Aspects of the chemistry and toxicology of indole-diterpenoid mycotoxins involved in tremorganic disorder of livestock

A method for the large-scale extraction ofNeotyphodium lolii (formerlyAcremonium lolii) infectedLolium perenne (perennial ryegrass) seed was optimized so as to efficiently obtain lolitrem B, the tremorgenic indole-diterpenoid compound thought to be the principal causative agent of the neurotoxic disorder ryegrass staggers. In a period of two weeks, 30 kg of seed could be processed yielding 125mg of pure, crystalline lolitrem B. In addition to lolitrem B, fifteen lolitrem analogues were isolated from the seed extracts and their structures determined by mass spectrometry and one- and two-dimensional NMR techniques. All but one of these analogues were previously unknown. Furthermore, three compounds related to paxilline, thought to be a precursor of lolitrem B were isolated fromPenicillium paxilli cultures. The isolation of the lolitrem and paxilline analogues provided much information on the likely pathways of lolitrem biosynthesis, and a detailed pathway is proposed. Paspaline was identified as the crucial intermediate in indole-diterpenoid biosynthesis, rather than paxilline, as previously thought. It is proposed that new endophyte incapable of biosynthesizing this substance will not produce any indole-diterpenoid tremorgens. Many of the lolitrem and paxilline derivatives isolated in the course of this study were tested for tremorgenic action in mice. These studies have identified a number of strucural features which are necessary for tremorgenic effect. Slow onset and long-duration tremors are characteristic of lolitrem compounds. For this effect, the acetal-linked isoprene unit and the stereochemistry at the A/B ring junction were identified as important. In contrast, paxilline compounds induce fast-onset and short-duration tremors. For this effect a C-13 α-orientated hydroxyl group is essential and the C-10 functionality is important. The extensive study of structure-activity relationships described in this thesis also suggests that lolitrem and paxilline compounds may exert their effects by binding to different target sites. Others have proposed that the more readily available paxilline be used as a model for the lolitrems in trials to select for animals resistant to ryegrass staggers, but the above observations suggest, however, that such an approach would not be successful.     

Interrelationships between Acremonium lolii, Peramine, and Lolitrem B in Perennial Ryegrass

Perennial ryegrass (Lolium perenne L.) is commonly infected with the endophytic fungus Acremonium lolii in a mutualistic relationship. The fungus produces a number of alkaloids, some of which are responsible for causing livestock disorders and/or for conferring insect resistance to the host grass. Little is known about the interrelationship between fungal growth and alkaloid production in the ryegrass plant and how this varies throughout the year. The concentrations of A. lolii and two of its alkaloid metabolites, lolitrem B and peramine, were monitored in basal (mainly leaf sheath) and upper (mainly leaf blade) parts of 17 endophyte-infected ryegrass plants on a monthly basis for 1 year. A. lolii, lolitrem B, and peramine concentrations were lowest in winter. The highest A. lolii concentrations were recorded in early summer, which coincided with the development of plant reproductive structures. Lolitrem B concentrations were highest from summer to early autumn and were consistently highest in the basal part of the plant. Peramine concentrations were generally highest in the upper part of the plant. Individual plants contained different levels of A. lolii, lolitrem B and peramine. These differences were generally maintained throughout the year. Although data for each month were variable, regression analyses showed that yearly mean concentrations of lolitrem B and peramine in individual plants were closely related to, and therefore probably largely determined by, yearly mean concentrations of A. lolii.     

Distribution of the fungal endophyte Neotyphodium lolii is not a major determinant of the distribution of fungal alkaloids in Lolium perenne plants

The relationships of the distributions of the insect and mammalian mycotoxins, lolitrem B and ergovaline, and the insect-feeding deterrent, peramine, with the distribution of fungal mycelium were investigated in three genotypes of perennial ryegrass (Lolium perenne L.) infected with the endophyte Neotyphodium lolii. In planta levels and distribution of the endophyte and of the three alkaloids were assessed in parallel, and different spatial or temporal concentration gradients were observed for each. Variation in the tissue distribution of the endophyte accounted only for 20%, 6%, and 31% of the variation in ergovaline, lolitrem B, and peramine, respectively. Alkaloid-endophyte ratios, determined in individual grass tissues, showed distinct in planta distribution patterns for each alkaloid and differed in magnitude among genotypes. The ergovaline-endophyte ratio was higher in the very basal plant tissues than in the apical tissues, while the lolitrem B and peramine ratios tended to be higher in apical tissues. The lolitrem B-endophyte ratio increased with leaf age, while no consistent temporal trends were detected for the other alkaloids. The results indicate that endophyte colonisation is a minor determinant of alkaloid levels, and that accumulation of the alkaloids relative to the endophyte mycelium is affected by plant genotype and tissue in a manner specific to each alkaloid. Possible factors in the regulation of alkaloid levels in the grass plant are discussed.     

Transformation of the ryegrass endophyte Neotyphodium lolii can alter its in planta mycelial morphology

The fungus Neotyphodium lolii grows in the intercellular spaces of perennial ryegrass as a mutualistic endosymbiont. One of the benefits it conveys to the plant is the production of alkaloids toxic to herbivores. We wanted to determine in planta expression patterns of the N. lolii 3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA reductase) gene, believed to be involved in the synthesis of two of these alkaloid toxins, lolitrem B and ergovaline. We transformed the N. lolii strain Lp19 with plasmids, in which DNA fragments upstream of the open reading frame of the N. lolii HMG CoA reductase gene controlled expression of the GUS (gusA; Escherichia coli β-glucuronidase) reporter gene. In exponentially growing cultures, the GUS gene was not expressed if the length of upstream sequence was less than 400 bp, and >1100 bp were required for maximum expression. When reintroduced into ryegrass plants, transformants often showed highly increased hyphal branching compared to the wild-type parent strain, although in culture their growth kinetics and morphology were indistinguishable from that of the wild-type. Deterioration of hyphae and the hypha–plant interface occurred and in one transformant reduced tillering (formation of new plants, referred to in agronomy as tillers) and death of infected plants. We found no evidence that these abnormalities were caused by interference of the construct with the function of the native gene, as judged by analysis of the site of integration of the promoter-GUS cassette, expression of the native gene and lolitrem B and ergovaline levels in infected plants. However, there was some correlation between GUS expression and the degree of hyphal branching, suggesting that high levels of β-glucuronidase may disturb the symbiotic interaction. Levels of another alkaloid, peramine, were also not significantly affected by transformation. In previous studies increased in planta branching of the endophyte has been shown to be associated with a severe reduction of alkaloid production. Our results show that a plant–endophyte association in which increased branching occurs is still able to produce alkaloids.     

Fungal endophytes of a forage grass reduce faecal degradation rates

Mutualisms between fungal endophytes and forage grasses can exert broad-reaching effects on grassland communities and ecosystem processes. We hypothesised that endophytes of grasses would retard the process of faecal degradation since grazing animals consume primarily live plant material and excrete a large portion of the herbage they consume as faeces. We examined the degradation rates of faeces from sheep that had consumed pure swards of perennial ryegrass containing a range of unique strains (AR1, AR37, or Wild type) of the fungal endophyte, Neotyphodium lolii, or no endophyte. Ultimately, the presence of endophytes in perennial ryegrass resulted in slower faecal decay rates compared to the nil endophyte treatment, although only consistently for the C concentration decay rates that were approximately 2× to 4× slower in the endophyte-derived faecal matter. The decay rate of dry matter content was significantly slower (ca. 1.5×) in the novel endophyte-derived faeces (AR1 and AR37) compared to the nil endophyte-derived faeces. The N decay rates differed significantly only in the AR1 treatment that was approximately 4× slower than the nil endophyte group. The reduced decay rates are attributed to the presence of endophyte-derived alkaloids in the faeces, and a greater proportion of more easily degraded hemicellulose in faeces from sheep that consumed the endophyte-free grass. There were no significant differences in the faecal carbon and nitrogen decay rates among the three endophyte strain treatments. This suggests that all the strain-specific alkaloids might have similar effects, or that N. lolii has a general effect that is not strain-specific, such as altered fibre composition, as reported here. This is the first report of a fungal endophyte affecting the rate of faecal degradation, and the first report of the alkaloids peramine, lolitrem B and epoxy–janthitrems in faecal matter. This study shows that a common agronomic grass–endophyte mutualism can have effects on ecosystem processes that have not previously been considered.     

A complex gene cluster for indole-diterpene biosynthesis in the grass endophyte Neotyphodium lolii

Lolitrems are a structurally diverse group of indole-diterpene mycotoxins synthesized by Epichloë/Neotyphodium endophytes in association with Pooid grasses. Using suppression subtractive hybridization combined with chromosome walking, two clusters of genes for lolitrem biosynthesis were isolated from Neotyphodium lolii, a mutualistic endophyte of perennial ryegrass. The first cluster contains five genes, ltmP, ltmQ, ltmF, ltmC, and ltmB, four of which appear to be orthologues of functionally characterized genes from Penicillium paxilli. The second cluster contains two genes, ltmE and ltmJ, that appear to be unique to lolitrem biosynthesis. The two clusters are separated by a 16 kb AT-rich sequence that includes two imperfect direct repeats. A previously isolated ltm cluster composed of ltmG, ltmM, and ltmK, is linked to these two new clusters by 35 kb of AT-rich retrotransposon relic sequence. All 10 genes at this complex LTM locus were highly expressed in planta but expression was very low or undetectable in mycelia. ltmM and ltmC were shown to be functional orthologues of P. paxilli paxM and paxC, respectively. This work provides a genetic foundation for elucidating the metabolic grid responsible for the diversity of indole-diterpenes synthesized by N. lolii.     

Evaluation of endophyte toxin production and its interaction with transgenic perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) with altered expression of fructosyltransferases

Alkaloid concentration of perennial ryegrass herbage is affected by endophyte strain and host plant genotype. However, previous studies suggest that associations between host and endophyte also depends on environmental conditions, especially those affecting nutrient reserves and that water-soluble carbohydrate (WSC) concentration of perennial ryegrass plants may influence grass-endophyte associations. In this study a single transgenic event, with altered expression of fructosyltransferase genes to produce high WSC and biomass, has been crossed into a range of cultivar backgrounds with varying Epichloë endophyte strains. The effect of the association between the transgenic trait and alkaloid production was assessed and compared with transgene free control populations. In the vast-majority of comparisons there was no significant difference between alkaloid concentrations of transgenic and non-transgenic plants within the same cultivar and endophyte backgrounds. There was no significant difference between GOI+?(gene of interest positive) and GOI? (gene of interest negative) populations in Janthritrem response. Peramine concentration was not different between GOI+?and GOI? for 10 of the 12 endophytes-cultivar combinations. Cultivar Trojan infected with NEA6 and Alto with SE (standard endophyte) exhibited higher peramine and lolitrem B (only for Alto SE) concentration, in the control GOI? compared with GOI+. Similarly, cultivar Trojan infected with NEA6 and Alto with NEA3 presented higher ergovaline concentration in GOI?. Differences in alkaloid concentration may be attributable to an indirect effect in the modulation of fungal biomass. These results conclude that the presence of this transgenic insertion, does not alter the risk (toxicity) of the endophyte–grass associations. Endophyte–host interactions are complex and further research into associations with high WSC plant should be performed in a case by case basis.     

Surrogate transformation of perennial ryegrass, Lolium perenne, using genetically modified Acremonium endophyte.

Summary Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyteAcremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700–800 transformants/g DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The -glucuronidase (GUS) gene,uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl -D-glucuronic acid and by enzyme assays of mycelial extracts. Severalhph- anduidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass. Molecular analysis of fungal isolates from the leaf sheath confirmed that the pattern of pAN7-1 and pNOM-2 hybridizing fragments was identical to that observed in the fungus used as inoculum.     

Functional analysis of an indole-diterpene gene cluster for lolitrem B biosynthesis in the grass endosymbiont Epichloë festucae

Epichloë festucae Fl1 in association with Lolium perenne synthesizes a diverse range of indole-diterpene bioprotective metabolites, including lolitrem B, a potent tremorgen. The ltm genes responsible for the synthesis of these metabolites are organized in three clusters at a single sub-telomeric locus in the genome of E. festucae. Here we resolve the genetic basis for the remarkable indole-diterpene diversity observed in planta by analyzing products that accumulate in associations containing ltm deletion mutants of E. festucae and in cells of Penicillium paxilli containing copies of these genes under the control of a P. paxilli biosynthetic gene promoter. We propose a biosynthetic scheme to account for this metabolic diversity.     

Importance of host plant species, Neotyphodium endophyte isolate, and alkaloids on feeding by Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae

Three grass host species--tall fescue, Festuca arundinacea Schreber; meadow fescue, Festuca pratensis Hudson; and perennial ryegrass, Lolium perenne L.--each infected with a number of different Neotyphodium endophyte isolates, were investigated for their effects on fall armyworm, Spodoptera frugiperda (J.E. Smith). Alkaloid profiles varied among associations. Choice and no-choice tests comparing feeding and early development of S. frugiperda larvae on endophyte-infected and endophyte-free leaf blade material were performed. Endophyte-mediated resistance to S. frugiperda was greatest in meadow fescue and weakest in tall fescue. Some endophyte isolates, particularly in perennial ryegrass and meadow fescue, had a major effect on feeding and development of S. frugiperda, whereas others had no effect or were only weakly efficacious. In tall fescue, some associations deterred S. frugiperda from feeding in choice tests but had no effect on development, whereas larvae reared on other associations weighed significantly more than control larvae fed endophyte-free grass. It was concluded that the deleterious consequences of endophyte infection were easily masked by other factors in tall fescue. Relative leaf age had no effect on feeding preferences in the three host species. Chemical analysis of herbage from the plants used, and results from a no-choice study using spiked artificial diets, failed to individually implicate any of the major known alkaloids (peramine, lolitrem B, ergovaline, and lolines) in the observed effects on S. frugiperda. Hypotheses explaining these observations, and their impact on creating desirable grass-endophyte associations for use in pastures, are discussed.     

Sympatric Epichloë species and chemotypic profiles in natural populations of Lolium perenne

The distribution of different Epichloë species within eight natural populations of Lolium perenne was studied. In total, 40.2% of the asymptomatic plants were infected by Epichloë festucae var. lolii or by Epichloë typhina. Both species occurred in sympatry in seven grass populations, and some plants had dual infections by both taxa. No hybrid taxa such as Epichloë hybrida were detected. Epichloë festucae strains were classified into two morphotypes, M1 and M3, according to culture characters, both morphotypes occurred in sympatry in seven populations. Plants bearing stromata produced by Epichloë typhina were observed, but asymptomatic plants infected by this species also occurred in seven populations. The alkaloid profile of Lolium perenne plants was related to the morphotype of their infecting strains: most plants infected by M3-strains were characterized by lolitrem, and those with M1-strains contained either ergovaline or lolitrem. Plants infected by Epichloë typhina were characterized by high peramine content.     

Endophytes of native grasses from South America: Biodiversity and ecology

We review and present preliminary results of studies on cool-season grass endophytes native to South America. These fungi have been studied only in Argentina, where they have been detected in 36 native grass species. The hybrid Neotyphodium tembladerae is present in an extremely wide host range found in diverse environmental conditions, but some other endophytes seem to be strictly associated with one host species in a particular environment. In host species that inhabit different environments, the incidence of endophytes is highly variable among populations and in most of the cases is clearly associated with environmental conditions. In these native grasses, Neotyphodium presents a mutualistic behaviour, conferring enhanced growth, promoting the symbiosis with arbuscular mycorrhizal fungi, and inhibiting growth of pathogenic fungi. In native forage grasses, preliminary analyses indicate that some Argentinian endophytes can produce lolines but are unlikely to produce lolitrem B or ergot alkaloids.     

Development and implementation of a multiplexed single nucleotide polymorphism genotyping tool for differentiation of ryegrass species and cultivars

Perennial ryegrass (Lolium perenne L.) and Italian ryegrass (Lolium multiflorum Lam.) are important temperate forage grasses which are closely related, generating fertile interspecific hybrids. All groups are represented by multiple cultivars in the commercial pasture seeds market. Due to the close taxonomic relationship between the two species, differentiation based on morphophysiological criteria is not always readily achievable. In addition, an obligate outbreeding reproductive habit produces high levels of individual heterozygosity and intrapopulation diversity, which presents problems for discrimination between cultivars. Molecular genetic marker polymorphism provides an effective means of addressing these challenges. An iterative process of resequencing from loci distributed across the perennial ryegrass genome was used to identify single nucleotide polymorphism (SNP) markers, which were then validated and formatted in a highly multiplexed (384-plex) assay system. SNP genotyping was then performed across samples of 48–192 individuals from a total of 27 ryegrass cultivars (19 of perennial ryegrass, seven of Italian ryegrass and one hybrid cultivar). SNP markers from perennial ryegrass exhibited a high level of transfer to Italian ryegrass. Data analysis permitted quantification of intra- and inter-species diversity, as well as discrimination between cultivars within each species, including diploid and autotetraploid cultivars of perennial ryegrass. Lower levels of SNP-based diversity were detected in Italian ryegrass than in perennial ryegrass. A neighbour-joining tree based on genetic distance analysis located a hybrid cultivar to an intermediate position between the two species-specific cultivar groups. The resulting catalogue of ryegrass cultivars will provide support for the processes of cultivar accreditation and quality assurance.     

A Role for BK Channels in Heart Rate Regulation in Rodents

The heart generates and propagates action potentials through synchronized activation of ion channels allowing inward Na⁺ and Ca²⁺ and outward K⁺ currents. There are a number of K⁺ channel types expressed in the heart that play key roles in regulating the cardiac cycle. Large conductance calcium-activated potassium (BK) ion channels are not thought to be directly involved in heart function. Here we present evidence that heart rate can be significantly reduced by inhibiting the activity of BK channels. Agents that specifically inhibit BK channel activity, including paxilline and lolitrem B, slowed heart rate in conscious wild-type mice by 30% and 42%, respectively. Heart rate of BK channel knock-out mice (Kcnma1^−/−) was not affected by these BK channel inhibitors, suggesting that the changes to heart rate were specifically mediated through BK channels. The possibility that these effects were mediated through BK channels peripheral to the heart was ruled out with experiments using isolated, perfused rat hearts, which showed a significant reduction in heart rate when treated with the BK channel inhibitors paxilline (1 µM), lolitrem B (1 µM), and iberiotoxin (0.23 µM), of 34%, 60%, and 42%, respectively. Furthermore, paxilline was shown to decrease heart rate in a dose-dependent manner. These results implicate BK channels located in the heart to be directly involved in the regulation of heart rate.     

Effects of Chicory/Perennial Ryegrass Swards Compared with Perennial Ryegrass Swards on the Performance and Carcass Quality of Grazing Beef Steers

An experiment investigated whether the inclusion of chicory (Cichorium intybus) in swards grazed by beef steers altered their performance, carcass characteristics or parasitism when compared to steers grazing perennial ryegrass (Lolium perenne). Triplicate 2-ha plots were established with a chicory/ryegrass mix or ryegrass control. Forty-eight Belgian Blue-cross steers were used in the first grazing season and a core group (n = 36) were retained for finishing in the second grazing season. The experiment comprised of a standardisation and measurement period. During standardisation, steers grazed a ryegrass/white clover pasture as one group. Animals were allocated to treatment on the basis of liveweight, body condition and faecal egg counts (FEC) determined 7 days prior to the measurement period. The measurement period ran from 25 May until 28 September 2010 and 12 April until 11 October 2011in the first and second grazing year. Steers were weighed every 14 days at pasture or 28 days during housing. In the first grazing year, faecal samples were collected for FEC and parasite cultures. At the end of the first grazing year, individual blood samples were taken to determine O. ostertagi antibody and plasma pepsinogen levels. During winter, animals were housed as one group and fed silage. In the second grazing year, steers were slaughtered when deemed to reach fat class 3. Data on steer performance showed no differences in daily live-weight gain which averaged 1.04 kg/day. The conformation, fat grade and killing out proportion of beef steers grazing chicory/ryegrass or ryegrass were not found to differ. No differences in FEC, O. ostertagi antibody or plasma pepsinogen levels of beef steers grazing either chicory/ryegrass or ryegrass were observed. Overall, there were no detrimental effects of including chicory in swards grazed by beef cattle on their performance, carcass characteristics or helminth parasitism, when compared with steers grazing ryegrass.     

Nitrous oxide emission factors for urine and dung from sheep fed either fresh forage rape (Brassica napus L.) or fresh perennial ryegrass (Lolium perenne L.)

In New Zealand, agriculture is predominantly based on pastoral grazing systems and animal excreta deposited on soil during grazing have been identified as a major source of nitrous oxide (N₂O) emissions. Forage brassicas (Brassica spp.) have been increasingly used to improve lamb performance. Compared with conventional forage perennial ryegrass (Lolium perenne L.), a common forage in New Zealand, forage brassicas have faster growth rates, higher dry matter production and higher nutritive value. The aim of this study was to determine the partitioning of dietary nitrogen (N) between urine and dung in the excreta from sheep fed forage brassica rape (B. napus subsp. oleifera L.) or ryegrass, and then to measure N₂O emissions when the excreta from the two different feed sources were applied to a pasture soil. A sheep metabolism study was conducted to determine urine and dung-N outputs from sheep fed forage rape or ryegrass, and N partitioning between urine and dung. Urine and dung were collected and then used in a field plot experiment for measuring N₂O emissions. The experimental site contained a perennial ryegrass/white clover pasture on a poorly drained silt-loam soil. The treatments included urine from sheep fed forage rape or ryegrass, dung from sheep fed forage rape or ryegrass, and a control without dung or urine applied. N₂O emission measurements were carried out using a static chamber technique. For each excreta type, the total N₂O emissions and emission factor (EF3; N₂O–N emitted during the 3- or 8-month measurement period as a per cent of animal urine or dung-N applied, respectively) were calculated. Our results indicate that, in terms of per unit of N intake, a similar amount of N was excreted in urine from sheep fed either forage rape or ryegrass, but less dung N was excreted from sheep fed forage rape than ryegrass. The EF3 for urine from sheep fed forage rape was lower compared with urine from sheep fed ryegrass. This may have been because of plant secondary metabolites, such as glucosinolates in forage rape and their degradation products, are transferred to urine and affect soil N transformation processes. However, the difference in the EF3 for dung from sheep fed ryegrass and forage rape was not significant.     

Lipid metabolism in mixtures of red clover (Trifolium repens) and perennial ryegrass (Lolium perenne) in lab scale silages and in vitro rumen incubations

Most often, farmers consider red clover an unattractive forage because of its low ensilability. Nevertheless, several in vivo and in vitro experiments also showed advantages of red clover silages such as decreased rumen biohydrogenation of polyunsaturated fatty acids. This has been attributed to a possible protective role of protein-bound phenols, with polyphenol oxidase playing a key role in their formation. This enzyme is active in red clover, but not in other green forages, such as, for example, perennial ryegrass. Therefore, the aim was to study the lipid metabolism within red clover/ryegrass mixtures in lab scale silages and during in vitro rumen batch incubations. Ensilability of red clover increased with higher proportions of ryegrass in the silage mixture. However, the lipid-protecting mechanism of red clover does not seem to occur in the co-ensiled ryegrass as lipolysis of polar lipids linearly increased with increasing proportions of ryegrass (86.0%, 91.6%, 89.9%, 93.1% and 95.6% in 60-day-old silages with 100/0, 75/25, 50/50, 25/75 and 0/100 red clover/ryegrass, respectively). Rumen lipolysis and biohydrogenation of C18:3n-3 and C18:2n-6 were negatively related to red clover proportions in the silage mixtures. The lipid-protective mechanism in red clover silages is confirmed, but it seems not to be transferred to lipids in co-ensiled forages.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum) : II. Chlorsulfuron Resistance Involves a Wheat-Like Detoxification System

Lolium rigidum Gaud. biotype SLR31 is resistant to the herbicide diclofop-methyl and cross-resistant to several sulfonylurea herbicides. Wheat and the cross-resistant ryegrass exhibit similar patterns of resistance to sulfonylurea herbicides, suggesting that the mechanism of resistance may be similar. Cross-resistant ryegrass is also resistant to the wheat-selective imidazolinone herbicide imazamethabenz. The cross-resistant biotype SLR31 metabolized [phenyl-U-¹⁴C]chlorsulfuron at a faster rate than a biotype which is susceptible to both diclofop-methyl and chlorsulfuron. A third biotype which is resistant to diclofop-methyl but not to chlorsulfuron metabolized chlorsulfuron at the same rate as the susceptible biotype. The increased metabolism of chlorsulfuron observed in the cross-resistant biotype is, therefore, correlated with the patterns of resistance observed in these L. rigidum biotypes. During high performance liquid chromatography analysis the major metabolite of chlorsulfuron in both susceptible and cross-resistant ryegrass coeluted with the major metabolite produced in wheat. The major product is clearly different from the major product in the tolerant dicot species, flax (Linium usitatissimum). The elution pattern of metabolites of chlorsulfuron was the same for both the susceptible and cross-resistant ryegrass but the cross-resistant ryegrass metabolized chlorsulfuron more rapidly. The investigation of the dose response to sulfonylurea herbicides at the whole plant level and the study of the metabolism of chlorsulfuron provide two independent sets of data which both suggest that the resistance to chlorsulfuron in cross-resistant ryegrass biotype SLR31 involves a wheat-like detoxification system.