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Mammalian toxins produced by the wild-type endophyte, Neotyphodium lolii, in perennial ryegrass (PRG) pasture cause production losses and animal health and welfare problems in livestock. Managing this risk is limited by the lack of information on fluctuations in the concentration of toxin in Australian pasture. We investigated how the toxin concentrations may be related to recent observations of weather. Swards sown from common seedlots of two cultivars of wild endophyte-infected PRG, grazed short by sheep, were sampled at 2–4 weekly intervals from spring to autumn at two sites in Victoria. The highest concentration of ergovaline and lolitrem B was observed at Hamilton, the site with the longer-growing season. The concentration of ergovaline peaked in early summer, coinciding with seed development, and declined through summer, before increasing with the commencement of autumn growth. The concentration of lolitrem B remained low in summer, then rose in autumn. Variation between the two cultivars in the concentration of toxins was small and rarely significant. The concentration of ergovaline declined as the mean daily maximum temperature over the preceding 1–5 days increased. Similarly, for lolitrem B, the concentration declined over the temperature range 12−20°C, from 1.3 to 0.3 mg/kg. At Hamilton, where solar radiation and soil temperature were recorded, both were superior to maximum temperature for predicting lolitrem B. Serial sampling of PRG from old naturalised pasture on seven farms across south-eastern Australia found two seasonal peaks for both alkaloids in most pastures. The concentration of ergovaline reached or exceeded tolerance levels for livestock in 23 of 43 samples, compared with 5 of 43 for lolitrem B. Ergovaline concentrations initially peaked (at 1.0–1.6 mg/kg) when mature reproductive material was present (coinciding with peduncle elongation and seed development). In pastures with low grazing pressure, i.e. where growth was allowed to continue through summer, ergovaline concentration was relatively low (<0.7 mg/kg) but in a hard-grazed pasture (sward height 3 cm), the ergovaline concentration was greater (up to 1.1 mg/kg). Concentration of lolitrem B also peaked in December, except on pasture where growth continued through summer. High concentrations of lolitrem B associated with neurotoxic signs in sheep (viz. 2.4–3.9 mg/kg) were observed only in mid-summer and autumn, and only if conditions favoured growth or where close grazing by sheep left the crown as the dominant source of herbage.

Severe outbreaks of toxicosis caused by the natural endophyte Neotyphodium lolii in perennial ryegrass (Lolium perenne L.) have occasionally resulted in heavy loss of livestock in Victoria and Tasmania. Meteorological records were examined from locations where acute toxicosis was recorded. High rainfall (>350 mm) in spring–summer was an obvious common feature; typically, it prolonged the period of high growth rate of pasture, increasing predicted spring–summer (September–March) growth (by ~36%) and increasing the predicted digestibility of organic matter of summer pasture (1 February) by 2.7% units (up to 55.6%). In years when severe toxicosis occurred, such rainfall was accompanied by dry, warm conditions in March and April, viz. mean maximum daily temperatures on the mainland of ≥21°C (March) and ≥18°C (April). During summer–autumn 2002, pastures from 10 Victorian farms on which acute perennial ryegrass toxicosis resulted in the loss of >7000 sheep/deer, were found to be dominated by perennial ryegrass. The ryegrass endophyte-produced toxins, ergovaline and lolitrem B, were detected in 74% and 100%, respectively, of composite faecal samples collected from acutely affected animals. The populations of perennial ryegrass all tested positive for Neotyphodium lolii endophyte, with a mean infection frequency of 90.5% (s.e.m. 1.81). The mean mycelial mass in basal tillers of perennial ryegrass was estimated at 66.00 (s.e.m. 3.40) from a sample of 20 plants, each of which was assessed on a scale of 1–5. Mycelial mass accounted for 19–20% of the variation in the concentration of ergovaline and lolitrem B in perennial ryegrass. Toxin concentrations in the plants where acute toxicosis was observed exceeded the tolerance levels for sheep (ergovaline 0.8 mg/kg; lolitrem B 1.8 mg/kg) in the minor (green) fraction of the plant in all but one affected pasture for ergovaline and all but two for lolitrem B. Maximum concentrations recorded were 4.3 and 4.6 mg/kg for ergovaline and lolitrem B respectively. Ergovaline increased in an asymptotic relationship with lolitrem B, which accounted for 45% of the variation in ergovaline. The concentrations of ergovaline and lolitrem B in the whole plant (green and dead fractions combined) were not greater than those recorded from randomly sampled pastures in earlier seasons, when only occasional mild cases of toxicosis were reported. Further examination of stored grass samples collected during the 2002 outbreak recently revealed unidentified peaks on the chromatograms for both ergovaline and lolitrem B; peaks that are not seen on chromatograms for toxic perennial ryegrass from New Zealand, where the expression of perennial ryegrass toxicosis is usually milder and rarely fatal.

Perennial ryegrass (PRG) was analysed for alkaloids associated with the expression of perennial ryegrass endophyte toxicosis (PRGT) in south-east Australia. Over two seasons, the PRG cultivar Samson (‘high endophyte’, viz. naturally infected with a wild-type strain of Epichloë festucae var. lolii) was sampled on five occasions during November to May at four farms in Victoria and at Lincoln, New Zealand. Endophyte frequency in the populations was 77–100%. PRG was also sampled from 20 Victorian and Tasmanian farm pastures where stock were experiencing PRGT (endophyte infection frequencies of 87–100%). The Victorian summer of 2010–11 was atypically moist; pasture remained green. Lolitrem B was consistently high at Lincoln and 2–3 times that observed in Victorian samples of isogenetic PRG, or in PRG causing PRGT; it was the dominant toxin in 2011 with concentrations commonly exceeding the tolerance level of 1.8 mg/kg. In the following year, one with a more typical summer, ergovaline was the dominant toxin. Liquid Chromatography–Mass Spectrometry/Mass Spectrometry (LC-MS/MS) was carried out to determine indole diterpene intermediates in the lolitrem B biosynthesis pathway and for ergot alkaloid intermediates in the ergovaline pathway. The values for lolitrem B determined by LC-MS/MS correlated strongly with those obtained using high pressure liquid chromatography. In both Years 1 and 2, significantly higher expression was observed in the Lincoln relative to Victorian samples of PRG for paspaline, terpendole C, lolitrem E, lolitrem B and lolitrem F. For the ergot alkaloids, significant differences were not apparent between Victorian and Lincoln samples in Year 1. In Year 2, LC-MS/MS results showed ergovaline concentrations were greater in Victorian samples. In addition to endophyte-produced toxins, ergot alkaloids produced by Claviceps purpurea (ergotamine, ergocryptine and ergocornine) were detected in grass samples on 6/27 occasions. Some unidentified metabolites were noted in both Victorian and Lincoln samples. The effects of ingested vaso-constrictive ergot alkaloids combined with that of high solar radiation on ruminants’ heat load are considered most important with respect to the occasionally severe expression of PRGT in Australia.

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