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A comparison of the voluntary intake and digestion of a range of forages at different times of the year by the sheep and the red deer (Cervus elaphus).

1. Comparisons were made between castrated male Scottish Blackface sheep and red deer (Cervus elaphus) of voluntary forage intake (VFI), digestibility and the mean retention time (MRT) of a particulate-phase marker (103Ru-phenanthroline) in the alimentary tract, when a range of forages: dried-grass pellets, chopped dried grass, fresh-frozen Agrostis-Festuca spp. and heather (Calluna vulgaris, L. Hull) were given at different times of the year. 2. On both the dried-grass-pellet and chopped dried-grass diets the red deer and sheep ate similar quantities. Both species had a higher VFI of dried-grass pellets in July than in November. The sheep digested the dried-grass-pellet diet better than the red deer and this was associated with a longer MRT of the particulate-phase marker in the alimentary tract. 3. The VFI of Agrostis-Festuca spp. and heather by the red deer was twice that of the sheep. The VFI of heather by the sheep increased by 32% between January and April, and the VFI of both the Agrostis--Festuca spp. and heather diets by the red deer increased by 65--70%. The sheep digested the Agrostis--Festuca spp. better than the red deer but the red deer digested the heather slightly better than the sheep. MRT of the particulate-phase marker was greater for the sheep than for the red deer on both the diets. The digestibility and MRT of both diets in the red deer did not decrease with the seasonal increase in VFI, suggesting a possible hypertrophy of the alimentary tract.

Animal Feed↗

Nitrogen digestion in sheep given poor-quality indigenous hill herbages.

1. In two experiments, the sites of digestion of non-ammonia nitrogen (NAN) and the amounts of urea N recycled to the rumen were measured in mature wether sheep given diets of indigenous hill herbage (Agrostis-Festuca and heather). 2. Duodenal and ileal flow values were obtained using 103Ru-phenanthroline and 51Cr-EDTA markers in animals prepared with simple (T-shaped) cannulas. Amounts of urea N recycled to the rumen were estimated from measurements of the transfer of plasma urea carbon into rumen bicarbonate and the production rate of rumen bicarbonate using 14C-labelled urea and bicarbonate respectively. 3. The flows of NAN at the duodenum and ileum were linearly related to the intake of herbage (P less than 0.001). There was a net gain of non-ammonia N anterior to the duodenum on both diets (at an intake of 460 g organic matter (OM)/d, 3.7 g NAN/d on Agrostis-Festuca and 3.3 g NAN/d on heather). 4. Net digestibility of NAN entering the small intestine was within a normal range on the Agrostis-Festuca (0.58 at 460 g OM intake) diet but low on the heather diet (0.43 at 460 g OM intake). 5. It was calculated that at 460 g OM intake only 0.9 and 1.1 g/d respectively of the duodenal NAN on the Agrostis-Festuca and heather diets could have been derived from urea-N recycled to the rumen. Thus 2.8 g and 2.2 g/d had to be accounted for as non-urea endogenous NAN.

Animal Feed↗

Metabolic fates of U-14C-labelled monosaccharides and an enzyme-treated cell-wall substrate in the fowl.

A major benefit of supplementing non-ruminant feedstuffs with exogenous enzymes is presumed to be the degradation of plant cell-wall polysaccharides to metabolizable monosaccharide residues. In the present study, metabolic fates of (U-14C-labelled, 10 mM) glucose, galactose, mannose, xylose and arabinose were compared in the fowl, by measuring recoveries of 14C radioactivity in exhaled carbon dioxide excreta and body tissues after administration either by wing vein (iv) or into the crop (ic). A further comparison was made with a tube-fed, enzyme-treated, U-14C-labelled cell-wall substrate, Festuca arundinacea, and a final experiment tested possible competition for absorption between different cell-wall residues. Delays between iv and ic treatments in recovery of 14C in CO2, which were assumed to reflect intestinal absorption, indicated that xylose was absorbed more slowly than glucose and galactose, but faster than mannose and arabinose. Total recoveries of 14C in CO2 and excreta over the whole test period indicated that metabolizabilities were highest with glucose, galactose and mannose, and lowest with arabinose. After testing, 14C recovery in caecal contents was highest with ic arabinose, and recoveries in body tissues, with all sugar treatments, were in the order liver greater than breast and leg muscle greater than abdominal fat greater than plasma. Results with the Festuca substrate showed similar patterns of recovery in body tissues and confirmed an increase in metabolizability with addition of enzyme. The timing of the 14CO2 response with Festuca and a wet enzyme pretreatment was broadly similar to a 'predicted Festuca' response, based on the composition of the substrate and the measured responses with individual (ic) monosaccharides. There was no evidence of any competition for absorption or metabolism among cell-wall residues. It was concluded that glucose release from cellulose is potentially the most important product of cell-wall degradation to contribute to enzyme enhancement of metabolizable energy.

Animals↗

Relationships among non-Acremonium sp. fungal endophytes in five grass species.

Many cool-season grasses (subfamily Pooideae) possess maternally transmitted fungal symbionts which cause no known pathology and often enhance the ecological fitness and biochemical capabilities of the grass hosts. The most commonly described endophytes are the Acremonium section Albo-lanosa spp. (Acremonium endophytes), which are conidial anamorphs (strictly asexual forms) of Epichloë typhina. Other endophytes which have been noted are a Gliocladium-like fungus in perennial ryegrass (Lolium perenne L.) and a Phialophora-like fungus in tall fescue (Festuca arundinacea Schreb.). Here, we report the identification of additional non-Acremonium sp. endophytes (herein designated p-endophytes) in three more grass species: Festuca gigantea, Festuca arizonica, and Festuca pratensis. In each grass species, the p-endophyte was cosymbiotic with an Acremonium endophyte. Serological analysis and sequence determinations of variable portions of their rRNA genes indicated that the two previously identified non-Acremonium endophytes are closely related to each other and to the newly identified p-endophytes. Therefore, the p-endophytes represent a second group of widely distributed grass symbionts.

Base Sequence↗

Tall fescue EST-SSR markers with transferability across several grass species.

Tall fescue (Festuca arundinacea Schreb.) is a major cool season forage and turf grass in the temperate regions of the world. It is also a close relative of other important forage and turf grasses, including meadow fescue and the cultivated ryegrass species. Until now, no SSR markers have been developed from the tall fescue genome. We designed 157 EST-SSR primer pairs from tall fescue ESTs and tested them on 11 genotypes representing seven grass species. Nearly 92% of the primer pairs produced characteristic simple sequence repeat (SSR) bands in at least one species. A large proportion of the primer pairs produced clear reproducible bands in other grass species, with most success in the close taxonomic relatives of tall fescue. A high level of marker polymorphism was observed in the outcrossing species tall fescue and ryegrass (66%). The marker polymorphism in the self-pollinated species rice and wheat was low (43% and 38%, respectively). These SSR markers were useful in the evaluation of genetic relationships among the Festuca and Lolium species. Sequencing of selected PCR bands revealed that the nucleotide sequences of the forage grass genotypes were highly conserved. The two cereal species, particularly rice, had significantly different nucleotide sequences compared to the forage grasses. Our results indicate that the tall fescue EST-SSR markers are valuable genetic markers for the Festuca and Lolium genera. These are also potentially useful markers for comparative genomics among several grass species.

Base Sequence↗

Field evaluation of in situ remediation of a heavy metal contaminated soil using lime and red-mud.

We evaluated the effectiveness of lime and red mud (by-product of aluminium manufacturing) to reduce metal availability to Festuca rubra and to allow re-vegetation on a highly contaminated brown-field site. Application of both lime and red mud (at 3 or 5%) increased soil pH and decreased metal availability. Festuca rubra failed to establish in the control plots, but grew to a near complete vegetative cover on the amended plots. The most effective treatment in decreasing grass metal concentrations in the first year was 5% red mud, but by year two all amendments were equally effective. In an additional pot experiment, P application in combination with red mud or lime decreased the Pb concentration, but not total uptake of Pb in Festuca rubra compared to red mud alone. The results show that both red mud and lime can be used to remediate a heavily contaminated acid soil to allow re-vegetation.

Aluminum Silicates↗

The large subunit determines catalytic specificity of barley sucrose:fructan 6-fructosyltransferase and fescue sucrose:sucrose 1-fructosyltransferase.

Plant fructosyltransferases are highly homologous in primary sequence and typically consist of two subunits but catalyze widely different reactions. Using functional expression in the yeast Pichia pastoris, we show that the substrate specificity of festuca sucrose:sucrose 1--beta-D-fructosyltransferase (1-SST) and barley sucrose:fructan 6--beta-D-fructosyltransferase (6-SFT) is entirely determined by the large subunit. Chimeric enzymes with the large subunit of festuca 1-SST (LSuB) and the small subunit of barley 6-SFT have the same catalytic specificity as the native festuca 1-SST and vice versa. If the LSuB is expressed alone, it does not yield a functionally active enzyme, indicating that the small subunit is nevertheless essential.

Amino Acid Motifs↗

Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass.

Lolitrems are potent tremorgenic mycotoxins that are synthesised by clavicipitaceous fungal endophytes of the Epichloë/Neotyphodium group in association with grasses. These indole-diterpenes confer major ecological benefits on the grass-endophyte symbiotum. A molecular signature for diterpene biosynthesis is the presence of two geranylgeranyl diphosphate (GGPP) synthases. Using degenerate primers for conserved domains of fungal GGPP synthases, we cloned two such genes, ltmG and ggsA, from Neotyphodium lolii. Adjacent to ltmG are two genes, ltmM and ltmK, that are predicted to encode an FAD-dependent monooxygenase and a cytochrome P450 monooxygenase, respectively. The cluster of ltm genes is flanked by AT-rich retrotransposon DNA that appears to have undergone extensive repeat induced point (RIP) mutation. Epichloë festucae, the sexual ancestor of N. lolii, contains an identical ltm gene cluster, but lacks the retrotransposon "platform'' on the right flank. Associations established between perennial ryegrass and an E. festucae mutant deleted for ltmM lack detectable levels of lolitrems. A wild-type copy of ltmM complemented this phenotype, as did paxM from Penicillium paxilli. Northern hybridization and RT-PCR analysis showed that all three genes are weakly expressed in culture but strongly induced in planta. The relative endophyte biomass in these associations was estimated by real-time PCR to be between 0.3 and 1.9%. Taking this difference into account, the steady-state levels of the ltm transcripts are about 100-fold greater than the levels of the endogenous ryegrass beta-tubulin (beta -Tub1) and actin (Act1) RNAs. Based on these results we propose that ltmG, ltmM and ltmK are members of a set of genes required for lolitrem biosynthesis in E. festucae and N. lolii.

Actins↗

Biological and physiological characteristics of Neotyphodium gansuense symbiotic with Achnatherum inebrians.

Biological and physiological characteristics of Neotyphodium gansuense were compared with Neotyphodium coenophialum and Epichloë festucae at a range of temperatures and pH values, and on carbon and nitrogen amended media. N. gansuense was able to grow at 10-30 degrees C, but not at 5 degrees C, and slowly at 35 degrees C. The optimal temperature for both N. gansuense and N. coenophialum was 25 degrees C, but that of E. festucae was 20-25 degrees C. The optimal pH ranges for mycelial growth of N. gansuense, N. coenophialum and E. festucae were 5-9, 5-9 and 5-7, respectively. The Neotyphodium and Epichloë endophytes varied in their ability to grow on media containing different carbon and nitrogen nutrients. The preference of N. gansuense for carbon source was sucrose>glucose, lactose, sorbitol, inulin, maltose, mannitol, starch, fructose>xylose. Growth of all three endophytes tested was significantly improved by peptone, tryptone, casein, yeast extract and l-proline. Yeast extract, peptone, casein, tryptone, l-proline, potassium nitrate, ammonium oxalic acid and l-leucine significantly improved growth of N. gansuense. However, ammonium nitrite was not utilized at all by any tested endophyte. N. gansuense grew significantly better on potato dextrose agar (PDA) and oat meal agar (OMA) than on corn meal agar (CMA) and drunken-horse-grass agar (DA), and most slowly on water agar (WA) and saltwater nutrient agar (SNA).

Carbon↗

[Investigations on the fermentation quality of silage from low-nitrate green forage].

In 3-year-experiments green forage from Dactylis glomerata and Festuca species, produced by differentiated cutting frequencies, was ensiled under laboratory conditions. The forage was almost without nitrate. Because of the higher content of water-soluble carbohydrates the fermentability coefficients of Festuca species were above of Dactylis glomerata. The fermentability coefficients of both plant species were inferior in the second and third cut, even if the first cut was delayed until July. Despite of similar fermentability coefficients of the two plant species silages contained different amounts of lactic and butyric acid. The portion of lactic acid amounted to 52 to 57% and 60 to 73% of total acids, the proportion of butyric acid 30 to 35% and 18 to 25% for Dactylis glomerata and Festuca species, respectively. According to the high fermentability coefficients silages free of butyric acid would be expected, however, by both species butyric acid always was formed, in some cases at high concentrations. In the contrary the amounts of ammonia, acetic acid and alcohol as well as the pH-value were almost extremely low. It can be concluded that the quality of silages produced of green forages with low nitrate cannot be assessed adequately by the current evaluation key.

Acetic Acid↗

Endophytic fungal beta-1,6-glucanase expression in the infected host grass.

Mutualistic fungal endophytes infect many grass species and often confer benefits to the hosts such as reduced herbivory by insects and animals. The physiological interactions between the endophytes and their hosts have not been well characterized. Fungal-secreted proteins are likely to be important components of the interaction. In the interaction between Poa ampla and the endophyte Neotyphodium sp., a fungal beta-1,6-glucanase is secreted into the apoplast, and activity of the enzyme is detectable in endophyte-infected plants. Sequence analysis indicates the beta-1,6-glucanase is homologous to enzymes secreted by the mycoparasitic fungi Trichoderma harzianum and Trichoderma virens. DNA gel-blot analysis indicated the beta-1,6-glucanase was encoded by a single gene. As a secreted protein, the beta-1,6-glucanase may have a nutritional role for the fungus. In culture, beta-1,6-glucanase activity was induced in the presence of beta-1,6-glucans. From RNA gel blots, similar beta-1,6-glucanases were expressed in tall fescue (Festuca arundinacea Schreb.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman) infected with the endophyte species Neotyphodium coenophialum and Epichloë festucae, respectively.

Amino Acid Sequence↗

Reactive oxygen species play a role in regulating a fungus-perennial ryegrass mutualistic interaction.

Although much is known about the signals and mechanisms that lead to pathogenic interactions between plants and fungi, comparatively little is known about fungus-plant mutualistic symbioses. We describe a novel role for reactive oxygen species (ROS) in regulating the mutualistic interaction between a clavicipitaceous fungal endophyte, Epichloë festucae, and its grass host, Lolium perenne. In wild-type associations, E. festucae grows systemically in intercellular spaces of leaves as infrequently branched hyphae parallel to the leaf axis. A screen to identify symbiotic genes isolated a fungal mutant that altered the interaction from mutualistic to antagonistic. This mutant has a single-copy plasmid insertion in the coding region of a NADPH oxidase gene, noxA. Plants infected with the noxA mutant lose apical dominance, become severely stunted, show precocious senescence, and eventually die. The fungal biomass in these associations is increased dramatically, with hyphae showing increased vacuolation. Deletion of a second NADPH oxidase gene, noxB, had no effect on the E. festucae-perennial ryegrass symbiosis. ROS accumulation was detected cytochemically in the endophyte extracellular matrix and at the interface between the extracellular matrix and host cell walls of meristematic tissue in wild-type but not in noxA mutant associations. These results demonstrate that fungal ROS production is critical in maintaining a mutualistic fungus-plant interaction.

Amino Acid Sequence↗

A symbiosis expressed non-ribosomal peptide synthetase from a mutualistic fungal endophyte of perennial ryegrass confers protection to the symbiotum from insect herbivory.

While much is known about the biosynthesis of secondary metabolites by filamentous fungi their biological role is often less clear. The assumption is these pathways have adaptive value to the organism but often the evidence to support this role is lacking. We provide the first genetic evidence that the fungal produced secondary metabolite, peramine, protects a host plant from insect herbivory. Peramine is a potent insect feeding deterrent synthesized by Epichloë/Neotyphodium mutualistic endophytes in association with their grass hosts. The structure of peramine, a pyrrolopyrazine, suggests that it is the product of a reaction catalysed by a two-module non-ribosomal peptide synthetase (NRPS). Candidate sequences for a peramine synthetase were amplified by reverse transcription polymerase chain reaction. Four unique NRPS products were identified, two of which were preferentially expressed in planta. One of these hybridized to known peramine producing strains. This clone was used to isolate an Epichloë festucae cosmid that contained a two-module NRPS, designated perA. Nine additional genes, which show striking conservation of microsynteny with Fusarium graminearum and other fungal genomes, were identified on the perA-containing cosmid. Associations between perennial ryegrass and an E. festucae mutant deleted for perA lack detectable levels of peramine. A wild-type copy of perA complemented the deletion mutant, confirming that perA is a NRPS required for peramine biosynthesis. In a choice bioassay, plant material containing the perA mutant was as susceptible to Argentine stem weevil (ASW) (Listronotus bonariensis) feeding damage as endophyte-free plants confirming that peramine is the E. festucae metabolite responsible for ASW feeding deterrent activity.

Animals↗

A clearing technique for detecting the fungal endophyte Acremonium sp. in grasses.

Leaf tissue of tall fescue Festuca arundinacea Schreb., hard fescue Festuca ovina L., red fescue Festuca rubra L. and perennial ryegrass Lolium perenne L. was stained with rose Bengal or aniline blue to detect the presence of the fungal endophyte Acremonium sp.. Specimens were cleared using methyl salicylate, an optical clearing agent, and viewed using bright field microscopy. Tissue was preserved as dried tissue or stored in 70% aqueous ethyl alcohol before staining and clearing. Tissue was observed at 2, 4 and 12 weeks following clearing to check for stain retention. Staining with rose Bengal was inferior to aniline blue when followed by the clearing agent methyl salicylate. Fungal mycelia stained lighter with rose Bengal and were more difficult to detect than mycelia stained with aniline blue. The results illustrate the usefulness of combining staining and methyl salicylate clearing for detecting fungal endophytes.

Acremonium↗

Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts.

Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMV(A/G) (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

Bromovirus↗

Amino acids as a nitrogen source in temperate upland grasslands: the use of dual labelled ((13)C, (15)N) glycine to test for direct uptake by dominant grasses.

It is becoming increasingly apparent that soil amino acids are a principal source of nitrogen (N) for certain plants, and especially those of N-limited environments. This study of temperate upland grasslands used glycine-2-(13)C-(15)N and ((15)NH4)(2)SO(4) labelling techniques to test the hypothesis that plant species which dominate 'unimproved' semi-natural grasslands (Festuca-Agrostis-Galium) are able to utilise amino acid N for growth, whereas those plants which dominate 'improved' grasslands (Lolium-Cynosurus), that receive regular applications of inorganic fertiliser, use inorganic N forms as their main N source. Data from field experiments confirmed that 'free' amino acids were more abundant in 'unimproved' than 'improved' grassland and that glycine was the dominant amino acid type (up to 42% of total). Secondly, the injection of representative amounts of glycine-2-(13)C-(15)N (4.76 and 42.86 mM) into intact soil cores from the two grassland types provided evidence of direct uptake of glycine by plants, with both (15)N and (13)C being detected in plant material of both grasslands. Finally, a microcosm experiment demonstrated no preferential uptake of amino acid N by the grasses which dominate the grassland types, namely Holcus lanatus, Festuca rubra, Agrostis capillaris from the 'unimproved' grassland, and Lolium perenne from the 'improved' grassland. Again, both (13)C and (15)N were detected in all grass species suggesting uptake of intact glycine by these plants.

Amino Acids↗

Vegetation establishment on a deposit of zinc mine wastes.

Field trials concerning the establishment of plant cover on a deposit of wastes from the Ammeberg zinc mine in central Sweden were carried out during 1976-1985. Different soil conditioners and manures were applied and plant species cultivars were evaluated with regard to plant biomass, vigour, durability and content of zinc, lead and cadmium. Sewage sludge and topsoil led to better establishment of grasses than did municipal waste, straw and hydraulic seeding. After 2 years, Festuca rubra and Poa pratensis dominated the swards. Other species (Dactylis glomerata, Bromus inermis, Lolium perenne, Phleum nodosum, Festuca pratensis and F. arundinacea) constituted only a minor part of the stand. After 10 years, F. rubra was the most dominant species, while native Agrostis tenuis had invaded 20-50% of the area within the plots. Merlin was the clearly dominant red fescue cultivar. The concentration of zinc in shoots (616 mg kg(-1) dw) was about 10% of that in the soil. Zinc concentration decreased with increasing biomass above ground. It increased with age in Scots pine needles and was very high in birch leaves. Grasses survived longer than legumes in the zinc sand waste. Among the surviving grasses was a group with high (3800 mg kg(-1) dw) and a group with low (320 mg kg(-1) dw) zinc concentrations. The low group included Merlin red fescue and Sobel creeping bent. The cultivar Merlin contained a much lower zinc concentration than the other cultivars of red fescue (375 and 624 mg kg(-1) dw, respectively). A large amount of root biomass was present in plots with dominating Merlin red fescue (1715 g m(-2)), 97% of which was concentrated in the top 10 cm of the soil. The concentration of zinc in the roots was very high (13 000-25 000 mg kg(-1) dw). Nitrate fertilizer, especially ammonium nitrate, and acidic water (pH 4.3) increased zinc leaching.

Journal Article↗