Endophyte-infected Perennial Ryegrass Research Articles

Phosphorus fertiliser is associated with reduced grass grub (Costelytra giveni) fitness in Epichloë endophyte-infected meadow fescue and perennial ryegrass.

Fertiliser applications are well-established tools in pasture-based agricultural landscapes. This study focuses on the impact of phosphorus (P) fertiliser on grass grub (Costelytra giveni), a major pasture pest. This research investigates the interplay between P, plant growth, and grass grub fitness in Epichloë endophyte-infected perennial ryegrass (Epichloë sp. LpTG-3 strain AR37) and meadow fescue infected with E. uncinata (strain MaxR; AR1017), alongside their endophyte-free counterparts. In a glasshouse trial, plants were grown in P-enriched soil with varying Olsen P levels (9, 18, 28 or 78 mg L-1), and grass grubs were introduced. Their survival and weight gain, and plant performance were measured. In a bioassay, grass grubs were placed in specimen vials with P-enriched soils (Olsen P levels 9, 18, 28 and 78 mg L-1) and provided with identical plant material to assess their diet consumption and weight gain. In the glasshouse trial, results highlighted a notable decrease in the survival of grass grub on plants infected with MaxR endophyte, but not with AR37, as well as increasing soil Olsen P levels in both plant species. While grass grub decreased plant performance at the low Olsen P level (9 mg L-1), this effect diminished with increasing P. Likewise, results from the bioassay showed a decrease in diet consumption with increasing soil Olsen P levels. In both trials increasing Olsen P levels correlated with diminished grass grub performance, revealing a nuanced relationship between soil fertility and pest dynamics. The study underscores the pivotal role of selected Epichloë endophyte-grass associations in mitigating grass grub damage across varying phosphorus levels. This study highlights the potential to integrate P applications for sustainable pest control against grass grub. Further field trials are required to validate these findings. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sub-acute feeding study of a tall fescue endophyte in a perennial ryegrass host using mice

Epichloë endophytes in grass associations express a myriad of secondary metabolites which can affect the health of grazing animals and reduce the impact of insect pests on pasture. The ideal endophyte-grass association must have a favourable chemical profile such that the impact on animal health is minimised while the beneficial, deterrent effect on insect pests is maximised. A number of endophyte-perennial ryegrass associations have been successfully commercialised but research is on-going to further improve production in farming systems. Secondary metabolites expressed by endophyte-infected tall fescue include lolines, an animal-safe class of compound which imparts a potent effect on insects. Since endophyte-infected perennial ryegrass does not express lolines, a tall fescue endophyte, AR501, was inoculated into perennial ryegrass in an attempt to improve the insect resistance of this pasture type. In addition to animal safety, it is imperative that consideration is given to the safety of humans consuming animal products derived from livestock grazing the novel pasture. Although pure loline alkaloids have previously been tested on mice it is essential that the entire AR501 endophyte-infected perennial ryegrass matrix is tested since this will result in the exposure of both known and unknown secondary metabolites to mice. Three treatment groups each containing 6 male and 6 female mice were fed diets containing AR501 endophyte-infected perennial ryegrass seed (30%), perennial ryegrass seed containing no endophyte (30%) or a diet without seed (control) for 3 weeks. Mice fed control diet ate more than either of the treatment groups fed a diet containing seed. Male mice fed diet containing Nil endophyte seed ate more than those eating AR501 endophyte-infected perennial ryegrass seed although there was no difference observed in the food intake of female mice. While a few statistically significant differences were observed in the haematology and serum biochemical data, in every instance the difference was restricted to only one gender so is considered unlikely to be of toxicological significance. Mice fed AR501 endophyte-infected perennial ryegrass seed remained healthy throughout the experimental period despite consuming 62,000 mg/kg lolines and 4600 mg/kg peramine per day as well as the wide array of other unknown secondary metabolites expressed by this endophyte. Although animal products may contain additional metabolites as a result of animal metabolism, this experiment raises no food safety concerns for AR501 endophyte-infected perennial ryegrass.

Determination of a sub-chronic threshold for lolitrem B and perennial ryegrass toxicosis in Angus cattle consuming endophyte-infected perennial ryegrass (Lolium perenne) straw over 64 days

Lolitrem B is a potent tremorgen responsible for the syndrome known as “ryegrass staggers” in livestock consuming perennial ryegrass (Lolium perenne) infected with the endophyte fungus Epichloë festucae var. lolii. We evaluated clinical signs in cattle continuously fed endophyte-infected perennial ryegrass straw at four concentrations of lolitrem B (LB)/ergovaline (EV) (groups: I-2256/638, II-1554/373, III-1012/259 or IV-<300/0 µg/kg LB/EV, n=5-6 steers/group) over 64 days to establish a sub-chronic threshold of toxicity for this mycotoxin; animals were supplemented with 1 kg grain mix/day. Most animals in groups I and II developed clinical signs consistent with ryegrass staggers by day 64; thus, it is recommended that a threshold level of 1554 µg/kg LB should not be exceeded for extended feeding regimens using endophyte-infected perennial ryegrass feed material alone. This toxicity threshold is lower than that observed with shorter feeding periods, i.e. 1800–2000 µg/kg LB when fed for less than 30 days. No clinical signs associated with ergovaline toxicosis (i.e. decreased weekly weight gain) were observed. As adipose tissue appears to be the main compartment where LB is deposited, the concentration of LB and its metabolic derivatives in fat samples was determined at 37 and 64 days. Lolitrem B and three previously defined metabolites bioaccumulated in a dose- and time-dependent manner throughout the feeding experiment in fat tissue, at levels similar to those found in past research. Ratios of metabolites to LB were consistent across time points and dose groups, indicating that the enzyme(s) responsible for LB metabolism were not saturated. In summary, LB toxicity thresholds are dependent upon specificities of the dietary program including the duration feed material is fed, and should be considered when formulating safe livestock feeding regimens.

Gene analysis reveals that leaf litter from Epichloë endophyte-infected perennial ryegrass alters diversity and abundance of soil microbes involved in nitrification and denitrification

Although Epichloë endophytes are present only in aboveground tissues of grasses they indirectly influence soil biological characteristics through increased litter incorporation and root exudation. Epichloë endophytes have been reported to affect the decomposition rates of litter by altering litter quality and microbial decomposers to affect soil characteristics. However, it is not well-studied that underlying effects on functional genes involved in nitrification and denitrification after incorporation of litter containing Epichloë endophytes into soil. We collected soil samples at S0 (first litter incorporation), S1 (second litter incorporation), S2 (third litter incorporation) and S3 (120 days after the third litter incorporation) times after incorporation of litter that was either infected with the fungal endophyte Epichloë festucae var. Lolii or free of Epichloë endophyte, respectively. . We sampled the soil fororganic carbon (OC), total nitrogen (TN), total phosphorus (TP), ammonium nitrogen (NH4+-N: AN), nitrate nitrogen (NO3−-N: NN), soil microbial biomass carbon (MBC), nitrogen (MBN), ammonia-oxidizing bacteria (AOB-amoA) and ammonia-oxidizing archaea (AOA-amoA) functional genes for nitrification, nitrite reduction (nirS and nirK) and nitrous oxide reduction (nosZ) functional genes for denitrification. We found that Epichloë-infected plant increased the soil properties (OC, TN, AN, NN) and microbial biomass C in S1 to S3 times, and microbial biomass N in S2 and S3 times, but reduced soil pH in S1 times and the C_N ratio in S2 and S3 times by litter incorporation. The absolute abundance of the AOB-amoA functional gene at S1 and S3 times and relative abundances of Planctomycetes, Methylobacterium, Nitosomonas, Gemmata, Geodermatophilus and Sinorhizobium in the nirK functional gene community were significantly higher in soil with Epichloë-infected litter compared to soil with Epichloë-free litter at S1 and S3 times. While the absolute abundance at S1 and S3 times and diversity at S3 times of the nirK functional gene, and the absolute abundance at S1 to S3 times and diversity at S1 times of the nosZ functional gene, and relative abundance of Proteobacteria in the AOB-amoA functional gene were significantly lower in soil with litter containing Epichloë endophyte than that of soil with litter without the endophyte. The soil moisture, and the contents of TP, OC, AN and MBN were the best explanatory variables for the altered microbial community. Our results provide a new understanding of the responses of soil nitrification and denitrification genes through incorporation of litter containing Epichloë endophytes into soil.

Correction: Fungal Alkaloid Occurrence in Endophyte-Infected Perennial Ryegrass during Seedling Establishment.

The original version of this article unfortunately contained a mistake. There is a mistake in the affiliation section and Figure 6. The correct affiliation and Figure 6 are shown here.

Fungal Alkaloid Occurrence in Endophyte-Infected Perennial Ryegrass during Seedling Establishment.

The symbiotic Epichloë festucae var. lolii endophyte produces alkaloids which can provide its host grass, perennial ryegrass (Lolium perenne L), with a selective advantage in both natural and agricultural managed ecosystems. This study focuses on understanding the alkaloid concentrations that occur in endophyte-infected perennial ryegrass during the early establishment phase. In a glasshouse experiment fungal alkaloid concentrations (peramine, lolitrem B, ergovaline, and epoxy-janthitrems) were measured in perennial ryegrass seedlings infected with E. festucae var. lolii proprietary strains AR1, AR37, NEA2, and NZ common toxic for 69days after sowing. The endophyte becomes metabolically active, starting alkaloid production, as early as 6days after sowing. Alkaloid concentrations peaked in 8- to 10- day-old seedlings due to a seedling growth slowdown. This study provides data showing that the loss of insect protection in endophyte-infected seedlings is linked to a reduction in chemical defence after seed-stored, maternally synthesised alkaloids are diluted by seedling dry matter accumulation.

A Simple LC-MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass.

The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë-infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography–mass spectrometry (LC–MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC–MS (for peramine). This work provides the first highly sensitive quantitative LC–MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.

Perennial Ryegrass Alkaloids Increase Respiration Rate and Decrease Plasma Prolactin in Merino Sheep under Both Thermoneutral and Mild Heat Conditions.

A study was undertaken to determine the effects of feeding two levels of perennial ryegrass alkaloids (nil vs. moderate) under two climatic conditions. Alkaloids were fed via endophyte-infected perennial ryegrass seed and hay. Twenty-four Merino ewe weaners (six months, initial BW 32 ± 1.7 kg) were used in a study that lasted for 21 days after 14 days of adaptation. Sheep were fed either a control or alkaloid (Alk, 110 μg/kg LW ergovaline and 75 μg/kg LW lolitrem B) supplemented diet. Sheep were exposed to either constant thermoneutral (TN, 21–22 °C, 49% RH) or mildly heated (HS, 33 °C 1000–1500 h, 28% relative humidity) conditions. Dietary Alk and HS reduced dry matter intake (DMI) (p < 0.001, p = 0.02, respectively) with the combination of both reducing DMI by 42%. Reductions in DMI resulted in a lower daily gain in the Alk treatment (p < 0.001). Feed digestibility was reduced in the combined treatment (p = 0.03). Rectal temperature, respiration rate, and skin temperature increased in the Alk treatment. Plasma prolactin concentrations were decreased by Alk and increased by mild HS. The data indicate that production is compromised in the presence of Alk and mild HS, with this effect being exacerbated by a combination of both.

Toxic Indole Diterpenes from Endophyte-Infected Perennial Ryegrass Lolium perenne L.: Isolation and Stability.

The most potent of the indole diterpenes, lolitrem B, is found in perennial ryegrass (Lolium perenne L.) infected with the endophyte Epichloë festucae var. lolii (also termed LpTG-1). Ingestion causes a neurological syndrome in grazing livestock called ryegrass staggers disease. To enable the rapid development of new forage varieties, the toxicity of lolitrem B and its biosynthetic intermediates needs to be established. However, most of these indole diterpenes are not commercially available; thus, isolation of these compounds is paramount. A concentrated endophyte-infected perennial ryegrass seed extract was subjected to silica flash chromatography followed by preparative HPLC and purification by crystallization resulting in lolitrem B and the intermediate compounds lolitrem E, paspaline and terpendole B. The four-step isolation and purification method resulted in a 25% yield of lolitrem B. After isolation, lolitrem B readily degraded to its biosynthetic intermediate, lolitriol. We also found that lolitrem B can readily degrade depending on the solvent and storage conditions. The facile method which takes into consideration the associated instability of lolitrem B, led to the purification of indole diterpenes in quantities sufficient for use as analytical standards for identification in pastures, and/or for toxicity testing in pasture development programs.

Plant age and seasonal timing determine endophyte growth and alkaloid biosynthesis

Abstract The systemic grass endophyte Epichloe festucae var. lolii produces alkaloids which can protect the host grass Lolium perenne from herbivory. Alkaloid concentrations depend on genetic predisposition of grass and endophyte, and are affected by the environment. However, the role of plant age and seasonal timing remains unknown. We monitored monthly endophyte and alkaloid concentrations in endophyte infected perennial ryegrass over 29 months in a common garden experiment in Germany. Climatic conditions in spring and summer enhanced endophyte growth and alkaloid production, explaining a dominant role of seasonal timing. Alkaloid concentrations also increased with plant age and exceeded the toxic threshold for invertebrates in the first summer and for livestock in the third summer. Our results highlight the key role of plant age and seasonal timing in affecting the toxicity of systemic fungal endophytes of grasses. Endophyte mediated livestock intoxications may increase on European grasslands with global warming.

Effect of moisture deficit on four perennial ryegrass cultivars

The effect of moisture deficit on herbage production, root growth, tiller density, leaf appearance, percentage dry matter, and water-soluble carbohydrate (WSC) concentration and content in the stubble of Epichloë endophyte-infected perennial ryegrass plants (cultivars Aberdart AR1, Bealey NEA2, One50 AR37, Trojan NEA2) was investigated in a glasshouse study. Water was withheld from half the plants for 6 weeks, followed by a 4-week recovery period, when all plants were watered. At the end of the treatment period, there were differences among cultivars in tiller density, moisture content of the herbage, WSC content of stubble, and root mass. However, there was little effect of moisture deficit on these variables or on root depth. At the end of the recovery period, previously moisture-stressed plants had increased leaf appearance by 25%, leafy-herbage mass by 29%, and low molecular-weight WSC content of stubble by 37% compared with well watered plants, but there was a decrease in root mass in some cultivars. There were differences among cultivars in tiller density, the moisture content of the herbage and stubble WSC content. The effects of previous moisture deficit were greatest during the recovery period, and differed among cultivars, for stubble, root and total plant biomass. Results suggested that avoiding overgrazing immediately after drought is just as critical for recovery as is management during a drought, given that the greatest responses to previous moisture deficit were detected during this period.

Lolitrem B and Indole Diterpene Alkaloids Produced by Endophytic Fungi of the Genus Epichloë and Their Toxic Effects in Livestock.

Different group of alkaloids are produced during the symbiotic development of fungal endophytes of the genus Epichloë in grass. The structure and toxicity of the compounds vary considerably in mammalian herbivores and in crop pests. Alkaloids of the indole-diterpene group, of which lolitrem B is the most toxic, were first characterized in endophyte-infected perennial ryegrass, and are responsible for “ryegrass staggers.” Ergot alkaloids, of which ergovaline is the most abundant ergopeptide alkaloid produced, are also found in ryegrass, but generally at a lower rate than lolitrem B. Other alkaloids such as lolines and peramine are toxic for crop pests but have weak toxicological properties in mammals. The purpose of this review is to present indole-diterpene alkaloids produced in endophyte infected ryegrass from the first characterization of ryegrass staggers to the determination of the toxicokinetics of lolitrem B and of their mechanism of action in mammals, focusing on the different factors that could explain the worldwide distribution of the disease. Other indole diterpene alkaloids than lolitrem B that can be found in Epichloë infected ryegrass, and their tremorgenic properties, are presented in the last section of this review.

Recent investigations of ergot alkaloids incorporated into plant and/or animal systems.

Recent investigations of ergot alkaloids incorporated into plant and/or animal systems.

Bovine hepatic differential gene expression in response to perennial ryegrass staggers

&gt;‘Perennial ryegrass staggers’ is a neurological condition characterised by muscular tremors and ataxia in livestock that ingest the indole diterpene lolitrem B from endophyte-infected perennial ryegrass. While the neurotoxic mechanism of action of ryegrass staggers has been defined, little is known about metabolic and other molecular processes that lolitrem B may affect in livestock. The objective of this study was to characterise differential gene expression in the liver of animals fed lolitrem B-containing feed over an extended period of time. Eighteen steers were fed one of three rations (n=6/group) containing lolitrem B (247, 1,554 or 2,256 μg/kg (dry matter basis)) over 64 days in a double-blind study. Microarray and confirmatory qPCR were performed to evaluate the hepatic gene expression profile from biopsies taken on days 0 and 64. Widespread perturbation of gene expression was observed in all groups receiving lolitrem B, with a total of 152 differential genes identified (false discovery rate ≤0.05). This suggests that chronic exposure to lolitrem B, even at levels below the current threshold of toxicity (2,000 μg/kg lolitrem B), can perturb many genes, biological processes and pathways. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that many of these genes were categorised under lipid/steroid biosynthesis/metabolism and oxidation-reduction. Specifically, genes involved in the biosynthesis pathway of ceramide, a sphingolipid molecule (ACSS2,LASS6 andSCD) and changes in neurosignaling through alteration of nitric oxide synthase activity (ARG1 andGPX4) were up-regulated. Future work should focus on the overall balance between ceramide and its metabolites and antioxidants/oxidants in a variety of body matrices in animals with perennial ryegrass staggers, to determine how these compounds contribute to the overall etiology of this disease.

Ergovaline and lolitrem B concentrations in perennial ryegrass in field culture in southern France: distribution in the plant and impact of climatic factors.

Perennial ryegrass (Lolium perenne) infected by Epichloë festucae var. lolii contains alkaloids that are responsible for toxicosis in several countries, but few cases are reported in Europe. Lolitrem B is generally the most abundant alkaloid and is recognized to be responsible for livestock staggers, whereas ergovaline is less frequently documented in perennial ryegrass. Lolitrem B and ergovaline were monitored over a three-year period in endophyte-infected perennial ryegrass 'Samson' sown in southern France. Alkaloid concentrations were strongly influenced by the stage of maturity of the plant; maximum concentrations were always measured at the fully ripe stage. Over the three years of analysis, variations in lolitrem B in the whole plant at the fully ripe stage were low (from 1296 to maximum 1871 μg/kg dry matter), whereas ergovaline varied considerably (from 526 to 2322 μg/kg dry matter), suggesting that abiotic factors play a key role in determining ergovaline levels in endophyte-infected perennial ryegrass.

Physiological effects of endophyte-infected perennial ryegrass straw on female camels in the Middle East

Recently, the United Arab Emirates has increased importation of perennial ryegrass (Lolium perenne) straw as part of the grass/hay ration for camels. Unfortunately, perennial ryegrass straw may be infected with the endophyte Neotyphodium lolii which produces lolitrem B, a toxic alkaloid responsible for the disease ‘ryegrass staggers.’ A range-finding study was conducted using 24 non-pregnant female camels fed four doses (0, 1111, 1478 and 2273 ppb) of lolitrem B-containing perennial ryegrass straw over 56 days to establish a threshold of toxicity in camels so that perennial ryegrass straw can be safely fed as part of their dietary ration. Physiological parameters were evaluated. Neurological deficits were evaluated by videotape and scored according to an established scale for the neurological syndrome ryegrass staggers. Camels in the endophyte-infected groups developed varying degrees of ataxia. Brain edema, degenerative renal and hepatic lesions as well as Purkinje cell vacuolar degeneration were observed in camels receiving the highest dose. To avoid clinical disease in camels, endophyte-infected perennial ryegrass straw should be fed at 500 ppb or lower lolitrem B; straw at 1000 ppb or higher should be diluted to achieve the 500 ppb threshold of toxicity.

Toxicological evaluation and bioaccumulation potential of lolitrem B, endophyte mycotoxin in Japanese black steers

Lolitrem B, a causative toxin for ryegrass staggers, is produced by Neotyphodium lolii infecting perennial ryegrass (Lolium perenne). Japanese black cattle have been suspected to be more sensitive to lolitrem B than to other strains, and there has been a concern about the public health hazard of eating beef contaminated with lolitrem B. We carried out a feeding experiment to examine the sensitivity of Japanese black cattle to lolitrem B and the residual level of lolitrem B in several animal tissues. Japanese black steers were fed a 0, 500, 750, 1000, 1500 or 2000 µg kg−1 diet of lolitrem B provided by endophyte-infected perennial ryegrass straw for 12 weeks. All six animals in the 2000 µg kg−1 diet group exhibited ryegrass staggers symptoms. Furthermore, two out of three animals in the 1500 µg kg−1 diet group, three out of six animals in the 1000 µg kg−1 diet group and one out of three animals in the 750 µg kg−1 diet group presented clinical signs of ryegrass staggers. These results suggest that a daily intake of 18 µg kg−1 body weight of lolitrem B can produce ryegrass staggers in Japanese black steers. Perirenal fat tissues of the steers from those groups having one or more animals exhibiting ryegrass staggers symptoms contained approximately 150 ng g−1 of lolitrem B, while only small amounts of lolitrem B were detected in muscle, liver and kidney. Because the residual amount of lolitrem B in tissues of Japanese black cattle is small, the exposure to lolitrem B in consumers of the beef is likely to be low.

FORAGES AND PASTURES SYMPOSIUM: Fungal endophytes of tall fescue and perennial ryegrass: Pasture friend or foe?12

Tall fescue [Lolium arundinaceum (Schreb.) Darbysh. syn. Festuca arundinacea Schreb.] and perennial ryegrass (Lolium perenne L.) are important perennial forage grasses utilized throughout the moderate- to high-rainfall temperate zones of the world. These grasses have coevolved with symbiotic fungal endophytes (Epichloë/Neotyphodium spp.) that can impart bioactive properties and environmental stress tolerance to the grass compared with endophyte-free individuals. These endophytes have proven to be very important in pastoral agriculture in the United States, New Zealand, and Australia, where forage grasses are the principal feed for grazing ruminants. In this review, we describe the biology of these grass-endophyte associations and implications for the livestock industries that are dependent on these forages. Endophyte alkaloid production is put in context with endophyte diversity, and we illustrate how this has facilitated utilization of grasses infected with different endophyte strains that reduce livestock toxicity issues. Utilization of tall fescue and use of perennial ryegrass in the United States, New Zealand, and Australia are compared, and management strategies focused predominantly on the success of endophyte-infected perennial ryegrass in New Zealand and Australia are discussed. In addition, we consider the impact of grass-endophyte associations on the sustainability of pasture ecosystems and their likely response to future changes in climate.

The evaluation of fungal endophyte toxin residues in milk

AIM: To determine the concentrations of fungal endophyte toxins in the milk of cows fed perennial ryegrass containing wild-type or AR37 endophyte. METHODS: Groups of 10 multiparous Holstein-Friesian cows were fed wild-type (containing lolitrem B) or AR37 (containing epoxy-janthitrems) endophyte-infected perennial ryegrass (Lolium perenneL.). Animals were kept indoors and fed for 12 days. Over this period, animals were regularly assessed for ryegrass staggers and herbage intake measured. At the conclusion of the 12-day indoor-feeding period, cows were grazed on AR1 (toxin-free) pastures for a further 8 days. Daily individual milk samples and milk yields were collected over the complete 20-day period. Milk samples were analysed for endophyte toxins using HPLC methods developed during this study. Daily herbage samples were also taken and concentrations of endophyte toxins measured. RESULTS: Methods were successfully developed for the analysis of lolitrem B and epoxy-janthitrems in milk which allowed the concentrations of these compounds in milk to be compared with the concentrations in feed consumed by the animals. Both toxin types could be detected in milk after only 1 day of exposure to respective treatment pastures. The maximum concentration of endophyte toxins in milk was 5 ng/mL lolitrem B and 109 ng/mL epoxy-janthitrems from cows fed wild-type and AR37 endophyte-infected ryegrass pastures, respectively. Concentrations of epoxy-janthitrems present in herbage were much higher than for lolitrem B (Day 1–12 average of 14.6 and 1.8 ppm, respectively). Despite the high concentrations of epoxy-janthitrems consumed by cows fed AR37 endophyte-infected pastures no signs of ryegrass staggers were observed over the experimental period, whereas those cows fed wild-type endophyte-infected pastures all showed signs of ryegrass staggers. This is consistent with the view that epoxy-janthitrems are low potency tremorgens. At the conclusion of the toxin feeding period, endophyte toxin concentrations in milk quickly dropped to almost zero after 8 days. A comparison of the quantities of lolitrem B and epoxy-janthitrems consumed by each cow with the quantities secreted in milk showed that only very low proportions of the total amount ingested are secreted in milk (0.23% lolitrem B and 0.49% epoxy-janthitrems). CONCLUSION: Lolitrem B and epoxy-janthitrems can be detected in the milk of cows consuming wild-type and AR37 endophyte-infected perennial ryegrass, respectively. Concentrations detected were low and changed quickly in association with the amounts being consumed by the cows. Available evidence gives no indication that these compounds may pose a threat to human health.

The evaluation of endophyte toxin residues in sheep fat

AIM: To monitor changes in concentrations of lolitrem B and epoxy-janthitrems in the fat of sheep grazing perennial ryegrass infected with wild-type- and AR37-endophyte, respectively, during the time of year when ryegrass staggers would be expected to be observed. METHODS: Ten 5-month-old lambs with no previous exposure to endophytes were grazed on either wild-type (containing lolitrem B, n = 5) or AR37 (containing epoxy-janthitrems, n = 5) endophyte-infected perennial ryegrass (Lolium perenne L.) pastures between October 2008 and June 2009. Animals were regularly assessed for ryegrass staggers using the Keogh scale (0 = no signs, 5 = severe tremors). When a score of > 3.5 was observed animals were removed from the treatment pastures for 1 month. Fat biopsy samples were taken from each animal at approximately monthly intervals and analysed for endophyte metabolites using high performance liquid chromatography (HPLC) methods developed during this study. Regular herbage samples were also taken and concentrations of endophyte metabolites measured. RESULTS: Efficient and reproducible methods to analyse both lolitrem B and epoxy-janthitrems in fat were developed. Concentrations of lolitrem B and epoxy-janthitrems in herbage and in sheep fat increased from late November to peak in mid-February. Ryegrass staggers was observed in both groups of sheep at this time. Following 1 month of grazing non-infected pasture mean concentrations in fat of lolitrem B decreased by 43% from 61.8 to 35.3 ppb, and of epoxy-janthitrems by 38% from 1032.0 to 639.5 ppb. Maximum concentrations in herbage of epoxy-janthitrems (35.7 ppm) were higher than of lolitrem B (3.4 ppm), but signs of staggers were less severe in sheep grazing pasture containing the former compared with the latter (median Keogh scores in late February were 2 and 3, respectively), consistent with epoxy-janthitrems being low potency toxins. CONCLUSION: This study demonstrated that concentrations of epoxy-janthitrems and lolitrem B in sheep fat increased quickly during the initial phase of the study when concentrations in pasture increased, and decreased when animals were removed from pastures containing these compounds. These data will be used in the risk assessment of the endophyte metabolites.