The effect of glyphosate, paraquat and paclobutrazol on lolitrem B levels in endophyte-infected perennial ryegrass

Two herbicides (glyphosate and paraquat) and a plant growth regulator (paclobutrazol) were applied to endophyteinfected (Acremonium lolii) perennial ryegrass swards. Subsamples of these swards were then chemically analysed at intervals up to 28 days later for lolitrem B, the compound responsible for perennial ryegrass staggers in domestic livestock. Glyphosate and paclobutrazol had no effect on lolitrem B concentrations. Paraquat applications decreased lolitrem B concentrations in the herbage. Because none of the chemicals tested increased the concentration of lolitrem B in the herbage, they are unlikely to be directly implicated in perennial ryegrass staggers in grazing animals.     

Young perennial ryegrass staggers in a dairy herd

Ryegrass staggers was diagnosed in a group of young stock on a dairy farm in the Netherlands. The cattle were fed on ryegrass hay originating from a farm in the region. The typical clinical signs observed in this herd were difficulty in rising, hypermetria of especially the front legs, incoordination, and tremors of the head and neck muscles. The morbidity of the illness was about 80 percent and there was no mortality. Four weeks after withdrawal of the hay, the animals recovered completely. The diagnosis was confirmed by demonstrating high concentrations of Lolitrem-B (1.7 ppm) in the hay. This neurotoxin is usually produced by the endophyte Neotyphodium lolii with which ryegrass may be infected. This is the first case of ryegrass staggers in cows reported in the Netherlands since 1992.     

Association between feeding perennial ryegrass (Lolium perenne cultivar Grasslands impact) containing high concentrations of ergovaline, and health and productivity in a herd of lactating dairy cows

Perennial ryegrasses are frequently infected with fungal endophyte (Neotyphodium lolii) to increase the resistance of the plant to insect damage. Unfortunately, a side effect of endophyte infection can be the production of alkaloids, including Lolitrem B and ergovaline, that produce toxic effects in animals. A significant 4.6 litre decrease in milk production in a herd of Holstein-Friesian dairy cows was associated with high concentrations of ergovaline in ryegrass silage. Simultaneously, milk SCC increased significantly over a comparable period and reproductive performance declined. Body condition score and coat condition of cows were adversely affected. Unique aspects of this Case report include; very stable production of the herd over a period of years before and after cessation of feeding silage containing high concentrations of ergovaline; the presence of high concentrations of ergovaline in the silage; and a controlled diet that reduced the risks of variation in feed availability and other sources of toxins. Veterinarians and other farm advisors should be aware of the potential for negative effects on animal health and production of fungal endophyte and the potential for Neotyphodium lolii to produce ergovaline.     

Staggers induced by consumption of perennial ryegrass in cattle and sheep from northern California

Staggers was diagnosed in sheep and cattle from the northern California coast. The diagnosis was made on the basis of history of ingestion of perennial ryegrass (Lolium perenne) stubble, clinical signs of transient ataxia, which was aggravated by stimulation, and nearly complete recovery after removal of ryegrass as the primary forage. Morbidity was high, but death did not occur in any affected animals. The toxic endophyte, Acremonium lolii, was in most lower leaf sheaths from the ryegrass. Injection of extracts of the ryegrass from affected farms into mice induced signs of toxicosis. Additionally, ryegrass from all 3 farms contained the tremorgenic mycotoxin, lolitrem-B.     

苇状羊茅与多年生黑麦草内生真菌菌丝基因组DNA提取的优化及PCR初步检测

感染内生真菌的禾草在牧草和草坪业上具有重要的生态和经济意义,家畜采食感染Neotyphodiumcoenophialum和N.lolii的苇状羊茅Festuca arundinacea和多年生黑麦草Lolium perenne会发生中毒。研究收集天津口岸1998年以来进境的部分苇状羊茅和多年生黑麦草种子,对经镜检确认带有内生真菌的种子进行分离培养,对疑似菌株的菌丝用改进的Moller等方法进行基因组DNA抽提,测定浓度及纯度,对照原方法,DNA的纯度有较大提高,浓度略有上升。Tubulin2基因的引物IS1~IS3扩增结果显示为单一的条带,结合形态学和序列比对,分离培养得到的菌株可以基本确定为N.coenophialum和N.lolii。根据Gen-bank中N.coenophialum和N.lolii的NC25基因序列设计出引物F1~R1,扩增得到能区分开N.coenophialum和N.lolii的单一条带(相差160 bp),建立了N.coenophialum和N.lolii的PCR检测方法,结果准确可靠。     

The evaluation of endophyte toxin residues in sheep fat

Abstract

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.     

Ergovaline does not alter the severity of ryegrass staggers induced by lolitrem B

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.     

披碱草内生真菌对三种草坪草种子与种苗的化感效应

以多年生黑麦草、高羊茅和草地早熟禾3种草坪草作为供试对象,用种子萌发法和盆栽法初步测定了披碱草内生真菌的化感效应。结果表明,披碱草内生真菌对3种草坪草种子萌发与生长具有一定的影响作用。种子室内滤纸萌发条件下,披碱草草粉对3种草坪草种子萌发有一定的抑制作用;且内生真菌显著增强了披碱草草粉对多年生黑麦草苗重、对高羊茅的发芽率、根长、苗长、苗重和对草地早熟禾发芽率的抑制作用(P<0.05),显著降低了披碱草草粉对多年生黑麦草的根长和苗长、对高羊茅的根重以及对草地早熟禾根长的抑制作用(P<0.05)。盆栽条件下,披碱草草粉对3种草坪草的生长均有一定的促进作用;且内生真菌显著增强了披碱草草粉对高羊茅初期发芽率的促进作用(P<0.05),显著降低了披碱草草粉对多年生黑麦草的初期发芽率和对草地早熟禾苗长的促进作用(P<0.05)。     

Amino acid neurotransmitter release from cerebrocortical synaptosomes of sheep with severe ryegrass staggers in New Zealand

Enhanced unstimulated release of the excitatory amino acid neurotransmitters aspartic acid and glutamic acid was measured in cerebrocortical synaptosomes prepared from sheep showing severe symptoms of ryegrass staggers. A similar pattern of deviation from neurologically normal animals was seen whether the sheep had contracted the disorder naturally or whether they had been deliberately caused to develop the disorder by grazing specific pastures rich in endophyte infected ryegrass. Deranged neurotransmitter release may reasonably be involved in the chemical pathology of ryegrass staggers and the findings are in accord with the putative synonymy of lolitrem toxicosis and ryegrass staggers.     

带内生真菌的野大麦草粉对3种草坪草种子萌发及生长的影响

以多年生黑麦草(Lolium perenne)、高羊茅(Festuca arundinacea)和草地早熟禾(Poa pratensis)为研究对象,用种子萌发法和盆栽法测定了野大麦(Hordeum brevisubulatum)内生真菌(Neotyphodium)对这3种草坪草种子萌发及生长的影响。结果表明,在种子萌发试验中,野大麦草粉对3种草坪草的生长均有一定的抑制作用,而且内生真菌显著增强了野大麦草粉对多年生黑麦草苗长、须根数和对草地早熟禾苗长的抑制作用（P<0.05）;显著降低了野大麦草粉对多年生黑麦草和高羊茅的发芽率及高羊茅苗长的抑制作用（P<0.05）。在盆栽试验中,野大麦草粉对3种草坪草的生长均有一定的促进作用,而且内生真菌显著增强了野大麦草粉对高羊茅和草地早熟禾的苗长和发芽率的促进作用（P<0.05）。     

Growth rate and physiology of steers grazing tall fescue inoculated with novel endophytes

Cattle grazing tall fescue (Festuca arundinacea Schreb.) often develop fescue toxicosis. This condition is thought to be caused by ergot alkaloids produced by the endophyte Neotyphodium coenophialum. Endophytes from wild tall fescue plants, which do not produce ergot alkaloids, were transferred into the endophyte-free tall fescue germplasm, HiMag. The novel associations also lacked the ability to produce ergot alkaloids. Our objective was to determine whether cattle grazing these novel endophyte associations showed signs of fescue toxicosis. At the Fayetteville, Arkansas location, tester steers (n = 72) were assigned to one of four pasture treatments: endophyte-free HiMag tall fescue (HiMag-); 'Kentucky-31' tall fescue infected with its native, toxic endophyte (KY+); and two novel endophyte-infected tall fescue associations, HiMag4 and HiMag9. At the Mount Vernon, Missouri location, steers (n = 54) were used to test three of the four cultivars (HiMag9 was not tested). Ergot alkaloid concentrations in the forage of HiMag4 and HiMag9 were low or undetectable. Respiration rate, rectal temperature, ADG, and hair scores were measured during the grazing period. Blood was collected via jugular venipuncture and used for prolactin, aspartate aminotransferase, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), cholesterol, triglyceride, and creatinine analysis. Weight gains by steers grazing HiMag4 and HiMag9 did not differ from those of steers grazing HiMag-, but were greater than gains (P < 0.05) by steers on the KY+ treatment. Steers grazing KY+ had higher (P < 0.05) respiration rates, rectal temperatures, and hair scores than did steers grazing novel endophyte and HiMag- pastures. Prolactin, ALP, cholesterol, LDH, and triglycerides all were suppressed (P < 0.05) in steers grazing KY+ compared with steers grazing novel endophyte and HiMag- pastures. Steers grazing the novel endophyte tall fescues did not suffer from the decreased weight gains and toxicities associated with fescue toxicosis, resulting in enhanced animal production.     

Animal performance and economic comparison of novel and toxic endophyte tall fescues to cool-season annuals

Increased costs of annual establishment of small grain pasture associated with fuel, machinery, and labor are eroding the profitability of stocker cattle enterprises. Interest has therefore increased in development of cool-season perennial grasses that are persistent and high quality. This study occurred on 24 ha (divided into thirty 0.81-ha paddocks) located at the University of Arkansas Division of Agriculture Livestock and Forestry Branch Station, near Batesville. Two tall fescue (Festuca arundinacea Schreb.) cultivars infected with novel endophytes (NE), Jesup infected with AR542 endophyte (Jesup AR542), and HiMag infected with Number 11 endophyte (HM11) were established in September 2002. Jesup AR542 and HM11 were compared with endemic endophyte Kentucky 31 (KY-31) tall fescue; wheat (Triticum aestivum L.) and cereal rye (WR, Secale cereale L.) planted in September 2003, 2004, and 2005; and annual ryegrass [RG, Lolium perenne L. ssp. multiflorum (Lam.) Husnot] planted in September 2004 and 2005. Each year, 3 steers (3.7 steers/ha) were placed on each pasture for fall and winter grazing, and 5 steers (6.2 steers/ha) were placed on each pasture for spring grazing. Animal performance is presented by year in the presence of a year x treatment interaction (P < 0.01). Body weight gain per hectare of steers grazing NE tall fescue was greater (P < 0.01) than those of KY-31 and WR during 2003 to 2004, whereas in 2004 to 2005, BW gain per hectare of steers grazing NE and RG did not differ (P < or = 0.29) and was greater (P < 0.01) than that of WR, which was greater (P < 0.01) than that of KY-31. During 2005 to 2006, BW gain per hectare was greater (P < 0.01) for steers grazing RG than those of NE and WR, which did not differ (P > or = 0.14). Body weight gain per hectare was least (P < 0.01) for steers grazing KY-31. Average net return of NE tall fescue was greater (P < 0.01) than KY-31, but profitability of NE did not consistently differ from cool-season annuals. Across the 3-yr study, NE tall fescue produced net returns per hectare of $219; this level of profitability would require 4 yr for a new planting of NE tall fescue to break even. Novel endophyte tall fescues offer potential benefits related to decreased risk of stand establishment of annual forage crops, longer growing season, and acceptable animal performance.     

Correlation of ergovaline and lolitrem B levels in endophyte-infected perennial ryegrass (Lolium perenne).

The varieties of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotiphodium lolii contain several classes of toxic alkaloids, including ergopeptide alkaloids and lolitrem alkaloids. Lolitrem B, a potent tremorgen, is generally considered to be the predominant alkaloid in endophyte-infected perennial ryegrass. Ergovaline, a vasoconstrictor normally associated with endophyte-infected tall fescue (Festuca arudinacea), is also present in endophyte infected perennial ryegrass. Clinical signs of animals ingesting endophyte-infected perennial ryegrass are consistent with the presence of lolitrem B. However, clinical signs normally associated with ergovaline poisoning are not usually observed in animals ingesting endophyte-infected perennial ryegrass. A survey was conducted to quantitate both lolitrem B and ergovaline in 459 perennial ryegrass straw samples received at the Oregon State University College of Veterinary Medicine. Samples were analyzed for each alkaloid using separate high-performance liquid chromatography analyses. A strong positive correlation between the 2 alkaloids (r2 = 0.7335) was observed, especially in the samples containing <3,000 ppb (ng/g) lolitrem B. The threshold levels above which clinical signs typically occur are 2,000 ppb lolitrem B and 300-400 ppb ergovaline. All of the samples analyzed contained <425 ppb ergovaline.     

Effects of the fungal endophyte Acremonium coenophialum in fescue on pregnant mares and foal viability.

Effects of the endophyte Acremonium coenophialum in tall fescue on pregnant mares and foal viability were evaluated. Twenty-two mature pregnant mares were randomly chosen to graze either Kentucky-31 tall fescue that was free from A coenophialum (endophyte-free, EF) or tall fescue infected with A coenophialum (endophyte-present, EP) after the first 90 days of pregnancy through parturition. Concentrations of pyrrolizidine and ergopeptine alkaloids were significantly greater in EP grass, compared with EF pasture. Ten of 11 mares grazing EP pasture had obvious dystocia. Mean duration of gestation was significantly greater for the EP group, compared with the EF group. Foal survivability was severely reduced among mares grazing EP fescue with only 1 foal surviving the natal period. Udder development and lactation were low in mares grazing EP grass. The absence of clinical problems in mares grazing EF grass implicated the endophyte as the causative agent of reproductive problems and perinatal foal mortality in pregnant mares grazing endophyte-infected fescue grass. Caution should be exercised in allowing pregnant mares to graze pastures infected with the endophyte A coenophialum.     

Incidence of ryegrass staggers in white rhinoceros (Ceratotherium simum) at Auckland Zoo

Abstract

Extract

Most perennial ryegrass (Lolium perenne) plants in pastures in New Zealand contain a naturally occurring fungal endophyte, Neotyphodium lolii. Endophyte-infected perennial ryegrass produces alkaloids that vary in concentration during the year. Some alkaloids enhance the persistence and productivity of ryegrass pastures by protecting them against insect attack. However, when other alkaloids are consumed they can reduce animal performance and lead to health problems. The alkaloid lolitrem B, for example, causes the neuromuscular disorder ryegrass staggers (Fletcher et al 1999 Fletcher, LR, Sutherland, BL and Fletcher, CG. 1999. “The impact of endophyte on the health and productivity of sheep grazing ryegrass-based pastures”. In Ryegrass endophyte: an essential New Zealand symbiosis Grassland Research and Practice Series Edited by: Woodfield, DR and Matthew, C. Vol. 7, 11–17.  [Google Scholar]). Clinical symptoms of ryegrass staggers in animals range from slight muscular tremors through to staggering and complete collapse. Severely affected animals create management problems and are prone to accidental death. Outbreaks occur sporadically, particularly in summer and autumn and affect sheep, cattle, deer, horses, llamas (Lama glama) and alpaca (Lama pacos). Lolitrem B appears to be a stable compound, which tends to be concentrated in the leaf sheath at the base of the ryegrass plant and in the seed-heads (di Menna et al 1992 di Menna, ME, Mortimer, PH, Prestidge, RA, Hawkes, AD and Sprosen, JM. 1992. Lolitrem B concentrations, counts of Acremo ni um lolii hyphae, and the incidence of ryegrass staggers in lambs on plots of A. lolii-infected perennial ryegrass. New Zealand Journal of Agricultural Research, 35: 211–217. [Taylor &; Francis Online], [Web of Science ®] , [Google Scholar]; Keogh et al 1996 Keogh, RG, Tapper, BA and Fletcher, RH. 1996. Distributions of the fungal endophyte Acremonium lolii, and of the alkaloids lolitrem B and peramine, within perennial ryegrass. New Zealand Journal of Agricultural Research, 39: 121–127. [Taylor &; Francis Online], [Web of Science ®] , [Google Scholar]). Hay made from endophyte-infected perennial ryegrass contains large numbers of seed-heads and lolitrem B concentrations can be high, especially if it is made in late summer or autumn and, consequently, animals fed such hay are at risk of developing ryegrass staggers.     

The role of Penicillia in ryegrass staggers

Tremorgenic strains of Penicillium verrucosum var cyclopium, P canescens, P janthinellum, P novaezeelandiae and P estinogenum were isolated from the faeces of 15 of 23 affected sheep and cattle in eight of nine field outbreaks of ryegrass staggers. One tremorgenic strain of P griseofulvum was isolated from the faeces of one of 25 sheep grazing in unaffected flocks. Tremorgenic strains of P verrucosum var cyclopium, P canescens, P janthinellum and P estinogenum were also isolated from the A horizon of New Zealand soils. Since a large proportion of experimentally dosed live P verrucosum var cyclopium died during passage through the gut, the faecal evidence from naturally staggering animals suggests that at least some outbreaks of ryegrass staggers are caused by tremorgenic Penicillia and that their source may be soil.     

Ryegrass staggers in Canadian wapiti deer

Extract

Sir:- We would like to report an outbreak of ryegrass staggers in Canadian wapiti (Cervus elaphus mannitobensis) which occurred at the Invermay Agricultural Research Centre, Mosgiel. As far as we are aware this is the first recorded case of ryegrass staggers in deer associated with grazing perennial rye grass (Lolium perenne L.).     

内生真菌侵染对盐胁迫下黑麦草种子萌发的影响

以低内生真菌侵染率和高内生真菌侵染率的多年生黑麦草（Lolium perenne）种子为材料,研究了在不同浓度盐胁迫下内生真菌侵染对种子萌发的影响。结果表明,高内生真菌侵染率和低内生真菌侵染率种子的相对发芽率、相对活力指数、相对根长、相对苗长、相对根苗比均随盐胁迫程度的增加而显著下降。中、低浓度盐胁迫下（≤170 mmol·L-1 NaCl）二者的相对发芽率、相对活力指数、相对根长、相对苗长的差异不显著(P>0.05);高浓度盐胁迫下（255 mmol·L-1 NaCl）,高内生真菌侵染率种子的相对发芽率、相对活力指数、相对苗长均显著大于低内生真菌侵染率的品种(P<0.05)。盐胁迫抑制了多年生黑麦草种子的萌发,但在高浓度盐胁迫下内生真菌对宿主具有增益作用。     

Gangrenous ergotism in cattle grazing fescue (Festuca elatior L.) in South Africa

The 1st outbreak of fescue toxicosis in South Africa was recently confirmed in a Brahman herd at Perdekop, near Standerton, in Mpumalanga province, South Africa. Within 3 weeks of being placed on a fescue pasture in mid-winter, 50 of 385 cattle developed lameness and/or necrosis of the tail. The farmer had established Festuca elatior L. (tall fescue, Iewag variety) on c. 140 ha for winter grazing. Fescue may be infected by an endophyte, Neotyphodium coenophialum, which produces ergot alkaloids, in particular ergovaline. Ergovaline concentrations in basal leaf sheaths and grass stems collected during the outbreak ranged from 1720-8170 ppb on a dry-matter basis.     

舟叶橐吾浸提液对三种牧草种子萌发及幼苗生长的影响

本试验以滇西北人工草地建设中常用的白三叶(Trifolium repens L.)、多年生黑麦草(Lolium perenne L.)、鸭茅(Dactylis glomerata L.)为试验材料，研究了舟叶橐吾(Ligularia cymbulifera (W. W. Smith) Hand.-Mazz.)地上部和根部的水浸提液对种子萌发和幼苗生长的影响。结果表明，不同部位、不同浓度的舟叶橐吾植株浸提液对3种供试牧草种子的发芽势、发芽率和发芽指数都有抑制作用，其中根部浸提液的抑制作用比地上部更为明显，且具有很强的浓度效应。另外，高、中浓度的提取液进一步抑制了供试牧草苗高和根的生长(P < 0.05)。根据化感效应指数和综合化感效应指数，供试牧草对舟叶橐吾植株浸提液的敏感程度也有差异，表现为：白三叶>鸭茅>多年生黑麦草。对舟叶橐吾叶片开展的非靶向分析一共发现了283种成分，主要是有机酸、酰胺类。由于舟叶橐吾有较强的化感作用，在人工草地建设中应尽可能去除舟叶橐吾，避免给生产带来损失。