Effects of condensed tannins in white clover flowers on their digestion in vitro

Protein in white clover (Trifolium repens L.) is poorly utilised by ruminants because of its extensive degradation to ammonia in the rumen. However, white clover produces condensed tannins (CT) in its flowers, which can reduce rumen proteolysis. Effects of increasing proportions of clover dry matter (DM) as flowers (and therefore floral CT) on soluble protein, ammonia and volatile fatty acid (VFA) concentrations were determined with in vitro incubations. Minced mixtures of 0, 250, 500, 750 and 1000 g/kg of DM as white clover flower (F) with the remainder as white clover leaf, were incubated in vitro and sampled after 0, 2, 4, 8, 12 and 24 h. Treatments contained 0, 13, 26, 39 and 52 g CT/kg DM, respectively. A further treatment with 500 g/kg DM as flower and 500 g/kg DM as leaf had polyethylene glycol added to remove effects of CT. Increasing the proportion of white clover as flowers from 0 to 1000 g/kg DM reduced net conversion of plant N to ammonia N from 290 to 120 mM/M at least partly due to reduced solubility of the protein. Treatments with 750 g/kg DM or more as clover flowers reduced ammonia concentrations to levels likely to limit microbial growth. Total VFA production was not affected by flower content, although the proportion of acetate to propionate increased. The contribution of CT to treatment effects was small compared to effects attributed to difference in chemical composition between flowers and leaves.     

The effect of nitrogen status on the regulation of plant-mediated proteolysis in ingested forage; an assessment using non-nodulating white clover

Production of nitrogenous waste by livestock agriculture is a significant environmental concern in terms of pollution of land and water. In the rumens of cattle and sheep, the excessive proteolysis which contributes to inefficiency of nutrient use involves both the rumen microbial population and the intrinsic plant proteases that can mediate protein degradation in ingested fresh forage on exposure to the environmental stresses of the rumen. Here, white clover (Trifolium repens) plants that do not form root nodules, and so are dependent on nitrate supplied to the roots, have been used to determine how nitrogen status of the plant affects the rate of plant‐mediated proteolysis in forage under conditions that simulate ingestion by grazing ruminants. Plants were grown from seed and supplied with nutrient solution containing 2.5, 5.0, 7.5 or 10 mM nitrate. Protein, free amino acid and protease activity were determined in leaves which had been placed in an in vitro system designed to simulate conditions experienced in the rumen (anaerobic phosphate buffer maintained at 39°C in the dark). Foliar protein content increased with increasing nitrate supply, while in vitro incubation of leaves resulted in time‐dependent decreases in protein concentration and increases in amino acid concentration. Regardless of nitrate supply, 50% of the protein was degraded in 6 h and 80% after 24 h. As the extent of protein decrease was determined by initial protein content, more protein degradation occurred in those plants grown with the highest nitrate supply: after 6 h, 130.7 mg g^?1 dry matter (DM) was degraded in leaves grown at 10 mM nitrate but only 52.3 mg g^?1 DM in leaves grown at 2.5 mM nitrate. Hence, although the percentage of proteolysis is independent of foliar protein concentration, the latter is critical to the quantity of protein degraded. Heat‐stable serine and cysteine proteases were active throughout the term of the in vitro incubation. Although proteolysis in ingested forage can continue for many hours, mediated by heat‐stable proteases, maximum amino acid accumulation accounted for less than 40% of initial protein. Therefore, it is proposed that continued and extensive proteolysis occurs following leaf excision and exposure to rumen conditions because amino acid accumulation is insufficient to initiate those feedback systems which sense cytoplasmic amino acid concentration and prevent excessive proteolysis during normal source–sink relations.     

Interaction between Sitona lepidus and red clover lines selected for formononetin content

Adult clover root weevil Sitona lepidus show a feeding preference for white clover Trifolium repens over red clover Trifolium pratense. The effects on S. lepidus of three red clover T. pratense lines, selected for high, medium, or low levels of the isoflavone formononetin in foliage, were compared in three experiments using white clover as a control. In a no‐choice slant board experiment, weevil larval weights were greater for larvae feeding on white clover roots than those feeding on roots of the red clovers. The effect of larval root herbivory on plant growth was similar for all four clovers. Following root herbivory, a large increase in root and shoot formononetin levels was observed in the high‐formononetin selection of red clover but little change in the low‐formononetin red clover. In a no‐choice experiment with sexually mature female adult weevils feeding on foliage of the four clovers, all the red clovers had increased weevil mortality. Female weevils eating the high‐formononetin red clover laid fewer eggs than weevils eating white clover. The red clover diet caused a large accumulation of abdominal fat and/or oil in the weevils, whereas weevils feeding on white clover did not accumulate fat/oil. When sexually immature adult weevils were given a choice of foliage from all four clovers, white clover was eaten preferentially, and the low‐formononetin red clover was preferred to the high‐formononetin red clover. The results suggest that formononetin and associated metabolites in red clover may act as chemical defences against adult S. lepidus and that distribution in forage legumes can be manipulated by plant breeding to improve root health.     

From plants to animals; the role of plant cell death in ruminant herbivores

Plant cell death occurring as a result of adverse environmentalconditions is known to limit crop production. It is less wellrecognized that plant cell death processes can also contributeto the poor environmental footprint of ruminant livestock production.Although the forage cells ingested by grazing ruminant herbivoreswill ultimately die, the lack of oxygen, elevated temperature,and challenge by microflora experienced in the rumen induceregulated plant stress responses resulting in DNA fragmentationand autolytic protein breakdown during the cell death process.Excessive ruminal proteolysis contributes to the inefficientconversion of plant to microbial and animal protein which resultsin up to 70% of the ingested nitrogen being returned to theland as the nitrogenous pollutants ammonia and urea. This constitutesa significant challenge for sustainable livestock production.As it is estimated that 25% of cultivated land worldwide isassigned to livestock production, it is clear that understandingthe fundamental biology underlying cell death in ingested foragewill have a highly significant role in minimizing the impactof human activities. This review examines our current understandingof plant metabolism in the rumen and explores opportunitiesfor exploitation of plant genetics to advance sustainable landuse. Key words: Anoxia, cell death, environment, heat, plant–microbe interactions, proteolysis Received 7 September 2007; Revised 21 November 2007 Accepted 23 November 2007     

Molecular Diversity of the Rumen Microbiome of Norwegian Reindeer on Natural Summer Pasture

The molecular diversity of the rumen microbiome was investigated in five semi-domesticated adult female Norwegian reindeer (Rangifer tarandus tarandus) grazing on natural summer pastures on the coast of northern Norway (71.00° N, 25.30° E). Mean population densities (numbers per gram wet weight) of methanogenic archaea, rumen bacteria and ciliate protozoa, estimated using quantitative real-time polymerase chain reaction (PCR), were 3.17 × 10⁹, 5.17 × 10¹¹ and 4.02 × 10⁷, respectively. Molecular diversity of rumen methanogens was revealed using a 16S rRNA gene library (54 clones) constructed using pooled PCR products from the whole rumen contents of the five individual reindeer. Based upon a similarity criterion of <97%, a total of 19 distinct operational taxonomic units (OTUs) were identified, nine of which are potential new species. The 16S rRNA sequences generated from the reindeer rumen exhibited a high degree of sequence similarity to methanogens affiliated with the families Methanobacteriaceae (14 OTUs) and Methanosarcinaceae (one OTU). Four of the OTUs detected belonged to a group of uncultivated archaea previously found in domestic ruminants and thought to be dominant in the rumen together with Methanobrevibacter spp. Denaturing gradient gel electrophoresis profiling of the rumen bacterial 16S rRNA gene and the protozoal 18S rRNA gene indicated a high degree of animal variation, although some bands were common to all individuals. Automated ribosomal intergenic spacer analysis (ARISA) profiling of the ruminal Neocallimastigales population indicated that the reindeer are likely to contain more than one type of anaerobic fungus. The ARISA profile from one animal was distinct from the other four. This is the first molecular investigation of the ruminal methanogenic archaea in reindeer, revealing higher numbers than expected based on methane emission data available. Also, many of the reindeer archaeal 16S rRNA gene sequences were similar to those reported in domesticated ruminants in Australia, Canada, China, New Zealand and Venezuela, supporting previous findings that there seems to be no host type or geographical effect on the methanogenic archaea community structure in ruminants.     

Influence of two selective factors on cyanogenesis polymorphism ofTrifolium repens L. in Darjeeling Himalaya

Cyanogenesis-the production of toxic hydrogen cyanide (HCN) by damaged tissue-inTrifolium repens L. (white clover), a type of most important pasture legume, has been studied at different elevations of Darjeeling Himalaya (latitude-27° 2′ 57″ N, longitude-88° 15′ 45″ E). Release of HCN takes place due to reaction between cyanogenic glucosides stored in vacuoles of the leaf cell and the corresponding enzyme β-glucosidase present in another compartment, often cell wall. Cyanogenesis, a defense system in plant, protects the clover from herbivore and inhibits grazing. Biochemical analysis showed the presence and absence of the cyanogenesis trait within the population in different proportions at different elevations. Acyanogenic individuals also showed variations with respect to presence or absence of either cyanogenic glucosides or β-glucosidase enzyme or both. The distribution of cyanogenic and acyanogenic plants was found in all places, but at lower altitudes (2084–2094 m) the dominating plants were cyanogenic whereas in higher altitude (2560 m) the dominating plants were acyanogenic. It was observed that blister beetle (Mylabris pustalata Thunb.) and the mollusc (Macrochlamys tusgurium Benson.) were the most common consumer of leaflets ofT. repens. Six categories of damage on white clover leaf by these animals were recorded. Our results suggest that the two selective factors or forces i.e. very cold temperature (harmful to cyanogenic plants) at higher altitude as well as indiscriminate but preferential predation (harmful to acyanogenic plants) interact to affect the system of cyanogenesis and also to cause the stable and protective polymorphism inT. repens rather than genotypic differences present among the plants.     

Evidence of a role for foliar salicylic acid in regulating the rate of post-ingestive protein breakdown in ruminants and contributing to landscape pollution

Ruminant farming is important to global food security, but excessive proteolysis in the rumen causes inefficient use of nitrogenous plant constituents and environmental pollution. While both plant and microbial proteases contribute to ruminal proteolysis, little is known about post-ingestion regulation of plant proteases except that activity in the first few hours after ingestion of fresh forage can result in significant degradation of foliar protein. As the signal salicylic acid (SA) influences cell death during both biotic and abiotic stresses, Arabidopsis wild-type and mutants were used to test the effect of SA on proteolysis induced by rumen conditions (39 °C and anaerobic in a neutral pH). In leaves of Col-0, SA accumulation was induced by exposure to a rumen microbial inoculum. Use of Arabidopsis mutants with altered endogenous SA concentrations revealed a clear correlation with the rate of stress-induced proteolysis; rapid proteolysis occurred in leaves of SA-accumulating mutants cpr5-1 and dnd1-1 whereas there was little or no proteolysis in sid2-1 which is unable to synthesize SA. Reduced proteolysis in npr1-1 (Non-expressor of Pathogenesis Related genes) demonstrated a dependence on SA signalling. Slowed proteolysis in sid2-1 and npr1-1 was associated with the absence of a 34.6 kDa cysteine protease. These data suggest that proteolysis in leaves ingested by ruminants is modulated by SA. It is therefore suggested that influencing SA effects in planta could enable the development of forage crops with lower environmental impact and increased production potential.     

The elimination of clover diseases by shoot tip culture

Shoot tip culture was used to eliminate white clover mosaic virus (WCMV) and red clover necrotic mosaic virus (RCNMV) from red clover, and clover phyllody disease (CP) and clover red leaf disease (CRL) from white clover. Shoot tips up to 2.4 mm (in some cases 3 mm) could regenerate plants free from the pathogens, but the efficiency of elimination, at least for WCMV and CRL, tended to decrease with increasing shoot tip size. The efficiency of plant regeneration from shoot tips generally improved with increasing tip size.     

Water deficit and plant competition effects on growth and water-use efficiency of white clover ( Trifolium repens,L.) and ryegrass (Lolium perenne,L.)

The combined effects of soil water deficit and above and below ground interspecific plant competition on the growth, water-use efficiency (WUE), and measured carbon isotopic composition (δ¹³C) values of white clover and ryegrass were studied. White clover and ryegrass were grown in specially designed crates 1) individually; 2) in shoot competition; or 3) in shoot + root competition and either well-watered or at a moderate or severe soil water deficit. The effects of shoot + root competition on shoot dry matter growth were substantial and benefited both white clover and ryegrass when well-watered or at a moderate soil water deficit, while severely reducing white clover shoot dry matter growth at severe soil water deficit. Plant competition did not affect the WUE of white clover or ryegrass. As soil water deficit increased, the WUE of white clover did not change whereas the WUE of ryegrass increased and was greater than that of white clover. This was attributed to the lower leaf water conductance of ryegrass which conserved water and maintained growth longer compared to white clover. A stronger correlation existed between soil water deficit and measured δ¹³C values for ryegrass at each plant competition level (P<0.001) than existed for white clover (individual: P<0.01; shoot + root: P<0.001; shoot: P<0.10). Unlike white clover, the relationship between measured δ¹³C values and shoot dry matter growth indicated that C assimilation for ryegrass was dependent on type of plant competition. That WUE remained constant for white clover while measured δ¹³C values increased as soil water deficit increased, suggests that the role below ground respiration rate played in determining δ¹³C values increased. The WUE of white clover appears to be independent of the nature of the competition between plants and the soil water deficit level at which it is grown, whereas for ryegrass, the addition of root competition to shoot competition should lead to increases in its WUE. This revised version was published online in June 2006 with corrections to the Cover Date.     

Symbiotically fixed nitrogen from field- grown white and red clover mixed with ryegrasses at low levels of15N-fertilization

A field study was carried out near Zürich (Switzerland) to determine the yield of symbiotically fixed nitrogen (¹⁵N dilution) from white clover (Trifolium repens L.) grown with perennial ryegrass (Lolium perenne L) and from red clover (Trifolium pratense L.) grown with Italian ryegrass (Lolium multiflorum Lam.). A zero N fertilizer treatment was compared to a 30 kg N/ha per cut regime (90 to 150 kg ha⁻¹ annually). The annual yield of clover N derived from symbiosis averaged 131 kg ha⁻¹ (49 to 227 kg) without N fertilization and 83 kg ha⁻¹ (21 to 173 kg) with 30 kg of fertilizer N ha⁻¹ per cut in the seeding year. Values for the first production year were 308 kg ha⁻¹ (268 to 373 kg) without N fertilization and 232 kg ha⁻¹ (165 to 305 kg) with 30 kg fertilizer N ha⁻¹ per cut. The variation between years was associated mainly with the proportion of clover in the mixtures. Apparent clover-to-grass transfer of fixed N contributed up to 52 kg N ha⁻¹ per year (17 kg N ha⁻¹ on average) to the N yield of the mixtures. Percentage N derived from symbiosis averaged 75% for white and 86% for red clover. These percentages were affected only slightly by supplemental nitrogen, but declined markedly during late summer for white clover. It is concluded that the annual yield of symbiotically fixed N from clover/grass mixtures can be very high, provided that the proportion of clover in the mixtures exceeds 50% of total dry mass yield.     

Influence of arbuscular mycorrhiza on clover and ryegrass grown together in a soil spiked with polycyclic aromatic hydrocarbons

 The effect of arbuscular mycorrhiza (AM) on white clover and ryegrass grown together in a soil spiked with polycyclic aromatic hydrocarbons (PAH) was assessed in a pot experiment. The soil was spiked with 500 mg kg^–1 anthracene, 500 mg kg^–1 chrysene and 50 mg kg^–1 dibenz(a,h)anthracene, representing common PAH compounds with three, four and five aromatic rings, respectively. Three treatments and two harvest times (8 and 16 weeks) were imposed on plants grown in spiked soil: no mycorrhizal inoculation, mycorrhizal inoculation (Glomus mosseae P2, BEG 69) and mycorrhizal inoculation and surfactant addition (Triton X-100). Pots without PAH were also included as a control of plant growth and mycorrhizal colonization as affected by PAH additions. The competitive ability of clover vis-à-vis ryegrass regarding shoot and root growth was enhanced by AM, but reduced by PAH and the added surfactant. This was reflected by mycorrhizal root colonization which was moderate for clover (20–40% of total root length) and very low for ryegrass (0.5–5% of total root length). Colonization of either plant was similar in spiked soil with and without the added surfactant, but the PAH reduced colonization of clover to half that in non-spiked soil. P uptake was maintained in mycorrhizal clover when PAH were added, but was reduced in non-mycorrhizal clover and in mycorrhizal clover that received surfactant. Similar effects were not observed on ryegrass. These results are discussed in the context of the natural attenuation of organic pollutants in soils. Accepted: 12 June 2000     

Root turnover in pasture species: chicory,lucerne, perennial ryegrass and white clover

For pastures, root turnover can have an important influence on nutrient and carbon cycling, and plant performance. Turnover was calculated from mini‐rhizotron observations for chicory (Cichorium intybus), lucerne (Medicago sativa), perennial ryegrass (Lolium perenne) and white clover (Trifolium repens) grown in the Manawatu, New Zealand. The species were combined factorially with four earthworm species treatments and a no‐earthworm control. Split plots compared the effects of not cutting and cutting the shoots at intervals. Observations were made c. 18 days apart for 2.5 years. This article concentrates on differences between plant species in root turnover in the whole soil profile to 40 cm depth. At this scale, earthworm effects were generally small and short lived. For ryegrass and white clover, root length and mass were linearly related (R² = 0.82–0.99). For chicory and lucerne, the relationships were poorer (R² = 0.38–0.77), so for those species length turnover may be a poor indicator of mass turnover. Standing root length, total growth and death generally decreased in the sequence ryegrass > lucerne > chicory = white clover. In length terms, scaled turnover (growth divided by average standing root length) generally followed the sequence lucerne > white clover > perennial ryegrass = chicory. Across species the scaled turnover rate averaged 3.4 per year or 0.9% per day. Cutting shoots reduced standing root length, growth and death, but increased scaled turnover. These results indicate fast and prolonged root turnover. For ryegrass and white clover, at least there is need to reappraise how to measure and model shoot : root ratios, dry matter production and carbon cycling.     

Population differences in<Emphasis Type="Italic"> Trifolium repens</Emphasis> L. response to ultraviolet-B radiation: foliar chemistry and consequences for two lepidopteran herbivores

White clover growing in New Zealand is experiencing increasing levels of ultraviolet-B (UV-B) radiation as a result of ozone depletion. We evaluated the effects of UV-B radiation on the foliar chemistry of two populations of white clover (Trifolium repens L.), ’Huia’ and ’Tienshan,’ and the consequences for the performance of armyworms (Spodoptera litura) and cutworms (Graphania mutans). Plants were grown in controlled environment rooms with and without supplemental UV-B radiation at a dose of 13.3 kJ m^–2 day^–1, corresponding to a 25% mid-summer ozone depletion above Palmerston North, New Zealand. In both white clover populations, UV-B radiation elicited changes in foliar chemistry, including slight increases in nitrogen concentrations and decreases in carbohydrate concentrations. In addition, the ’Huia’ population showed decreases in fiber concentrations and marked increases in cyanogenic activity. No change in UV-absorbing compounds was detected in either population. Long- and short-term feeding trials were conducted to assess dietary effects on insect growth, consumption, and food utilization. Changes in the performance of both insect species were generally small. The most pronounced effect was a 36% reduction in weight of S. litura after 2 weeks of feeding on Huia grown at high UV, but larval development times were only slightly prolonged and pupal weights were unaffected. S. litura short-term performance was affected by differences in white clover population. The long-term performance of G. mutans was not affected and its short-term performance (stadium duration and consumption rate) was only marginally affected by the high-UV treatment. We conclude that the effects of elevated UV-B radiation on white clover plant chemistry can be specific to certain plant populations. The differences in sensitivity of the two generalist insect species suggest that effects may also be specific to certain plant-herbivore associations. These results indicate that future UV-B herbivory studies should examine genotypic effects in both plants and animals. Received: 18 May 1999 / Accepted: 25 July 1999     

Effect of harvest time of red and white clover silage on chewing activity and particle size distribution in boli,rumen content and faeces in cows

The study examined the effects of harvest time of red and white clover silage on eating and ruminating activity and particle size distribution in feed boli, rumen content and faeces in cows. The clover crops were harvested at two stages of growth and ensiled in bales. Red clover crops had 36% and 45% NDF in dry matter (DM) at early (ER) and late (LR) harvest, respectively, and the white clover crops had 19% and 29% NDF in DM at the early (EW) and late (LW) harvest, respectively. The silages were fed restrictively (80% of ad libitum intake) twice daily to four rumen cannulated non-lactating Jersey cows (588 ± 52 kg) in a 4 × 4 Latin square design. Jaw movements (JM) were recorded for 96 h continuously. Swallowed boli, rumen mat, rumen fluid and faeces samples were collected, washed in nylon bags (0.01 mm pore size) and freeze-dried before dry sieving through 4.750, 2.360, 1.000, 0.500, 0.212 and 0.106 mm into seven fractions. The length (PL) and width (PW) values of rumen and faeces particles within each fraction were measured by use of image analysis. The eating activity (min/kg DM intake; P < 0.05) was higher in LR compared with the other treatments. The eating activity (min/kg NDF intake; P < 0.05) was affected by clover type with highest values for white clover silage. The mean ruminating time (min/kg DM), daily ruminating cycles (P < 0.001) and JM during ruminating (P < 0.05) were affected by treatment with increasing values at later harvest time. The proportion of washed particle DM of total DM in boli (P < 0.001), rumen mat (P < 0.001), rumen fluid (P < 0.01) and faeces was (P < 0.001) highest by feeding LR. There were identified two peaks (modes 1 and 2) on the probability density distribution (PDF) of PW values of rumen mat and faeces, but only one peak (mode 1) for PL values. There was no difference in the mean and mode 1 PW and PL value in rumen mat between the four treatments. The mean PL, mode PL, mode 2 PW and mean PW in faeces were highest for LR (P < 0.05). The mean particle size in boli measured by sieving was higher at white clover compared with red clover treatments (P < 0.001) and the highest value in faeces was found in LR (P < 0.01). The two peaks on PDF for width values of rumen mat and faeces particles are most likely related to the leaves and the stems/petioles. In conclusion, the mean total chewing activity per kg DM was lowest for the white clover silage and increased for both silages due to later harvest time. The mean particle size in boli was smallest for LR, whereas the mean PL and PW in faeces were highest for the LR.     

Suppression of myofibrillar proteolysis in chick skeletal muscles by α-ketoisocaproate

Summary. We previously reported that L-leucine suppresses myofibrillar proteolysis in chick skeletal muscles. In the current study, we compared the effects of L- and D-enantiomers of leucine on myofibrillar proteolysis in skeletal muscle of chicks. We also assessed whether leucine itself or its metabolite, α-ketoisocaproate (α-KIC), mediates the effects of leucine. Food-deprived (24 h) chicks were orally administered 225 mg/100 g body weight L-leucine, D-leucine or α-KIC and were sacrificed after 2 h. L-Leucine administration had an obvious inhibitory effect on myofibrillar proteolysis (plasma N^τ-methylhistidine concentration) in chicks while D-leucine and α-KIC were much more effective. We also examined the expression of the proteolytic-related genes (ubiquitin, proteasome, m-calpain and cathepsin B) by real-time PCR of cDNA in chick skeletal muscles. Ubiquitin mRNA expression was decreased by D-leucine and α-KIC but not L-leucine. Proteasome and m-calpain mRNA expressions as well as cathepsin B mRNA expression were likewise decreased by L-leucine, D-leucine and α-KIC. These results indicate that D-leucine and α-KIC suppress proteolytic-related genes, resulting in an decrease in myofibrillar proteolysis while L-leucine is much less effective in skeletal muscle of chicks, may be explain by conversion of D-leucine to α-KIC.     

Accumulation and Loss of Nitrogen in White Clover (Trifolium repens L.) Plant Organs as Affected by Defoliation Regime on Two Sites in Norway

In order to improve the basis for utilising nitrogen (N) fixed by white clover (Trifolium repens L.) in northern agriculture, we studied how defoliation stress affected the N contents of major plant organs in late autumn, N losses during the winter and N accumulation in the following spring. Plants were established from stolon cuttings and transplanted to pots that were dug into the field at Apelsvoll Research Centre (60°42′ N, 10°51′ E) and at Holt Research Centre (69°40′ N, 18°56′ E) in spring 2001 and 2002. During the first growing season, the plants were totally stripped of leaves down to the stolon basis, cut at 4 cm height or left undisturbed. The plants were sampled destructively in late autumn, early spring the second year and after 6 weeks of new spring growth. The plant material was sorted into leaves, stolons and roots. Defoliation regime did not influence the total amount of leaf N harvested during and at the end of the first growing season. However, for intensively defoliated plants, the repeated leaf removal and subsequent regrowth occurred at the expense of stolon and root development and resulted in a 61–85% reduction in the total plant N present in late autumn and a 21–59% reduction in total accumulation of plant N (plant N present in autumn + previously harvested leaf N). During the winter, the net N loss from leaf tissue (N not recovered in living nor dead leaves in the spring) ranged from 57% to 74% of the N present in living leaves in the autumn, while N stored in stolons and roots was much better conserved. However, the winter loss of stolon N from severely defoliated plants (19%) was significantly larger than from leniently defoliated (12%) and non-defoliated plants (6%). Moreover, the fraction of stolon N determined as dead in the spring was 63% for severely defoliated as compared to 14% for non-defoliated plants. Accumulation in absolute terms of new leaf N during the spring was highly correlated to total plant N in early spring (R² = 0.86), but the growth rates relative to plant N present in early spring were not and, consequently, were similar for all treatments. The amount of inorganic N in the soil after snowmelt and the N uptake in plant root simulator probes (PRS^TM) during the spring were small, suggesting that microbial immobilisation, leaching and gas emissions may have been important pathways for N lost from plant tissue.     

Neighbourhood effect of genotypes of Rhizobium leguminosarum biovar trifolii, Trifolium repens and Lolium perenne

 Ryegrass, white clover and Rhizobium isolated from the corresponding clover nodules, were harvested from a natural pasture in the Massif Central mountains (France). The specificity between Lolium, Trifolium and Rhizobium, and the genetic diversity of Rhizobium were examined. This study showed that: 1) Natural neighbouring combinations of white clover and ryegrass, re-planted together in pots, accumulated a higher biomass than non-neighbouring ones. This increase of mass is higher in the presence of the native strain of Rhizobium. 2) When white clover was inoculated with a mixture of Rhizobium strains, nodules were more often formed by its native strain. 3) The genetical diversity of the Rhizobium leguminosarum biovar trifolii was very high, as revealed by electrophoresis of esterases on seven substrates. These results support the hypothesis that there is a co-adaptation between white clover, ryegrass and Rhizobium Received: 25 March 1996 / Accepted: 13 September 1996     

Symbiotic growth of indigenons white clover (Trifolium repens) with local Rhizobium leguminosarum biovar trifolii

To study a possible adaptation of the symbiosis between white clover (Trifolium repens L.) and Rhizobium leguminosarum biovar trifolii with regard to light and temperature at northern latitudes, local seed populations of white clover and isolates of R. leguminosarum biovar trifolii from 3 different latitudes in Norway, 58°48'N, 67°20'N and 69°22'N, were used. The commercial cultivar Undrom was used as a reference plant. The experiments were done at 18 and 9°C under controlled conditions in a phytotron during the natural growing season at 69° 39'N. Growth of the plants was evaluated by number and size of leaves, dry matter production and total N-content. At 18°C the white clover plants were harvested twice while at 9°C there was only one growth period. The results from first harvest at 18°C and total growth at 9°C, showed that white clover populations from northern Norway had a lower growth potential than the population from the south and cv. Undrom. This difference was not apparent in the second growth period at 18°C. Growth of the plants from seeds to first harvest was enhanced by mineral nitrogen compared to plants dependent on Rhizobium only. However, after a second growth period dry weight and total nitrogen content of the plants with nitrogen fixation were comparable to the plants receiving mineral nitrogen. Statistical analysis showed that the most important factor for the variation in dry matter production was the plant population. Within the populations at 9°C and at first harvest at 18°C, there were no significant differences in dry matter production with different Rhizobium inoculum. In the second growth period at 18°C, different inoculum gave significantly different amount of dry matter within a population. The results showed a significant interaction between plant population and Rhizobium inoculum, and the results indicated that plants from the north gave higher yield when nodulated by Rhizobium from the north than from the south.     

Effect of pollen competition and stigmatic receptivity on seed set in white clover (Trifolium repens L.)

The effect of competition between incompatible and compatible pollen grains on the seed production of white clover was studied. Stigmatic receptivity was also studied. A selection line of white clover that has red leaves was used as a pollen donor and as a genetic marker to allow determination of the pollen donor responsible for ovule fertilisation. Results show that incompatible pollen did not inhibit compatible pollen grains from fertilising ovules and producing seeds, although it did slightly impair seed yield. At temperatures of 20/10°C (day/night) stigmas of white clover remained receptive to additional pollination up to 32 h after an initial compatible pollination and 40 h after an incompatible pollination. The results imply that factors other than inadequate pollination and the self-incompatibility system are responsible for failure of white clover to attain its potential seed yield. Received: 31 August 1999 / Revision accepted: 28 February 2000     

The Effect of Air Humidity on Photosynthesis of Ryegrass and White Clover at Three Temperatures

Increasing leaf-air vapour pressure deficit (VPD) decreasedthe stomatal conductance and the photosynthetic rate of leavesof ryegrass (Lolium perenne L.) and white clover (Trifolhimrepens L.) at light saturation and at lower irradiance. In ryegrassboth conductance and photosynthesis, and in clover photosynthesis,decreased less with increasing VPD in low irradiance than theydid at an irradiance which saturated photosynthesis. In ryegrass,relative to their values at 10 mb, photosynthesis and conductancedecreased less with increasing VPD at 25 °C than at 20 or16·5 °C. In white clover, relative conductance (butnot photosynthesis) was less reduced at 25 than at 16·5°C Measurements of VPD of air in the leaf canopy of a field-growncrop are combined with the observed responses of photosynthesisto VPD and temperature in a model. This shows that high VPDis likely to depress photosynthesis significantly and that,during a typical day, the rate of light saturated photosynthesismay remain fairly steady, because the depression of photosynthesisdue to rising VPD is offset by the stimulation due to risingtemperature Perennial ryegrass, Lolium perenne L., White clover, Trifolhim repens L., photosynthesis, leaf conductance, water vapour pressure deficit, temperature