline concentrations often increase in grass tissues regrown after defoliation and clipping of plants, suggesting an inducible defense response mechanism, involving both symbiotic partners. However, this increase appears to be due to higher loline levels in younger leaves compared to older leaves, but loline increases resembling inducible plant defenses have also been reported. Variation of loline concentration with the developmental stage of specific grass tissues suggests regulation of in planta loline distributions, providing greater protection of newly grown or embryonic tissues against attacks by insects. Surprisingly, exogenous application of the plant signaling compound, methyl jasmonate—which commonly signals predation by insects—decreases expression of the genes for the lolines. The factors that control loline production vary also among endophyte-infected grass tissues: whereas plant-supplied amino acids that are loline precursors limit accumulation of lolines in many grass tissues, their production in tissues that bear external mycelial growth for fungal reproduction (stromata) is regulated by the expression of loline genes.

Biosynthesis
The lolines are structurally similar to pyrrolizidine alkaloids produced by many plants, notably the necine ring containing a tertiary amine. This led to the early hypothesis that the biosynthesis of the lolines is similar to that of the plant pyrrolizidines, which are synthesized from polyamines. However, feeding studies with carbon isotope–labeled amino acids or related molecules in pure cultures of the loline-producing fungus N. uncinatum recently demonstrated that the loline alkaloid pathway is fundamentally different from that of the plant pyrrolizidines. The basic loline chemical structure is assembled in several biosynthetic steps from the amino acid precursors, L -proline and   L -homoserine. In the proposed first step in loline biosynthesis, these two amino acids are coupled in a condensation reaction linking the γ-carbon in homoserine to the secondary amine in proline in a PLP–type enzyme–catalyzed reaction to form the loline intermediate, N-(3-amino-3-carboxy)propylproline (NACPP). Further steps in loline biosynthesis are thought to proceed with sequential PLP-enzyme-catalyzed and oxidative decarboxylations of the carboxy groups in the homoserine and proline moieties, respectively, cyclization to form the core loline ring structure, and oxidation of the C-2 and C-7 carbons to give the oxygen bridge spanning the two pyrrolizidine rings.

Genetic studies agree with the biosynthetic routes established in the precursor-feeding experiments. AFLP-based studies using crosses between strains of the endophyte, Epichloë festucae, that differ in the capacity to produce lolines, show that loline production and protection of the grass, Lolium giganteum, from feeding by the aphid, Rhopalosiphum padi, segregate in a Mendelian fashion. The presence of a single locus for loline production was later confirmed by the finding that loline-producing epichloae endophytes contain a gene cluster (LOL cluster) of at least eleven genes. The LOL genes are greatly and coordinately upregulated during loline alkaloid production, and experimental genetic tests involving manipulation of selected LOL genes by RNA interference and gene knockout have directly confirmed the involvement of two of the LOL genes in loline biosynthesis. These tests and similarities in the peptide sequences of the proteins encoded by these genes to known enzymes indicate that one gene, termed lolC, is likely required for the first step in loline biosynthesis (condensation of L -proline and L -homoserine for NACPP formation), and another gene, LolP —likely encoding a cytochrome P450 monooxygenase—, for oxygenation of one methyl group on the C-1 amine of N-methylloline, which gives the most abundant loline in many grass–endophyte symbionts, N-formylloline.