Aims To assess animal health and production in sheep grazing perennial ryegrass (Lolium perenne) infected with a tall fescue endophyte (Epichloe sp.). Methods Three replicates of pure perennial ryegrass pastures infected with AR501 tall fescue endophyte (AR501 HE), AR1, AR37 or standard endophyte (STD) (all >85% infection) and a low endophyte control (AR501 LE) were grazed by 10 lambs for 7 weeks during late summer/early autumn, 2017. AR501 HE and AR501 LE were in the same tetraploid cultivar and the remaining treatments were in the same diploid cultivar. Lambs were weighed on Days 0, 19, 37 and 48 and assessed for ryegrass staggers on a 0-4 tremor scale nine times from Day 13. On Days 14 and 20, rectal temperatures and respiration rates were measured in lambs grazing AR501 HE, AR501 LE and STD under heat load. Pasture samples from each treatment were taken regularly (8 times) for analysis of known endophyte secondary metabolites. Results Peak mean ryegrass staggers scores for lambs grazing STD and AR37 treatments were 3.9 and 2.7, respectively, indicating environmental conditions were conducive to toxicity. Lambs grazing AR501 HE showed no ryegrass staggers at any date. The liveweight of STD lambs was up to 6.6 kg less than some or all other treatments on Days 19, 34 and 48, but there was no evidence of a difference between the liveweight of AR501 HE lambs and AR501 LE, AR37 or AR1 lambs on any date. Rectal temperatures and respiration rates of STD lambs were higher than AR501 HE lambs by 0.2 degrees C and 26 breaths/minute, respectively. Apart from lower rectal temperature of AR501 HE lambs on Day 14, there was no evidence of differences between the AR501 HE and AR501 LE lambs. N-Formylloline was the only loline present in AR501 HE herbage (350-860 mg/kg). Peramine in AR501 HE herbage (42-77 mg/kg) was greater than that in STD and AR1 herbage on five and seven of the eight sampling dates, respectively. Conclusion Lambs grazing AR501 HE showed no ryegrass staggers, exacerbated heat stress or suppressed liveweight gain compared with lambs grazing AR501 LE in a season when endophyte-induced toxicity was evident in STD and AR37 pastures. This suggests AR501 tall fescue endophyte does not produce toxic concentrations of secondary metabolites.

AIM:To investigate whether Mediterranean tall fescue (Lolium arundinaceum (Schreb.) Darbysh. (syn Festuca arundinacea)) infected with selected fungal endophytes (Epichloë coenophiala (formerly Neotyphodium coenophialum)) caused equine fescue oedema when grown in New Zealand, and to examine the pathological changes associated with this intoxication.METHODS:Horses were grazed on Mediterranean tall fescue that was infected with the endophytes AR542 (n=2), or AR584 (n=3), or Mediterranean tall fescue that was endophyte-free (n=2). Blood samples were taken up to 7 days after the start of feeding to detect changes in concentrations of total protein in serum and packed cell volume. Any horse showing clinical evidence of disease was subject to euthanasia and necropsy.RESULTS:Within 6 days, both horses grazing fescue infected with AR542 became depressed and lethargic. One horse grazing fescue infected with endophyte AR584 became depressed within a 5-day feeding period while another horse in this group died shortly after being removed from the AR584 pasture. The third horse in this group did not develop clinical signs within the 5-day feeding period. However, haemoconcentration and hypoproteinaemia was detected in all horses grazing Mediterranean tall fescue that was infected by AR542 or AR584 endophyte. No abnormalities were observed in horses grazing fescue that was endophyte-free. Necropsy examination was performed on two horses grazing fescue infected with AR542 and one horse grazing fescue infected with AR584. All three horses had marked oedema of the gastrointestinal tract. Histologically, the oedema was accompanied by large numbers of eosinophils, but no necrosis.CONCLUSIONS:Horses grazing Mediterranean tall fescue that was infected by AR542 or AR584 developed hypoproteinaemia and haemoconcentration, most likely due to leakage of plasma proteins into the gastrointestinal tract. This suggests that these selected endophytes produce a compound that is toxic to horses, although the toxic principle is currently unknown.CLINICAL RELEVANCE:Mediterranean tall fescue that is infected by AR542 or AR584 should not be fed to horses in New Zealand. This intoxication should be considered in horses in which a rapid onset of hypoproteinaemia and haemoconcentration is detected. This intoxication should also be considered if marked gastrointestinal oedema is observed.

AIM: To investigate a possible interaction between lolitrem B and ergovaline by comparing the incidence and severity of ryegrass staggers in sheep grazing ryegrass (Lolium perenne) containing lolitrem B or ryegrass containing both lolitrem B and ergovaline. METHODS: Ninety lambs, aged approximately 6 months, were grazed on plots of perennial ryegrass infected with either AR98 endophyte (containing lolitrem B), standard endophyte (containing lolitrem B and ergovaline) or no endophyte, for up to 42 days from 2 February 2010. Ten lambs were grazed on three replicate plots per cultivar. Herbage samples were collected for alkaloid analysis and lambs were scored for ryegrass staggers (scores from 0–5) weekly during the study. Any animal which was scored ≥4 was removed from the study. RESULTS: Concentrations of lolitrem B did not differ between AR98 and standard endophyte-infected pastures during the study period (p=0.26), and ergovaline was present only in standard endophyte pastures. Ryegrass staggers was observed in sheep grazing both the AR98 and standard endophyte plots, with median scores increasing in the third week of the study. Prior to the end of the 42-day grazing period, 22 and 17 animals were removed from the standard endophyte and AR98 plots, respectively, because their staggers scores were ≥4. The cumulative probability of lambs having scores ≥4 did not differ between animals grazing the two pasture types (p=0.41). CONCLUSIONS AND CLINICAL RELEVANCE: There was no evidence for ergovaline increasing the severity of ryegrass staggers induced by lolitrem B. In situations where the severity of ryegrass staggers appears to be greater than that predicted on the basis of concentrations of lolitrem B, the presence of other tremorgenic alkaloids should be investigated.

AIMS To determine if equine fescue oedema (EFO) induced by grazing Mediterranean-type tall fescue (Lolium arundinaceum) infected with selected endophytes (Epichloë coenophiala) could be prevented by treatment with the corticosteroid, methylprednisolone, and anti-histamine, cetirizine, and to determine concentrations of lolines, specifically N-acetyl norloline (NANL), in grasses grazed by horses that did and did not develop EFO. METHODS Four horses were grazed on AR542-infected Mediterranean tall fescue pasture (from Day 0) for 7 days prior to being subjected to euthanasia. Two of these horses were treated with 250 mg methylprednisolone and 300 mg cetirizine hydrochloride every 12 hours orally from Days 0-7. Two more horses grazed meadow fescue (Festuca pratensis) infected with the naturally-occurring, common endophyte (Epichloë uncinata) for 21 days before euthanasia. All horses were observed closely for signs of EFO, and blood samples were taken daily for measurement of concentrations of total protein (TP) in serum. Following euthanasia post-mortem examinations were conducted on all horses. Pasture samples of meadow fescue and Mediterranean tall fescue from the current study, and endophyte-infected Mediterranean tall fescue from a previous study that were associated with EFO, were analysed for concentrations of lolines using gas chromatography. RESULTS By Day 7, the treated and untreated horses grazing AR542-infected Mediterranean tall fescue all developed signs of EFO, and concentrations of TP in serum of all horses were <45 g/L. No signs of EFO were observed in horses grazing meadow fescue and concentrations of TP remained above 60 g/L. Necropsy showed marked oedema and eosinophilic inflammation in the intestines of all horses grazing Mediterranean tall fescue. In the sample of meadow fescue, concentrations of total lolines and N-acetyl norloline (NANL) were 2,402 and 543 mg/kg, respectively. In the three samples of Mediterranean tall fescue associated with EFO, concentrations of total lolines were 308, 629 and 679 mg/kg, and concentrations of NANL were 308, 614 and 305 mg/kg. CONCLUSIONS AND CLINICAL RELEVANCE In horses grazing Mediterranean tall fescue infected with the AR542 endophyte, treatment with methylprednisolone and cetirizine did not prevent development of EFO. Results of pasture analysis indicated that lolines or NANL are unlikely to be the causative agent of this disease.

AIMS: To compare ryegrass pastures infected with endophytes producing diverse alkaloids for their ability to cause ryegrass staggers in grazing lambs; to compare respiration rates and rectal temperatures of these lambs after exposure to heat stress, and to compare liveweight gains during the study period.METHODS: Ryegrass pastures of cultivar Trojan infected with NEA endophytes, branded NEA2 (T-NEA2), endophyte-free Trojan (T-NIL), Samson infected with standard endophyte (S-STD), Samson infected with AR37 endophyte (S-AR37) and endophyte-free Samson (S-NIL), were grazed by lambs (n=30 per cultivar) for up to 48 days in February and March of 2012 and 2013. Pasture samples were analysed for alkaloid concentrations and lambs were scored for ryegrass staggers at intervals during the study period. Liveweight was recorded at the start (Day 0) and end of the study, and rectal temperatures and respiratory rates were measured in lambs exposed to heat stress on Days 23 and 26, in 2012 and 2013, respectively.RESULTS: Concentrations of alkaloids were lower in 2012 than 2013, associated with warmer and drier conditions in 2013, and the prevalence of ryegrass staggers was low in 2012. In 2013, concentrations of ergovaline were similar in T-NEA2 and S-STD, but concentrations of lolitrem B were lower in T-NEA2 than S-STD. S-AR37 produced epoxy-janthitrems but no lolitrem B or ergovaline. In 2013, by Day 20, 9/30 (30%) sheep grazing S-STD had severe staggers (score 4), and by Day 47 all sheep had been removed from this cultivar due to severe staggers. By Day 47, 18/30 (60%), 4/30 (13%) and 0/30 (0%) sheep grazing S-AR37, T-NEA2 and T-NIL pastures, respectively, had severe staggers. There were no differences in mean daily weight gain of lambs between cultivars in either year. In both years, mean rectal temperature and respiration rate following exposure to heat stress were highest in sheep grazing S-STD and T-NEA2, and lowest in sheep grazing T-NIL.CONCLUSIONS: In lambs grazing different ryegrass pastures infected with endophytes, ryegrass staggers was most severe on S-STD, less severe on S-AR37 and least on T-NEA2. When under heat stress, lambs grazing ergovaline-producing S-STD and T-NEA2 pastures had increased respiration rates and rectal temperatures compared with lambs grazing T-NIL.CLINICAL RELEVANCE: If ambient temperatures are suitable, NEA2-branded endophytes have the potential to express concentrations of ergovaline sufficient to induce heat stress in grazing sheep.

AIM:To compare vasoconstriction of the auricular artery and characteristics of blood flow in the carotid arteries between lambs grazing perennial ryegrass that was either infected with the AR6 novel endophyte (AR6), wild-type endophyte or endophyte-free. METHODS:Sixteen Coopworth lambs, mean 23.7 (SD 1.8) kg, were randomly assigned to graze three, 0.10-ha pastures of perennial ryegrass (cultivar Extreme) located in Lincoln, New Zealand, that were infected with either the AR6 novel endophyte (n=5) or wild-type endophyte (n=6) or were endophyte-free (n=5), for 18 days until 16 March 2009. Lambs on AR6 pasture were then switched to endophyte-free pasture, and those on endophyte-free were switched to AR6 pasture, for 18 days. Lambs continued grazing the wild-type ryegrass during both phases of the study. Colour Doppler ultrasonography was used to monitor cross-sectional area of lumina in the auricular and carotid arteries as measurements of vasoconstriction, and to measure pulsatility indices, heart rate, systolic and diastolic velocities, and mean velocity in the carotid artery. Urine was sampled once during each phase, to measure the concentration of urinary alkaloids. A heat challenge (32°C) was imposed on the last day of the experiment, to determine treatment effects on rectal temperature and respiration rate. RESULTS:Vasoconstriction was detected in the auricular arteries of lambs grazing AR6 and wild-type pastures. Areas of lumina declined linearly over 9 days in lambs that were switched from endophyte-free to AR6 pastures (p<0.05), and areas increased linearly in lambs that were switched from AR6 to endophyte-free pasture (p<0.001). Resistance to blood flow in the carotid arteries decreased linearly in the 9 days after lambs were switched from AR6 to endophyte-free pasture(p<0.05), and tended to increase after lambs on endophyte-free pasture were switched to AR6 (p<0.10). The concentrations of urinary alkaloids decreased after switching lambs from AR6 to endophyte-free pasture, and increased after switching from endophyte-free to AR6 pasture (p<0.05). The concentrations of urinary alkaloids of lambs on wild-type pasture were similar between the conditioning and experimental phases. There were no treatment effects on rectal temperature and respiration rate during the heat challenge. CONCLUSIONS:These results indicated that management approaches are needed to overcome potential vulnerabilities to heat stress for sheep grazing AR6 perennial ryegrass. Furthermore, following grazing such pasture, lambs will need to graze endophyte-free ryegrass for >18 days, to completely clear ergot alkaloids from their vasculature, assuming that complete clearance can be achieved.

The major limitation to perennial ryegrass-based systems is low soil moisture which reduces perennial ryegrass growth. irrigation can maintain adequate soil moisture levels for pasture growth, however, the associated cost requires high levels of forage water use efficiency.

Over 10 years the health and production of sheep grazing ryegrass with AR37 fungal endophyte was compared to those grazing Standard and AR1 endophytes, and endophyte-free, under four different trial protocols, including short and long term grazing, cultivar effects and seasonal differences. Sheep developed some ryegrass staggers on AR37 treatments but it was less frequent and usually less severe than on Standard.

The discovery in 1981 that ryegrass endophyte is responsible for ryegrass staggers has required a total revision of our understanding of ryegrassbased pastures. The effects on grazing livestock range from obvious clinical disorders to chronic impairment of productivity. On the other hand, endophyte infection is essential to ryegrass persistence in most of New Zealand, protecting the plant from some invertebrate pests and from over-grazing, and perhaps promoting its tolerance of moisture stress. Vigorous, endophyte-infected pastures sometimes include less clover than pastures relatively free of endophyte. Active compounds produced by the endophyte have been identified, and conditions causing high levels of them have been documented in part. Endophyte strains which do not produce the compounds responsible for livestock toxicoses offer exciting new possibilities in grassland agriculture. Keywords: invertebrate pests, mycotoxicoses, Neotyphodium lolii, perennial ryegrass, ryegrass staggers

The health and production of sheep grazing perennial ryegrass with and without wild-type endophyte (Neotyphodium lolii) has been studied in several trials over a number of years. Lambs/ hoggets grazing predominantly perennial ryegrass swards with endophyte developed moderate to severe ryegrass staggers in summer and autumn, while those grazing endophyte-free ryegrass did not. Lambs/hoggets grazing ryegrass with endophyte also had more dags, lower growth rates, lower plasma prolactin levels, higher body temperatures and respiration rates under warm humid conditions. Most of these adverse responses were more severe in summer and autumn when endophyte toxin concentrations were highest. Many of these symptoms are similar to those described for the "autumn ill thrift" syndrome in New Zealand. Keywords: dags, endophyte, flystrike, growth rates, hyperthermia, Neotyphodium, perennial ryegrass, prolactin, ryegrass staggers, sheep

Two glasshouse experiments were conducted to evaluate possible allelopathic effects of perennial ryegrass/endophyte (Lolium perenne/Neotyphodium lolii) associations on white clover (Trifolium repens). The influence of between-species competition, environmental stress, and soil fauna was eliminated by the use of a sterile sand nutrient culture technique to sustain potted clover seedlings, to which aqueous extracts from moisture-stressed perennial ryegrass pseudostem were applied. In Experiment 1, extracts from two ryegrass cultivars ('Grasslands Pacific' and 'Grasslands Nui'), each hosting in separate seed lines two endophyte strains (ES, WT), suppressed clover growth (mean suppression 22% at 100% concentration) relative to extracts from endophyte-free lines of the same cultivars. The degree of suppression increased with increasing extract concentration, differed between cultivars, but did not differ between endophyte strains. In Experiment 2, extracts at 100% concentration from a third ryegrass cultivar ('Grasslands Ruanui'), hosting in separate seed lines three endophyte strains (AR4, ES, WT), suppressed clover growth by a mean of 27% relative to extracts from an endophyte-free line of this cultivar (range 11 to 47%), with significant differences between strains. The endophyte strains differed in three known endophyte alkaloids, viz peramine, ergovaline, lolitrem B, but in this study none of these alkaloids could solely account for the allelopathic response. The suppression of clover by all ryegrass cultivar/endophyte strain associations tested indicates that clover suppression is not confined to the specific associations used in previous studies, and that further testing of new associations is warranted as variation in the degree of allelopathy existed, being dependant on endophyte strain and host ryegrass cultivar.

Reduced plasma prolactin levels are one of the more consistent responses in animals grazing ryegrass and tall fescue with endophyte. The ergopeptine alkaloid, ergovaline is believed to be responsible. A selected endophyte, AR6 (formerly 187 BE), produces higher levels of ergovaline than its wild-type counterpart (Davies et al., 1993). Prolactin levels in healthy sheep tend to increase as photoperiod, ambient temperature and consequent heat load on the animal increase. However ambient temperature takes no account of the heat increment due to solar radiation or relative humidity.Black-globe temperature takes account of both ambient temperature and the heat increment due to solar radiation. (Chemineau and Ravault, 1984). Prolactin levels measured at a specific time, in an infrequent sampling regime, are often assumed to be representative of levels over a wider time period. Although this assumption may be correct under controlled conditions, it is not necessarily valid for uncontrolled field conditions. Optimum field sampling conditions need to be determined if we are to maximize the differential prolactin response in animals grazing endophyte-free and endophyte infected grasses and assess how representative any sample is.

This paper reviews some of the current practical and economic implications of Acremonium endophytes of ryegrass and tall fescue in New Zealand grassland agriculture. A historical perspective of their discovery and possible future implications of a new "novel" endophyte strain are also covered. Keywords Acremonium, endophyte, ryegrass, Lolium, tall fescue, staggers, Argentine stem weevil, clover, liveweight gains

Ryegrass seed lines with (+E) and without (-E) the fungal endophyte Acremonium lolii were sown with white clover. Fewer clover seedlings survived under +E ryegrasses irrespective of whether the plots were mown or grazed. The dominant contrast was an inverse competitive relationship between grass yield and clover seedling survival. A further component unexplained by effects of defoliation or competition is interpreted as an allelopathic effect. Two pot trials, using wheat, were established to determine it there was any residual effect of +E wards on following crops. Wheat grown in soil with a history of +E or-E ryegrass with clover produced higher yields from the-E than the +E soil. However, wheat grown on soil from previously clover-free pastures showed no difference between +E and -E. Residual effects on wheat crops are therefore interpreted as being related to lower nitrogen fertility, as a result of reduced clover growth in +E pastures. Keywords: Ryegrass, endophyte, white clover. competition, vigour. allelopathy, soil nitrogen, wheat yield, Acremonium lolii