Sitona weevils (Coleoptera: Curculionidae) as agents for rapid transfer of nitrogen from white clover (Trifolium repens L.) to perennial ryegrass (Lolium perenne L.)

The effects of root feeding by larvae of Sitona hispidulus (F.) (a common weevil pest of white clover) on the rate of transfer of nitrogen between plants of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.) were investigated using a nutrient slant board technique. Clover plants, labelled with ¹⁵N were grown adjacent to ryegrass plants and were either infested with Sitona larvae or not infested. Ryegrass plants associated with the infested clover plants had a significantly higher dry matter yield and nitrogen content (75% and 74% respectively) than the uninvested plants, after 33 days exposure to insect herbivory. It was concluded that root feeding insects could play an important role in the cycling of nitrogen in grass/clover swards.     

Effects of white clover (Trifolium repens L.) on plant and soil nitrogen and soil organic matter in mixtures with perennial ryegrass (Lolium perenne L.)

To increase our insight into the above- and belowground N flows in grass and grass-clover swards relations between crop and soil parameters were studied in a cutting trial with perennial ryegrass (Lolium perenne) monocultures and ryegrass–white clover (Trifolium repens) mixtures. The effects of clover cultivar on herbage yield, the amount of clover-derived nitrogen, apparent N transfer to companion grass, dynamics of N and organic matter in the soil were estimated.The grass monocultures had very low DM yields (<2.1 t ha^-1) and a low N concentration in the harvested herbage. During 1992–1995 the annual herbage DM yield in the mixtures ranged from 7.0 to 14.3 t ha^-1, the white clover DM yield from 2.4 to 11.2 t ha^-1 and the mean annual clover content in the herbage DM harvested from 34 to 78%. Mixtures with the large-leaved clover cv. Alice yielded significantly more herbage and clover DM and had a higher clover content than mixtures with small/medium-leaved cvs. Gwenda and Retor. Grass cultivar did not consistently affect yield, botanical composition or soil characteristics.The apparent N₂ fixation was very high, ranging from 150 to 545 kg N ha^-1 in the different mixtures. For each tonne of clover DM in the harvested herbage 49 to 63 kg N was harvested, while the apparent N transfer from clover to grass varied between 55 and 113 kg N ha^-1 year^-1.The net N mineralization rate was lower under monocultures than under mixtures. The C mineralization and the amounts of C and N in active soil organic matter fractions were similar for monocultures and mixtures, but the C:N ratio of the active soil organic matter fractions were higher under grass than under mixtures. This explains the lower N mineralization under grass.     

15N estimates of nitrogen fixation by white clover (Trifolium repens L.) growing in a mixture with ryegrass (Lolium perenne L.)

The ¹⁵N isotope dilution technique and the N difference method were used to estimate N₂ fixation by clover growing in a mixture with ryegrass, in a field experiment and a controlled environment experiment. Values obtained using N difference were approximately 25% lower than those estimated using ¹⁵N isotope dilution. In the field experiment there was a measured N benefit to grass growing with clover, equivalent to 42.7 kgN ha^-1. The grass in the mixture had a lower atom %¹⁵N content and a higher N content than grass in a monoculture; therefore values for N₂ fixation were different depending on choice of control plant i.e. monoculture or mixture grass. In the controlled environment experiment there were no significant differences between either the atom %¹⁵N contents or the N contents of monoculture grass and grass growing in a mixture with clover. It is concluded that there is a long term indirect transfer of N from clover to associated grass which can lead to errors in estimates of N₂ fixation.     

The nitrogen handling characteristics of white clover (Trifolium repens L.) cultivars and a perennial ryegrass (Lolium perenne L.) cultivar

Ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) have contrasting responses to soil mineral N availability and clover has the ability to fix atmospheric N(2) symbiotically. It has been hypothesized that these differences are the key to understanding grass-clover coexistence and vegetative dynamics in pastures. However, the whole plant response of clover and ryegrass to mineral N availability has not been fully characterized and inter-cultivar variability in the N-handling dynamics of clover has not been assessed. A detailed experimental study to address these issues was undertaken. For all clover cultivars and ryegrass, mass specific mineral N uptake rates (of whole plants) were similar saturating functions of mineral N availability. For all clover cultivars total N assimilation rates, whole plant C : N ratios and root : shoot ratios were independent of mineral N availability. Clover growth rates were also independent of mineral N availability except for a slight (<10%) reduction at very low N availability levels. Specific N(2) fixation rate (whole plant) was precisely controlled to ensure fixation balanced the deficit between mineral N uptake and the total N assimilation required to maintain constant whole plant C : N ratio. There was always a deficit between N uptake and the total N assimilation required to maintain C : N ratio. Consequently, some N(2) fixation remained engaged even at high mineral N availability levels. All inter-cultivar variation in N(2) fixation dynamics could be attributed to variations in growth rate. Clover mass specific growth rate declined as plant size increased. Ryegrass specific growth rate, whole plant C : N ratio and root : shoot ratio were dependent on N availability. Increased N availability led to increased growth rate and decreased C : N and root : shoot ratios. Specific growth rate was also dependent on plant size, growth rate declining as plant size increased. It is concluded that clover inter-cultivar variation in field performance is unlikely to be a consequence of variation in N-handling characteristics. Inter-cultivar differences in growth rate are likely to be a much more important source of variation.     

Ovule abortion in white clover (Trifolium repens L.)

Plants of white clover ( Trifolium repens L.) cv. Olwen were grown in an open glasshouse maintained at a mean temperature of 20^oC and ovule growth and seed production measured. Differences in the rate of growth of ovules within ovaries were observed as early as 2 days after pollination. Ovules reached a maximum size after 8 days with the smallest only half the size of the largest. After 8 days, the smallest ovules became flaccid and shrivelled. Ovule position within the ovary had little effect on the frequency of seed set and although there was an apparently higher probability that central ovules produced a seed than those nearer the peduncle or style this was not statistically significant. Inflorescence position and floret position on the inflorescence had a significant effect on the number of seeds per floret and seed weight; the first formed inflorescences and the first florets to be pollinated on each inflorescence had more seeds per floret and heavier seeds and fewer florets with no seed than later pollinated florets. There were also differences between florets within the same whorl. The role of a number of factors which may influence floret site utilisation are discussed.     

Inheritance of phosphorus response in white clover (Trifolium repens L.)

Genotypes of white clover that exhibited divergent responses to P were identified in a glasshouse pot trial. Six high P-responding genotypes were selected from previously identified high P-responding cultivars and 5 low P-responding genotypes were selected from previously identified low P-responding cultivars. These were crossed in a full diallel design without selfing and reciprocals were kept separate. The P-response of progeny lines was compared with parents. High P-response was dominant over low P-response with progeny from crosses between high and low P-response genotypes being similar to the high P-response parent. Reciprocal effects were not significant. The general combining abilities of high P-response genotypes were generally greater than that of the low P-response genotypes, although there were significant specific combining abilities. Narrow sense heritabilities for P response were moderate, 0.46 based on the linear coefficient and 0.33 based on the quadratic coefficient of the fitted response curves.The mode of inheritance, feasibility of manipulating differences in P response by breeding and future directions of this work are discussed.Deceased.Deceased.     

Response to soil aluminium of two white clover (Trifolium repens L.) genotypes

An aluminium (Al) tolerant genotype of white clover was compared with an Al susceptible genotype in artificial soil profiles in which exchangeable Al increased with depth. The tolerant genotype had a greater proportion of its root mass deeper in the soil than the susceptible genotype. Nitrogenase activity showed a similar pattern. Shoot Al concentration did not vary between the genotypes but root Al in the susceptible line was twice that in the tolerant genotype. Plant potassium content in the susceptible line was relatively less, probably in response to higher aluminium content.     

Interactions between perennial ryegrass ( Lolium perenne L.) and white clover (Trifolium repens L.) under contrasting nitrogen availability: productivity,seasonal patterns of species composition,N2 fixation,N transfer and N recovery

Nitrogen (N) fertiliser and clover cultivar choice affect competition and productivity in grass-clover mixtures. Pure stands and mixtures of perennial ryegrass and white clover cultivars with contrasting growth habits were examined. The aim of this work was to study the effect of repetitive nitrogen (N) application and cultivar combination on competition and productivity, N yield in the harvested herbage, N₂ fixation in mixtures and pure stands, and transfer of N from clover to the companion grass. Large-leaved white clover cultivar Alice and small-leaved cv. Gwenda and perennial ryegrass cvs. Barlet (erect) and Heraut (prostrate) were sown in pure stands and as four binary grass-clover mixtures on a sandy soil in 1995. In the mixtures, two levels of N fertiliser were applied: 0 (-N) and 150 and 180 kg ha^-1 y^-1 N (+N) in 1996 and 1997, respectively, while the grass monocultures received three N levels (0, 140/180 and 280/360 kg ha^-1) in 1996 and 1997, respectively. No N was applied to pure clover. The plots were cut five times during 1996 and six times during 1997. Fertiliser N was applied in early spring and after every harvest. The treatments were continued until the summer of 1999. In pure grass, the applied N was effectively recovered. In mixtures, N application affected competition by enhancing grass growth and the overall effect of N application was 17 kg DM per kg N applied in 1996. However, there was no yield response to N fertilizer in 1997, because this was compensated for by a higher clover production in unfertilised mixtures. In 1997, -N mixtures yielded more N than +N mixtures, owing to the higher clover content and N₂ fixation. Large-leaved clover cv. Alice was better able to withstand the negative effect of repetitive N application on clover production in mixtures and increased its proportion during the growing season of the second harvest year. In 1997, mixtures with Alice yielded more N than mixtures with Gwenda, but in pure clover swards, there was no cultivar effect on N yield. Also, during the autumn of 1998 and the spring of 1999, the clover content was highest in mixtures with Alice. Harvested N and apparent N₂ fixation were almost twice as high in 1997 as in 1996. N yield and apparent N₂ fixation were higher in pure clover than in mixtures. In mixtures, the apparent N₂ fixation in 1996 was 142 kg N ha^-1, irrespective of cultivar or N treatment. In 1997, it was on average 337 kg N ha^-1, and higher in -N mixtures and in mixtures with Alice. For each tonne of clover DM in the harvested herbage, 65 and 57 kg N was harvested in 1996 and 1997 in -N mixtures, respectively. The apparent transfer of clover-derived N to grass was on average 29 and 70 kg N ha^-1 yr^-1 in 1996 and 1997, respectively. It was highest in +N mixtures and highest in mixtures with Gwenda in 1997. In contrast to clover, the grass cultivars were very similar in their productivity and seasonal patterns, despite their contrasting growth habits. Seasonal trends in N yield, N transfer and N recovery are discussed in relation to fertilizer application regimes and variation in production patterns in mixtures and pure stands. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.     

Breeding of four-leaf white clover (Trifolium repens L.) through 60Co gamma-ray irradiation

Four-leaf white clover is not found easily in nature due to its low appearance rate (1 in 10,000). Because people believe that it brings good luck and like to either keep it or present it to a loved one, it has commercial and ornamental value. To breed four-leaf clover, we exposed its flowers to γ-rays at the pollination stage. The M₁ seeds produced following doses at 25–100 Gy showed an approximately 74% germination rate, with seedling survival at 46%. In the M₁ generation of plants irradiated within that dose range, we found an increased frequency of four leaflets. One of them, Jeju Lucky-1 (JL-1), had a frequency of about 60%. To see whether that mutation was somaclonal or genetic, we observed its M₂ generation and found that such a phenotype reappeared. Although our results demonstrated that the irradiation of fully mature flowers led to a higher frequency of 4-leaflets, we could not clearly explain the genetic mechanism involved. We suggest that JL-1 is valuable as a new variety, without further genetic fixation, because white clover can be propagated vegetatively by stolons. I.-J. Song and H.-G. Kang contributed equally to this work.     

The growth and asymmetry of neighbouring plants of white clover (Trifolium repens L.)

Summary Transplants of white clover (Trifolium repens L.) were grown isolated from each other and in pairs placed at different distances apart. The paired plants developed asymmetrically and at the interface between paired clones both the density of nodes and of stolons appeared to reach ceiling values that were of the same order as those achieved in isolated clones. It is argued that the growth of plants of T. repens is controlled by the local conditions experienced by the plant parts and not by integrated growth of the whole. Transplants of three different genotypes of T. repens, which differed in growth form, were grown as neighbouring pairs and the calculated asymmetry of the plants was used to compare their mutual aggressivenes. The more compact (phalangeal) genotypes induced greater asymmetry in their neighbours than the more diffuse forms.     

Detection of selection in populations of white clover (Trifolium repens L.)

Seed populations of white clover polymorphic for the presence/absence of both ovariogenic glucosides and the hydrolysing enzyme linamarase, were introduced into three natural populations. Over the first six months of life a significant increase in the frequency of linamarase containing individuals occurred. Estimated selection coefficients against plants lacking linamarase were in the region of 0.3. This result may have been due to selection at the enzyme locus alone, or to selection favouring cyanogenic individuals which possess both cyanogenic glucosides and enzyme.     

Effects of feed intake on enteric methane emissions from sheep fed fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne) forages

Published analyses of enteric methane (CH₄) emissions from sheep and cattle show an inverse relationship between feed intake and CH₄ yield (g CH₄/kg dry matter (DM) intake), which suggests opportunities for reducing CH₄ emissions from feed eaten and per unit of animal production. Most relationships between feed intake and CH₄ yield have been based on animals fed conserved feeds, especially silages and grains. Our research is a series of experiments with fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne; ryegrass) forages fed to sheep at a range of feed intake levels. This study was comprised of four experiments where good quality freshly harvested white clover or ryegrass were fed to sheep over a three-fold range in DM intake, and CH₄ emissions were measured in respiration chambers for two consecutive days in each experiment. Measurements were made from 16 sheep in Experiment 1 (fed at 1.6 × metabolizable energy requirements for maintenance; ME_m), 28 sheep in Experiment 2 (at 0.8 and 2.0 × ME_m), eight sheep and two measurement periods in Experiment 3 (at 1.6 × ME_m), and 30 sheep in Experiment 4 (fed at 0.8, 1.2, 1.6, 2.0 and 2.5 × ME_m). Prior to each experiment, sheep had a 10 d acclimatization period to diets. Apparent digestibility was measured over 7 d from sheep in Experiments 1, 3 and 4, along with collection of rumen digesta for volatile fatty acid (VFA) determination. Although CH₄ yields differed when sheep were fed white clover or ryegrass at similar intakes, the differences were inconsistent and mean values similar across all experiments. This, and a similar structure of all experiments, enabled combined analysis of data from all four experiments using the restricted maximum likelihood (REML) procedure to estimate effects of feed intake level on digestibility, digestible nutrient intake, gas emissions, and VFA concentrations in the rumen. The REML analysis showed that when DM intake increased from 0.40 to 1.60 kg/d, the predicted responses were an increase in CH₄ production (g/d) of 187% (12.4–35.6 g/d; P<0.001), and a decline in CH₄ yield of 21% (25.6–20.2 g/kg DM intake; P<0.001). High feed intake levels were associated with increased molar proportions (mM of total VFA) of propionate from 0.17 to 0.21 (P=0.038). Single and multiple regressions were completed on the data from all experiments, with organic matter (OM) intake predicting 0.87 of the variation in CH₄ production, and molar proportion of propionate predicting 0.60 of the variation in CH₄ yield. Increasing feed intakes by 1 kg/d of DM reduced CH₄ yield by 4.5 g/kg DM intake. Plant chemical composition was weakly related to CH₄ yield. High intakes of fresh forages will lower CH₄ yield from fermentation, but effects of feed composition on CH₄ emissions were minor. The interaction between effects of feed intake and rumen function requires further investigation to understand relationships with CH₄ emissions.     

Nitrogen relations of a low nitrate uptake inbred line of white clover (Trifolium repens L.)

The nitrogen relations of an inbred line of white clover (Trifolium repens L.) thought to exhibit an abnormally low capacity for NO₃ ^– uptake (line LNU) were compared with a line regarded as normal with respect to NO₃ ^– uptake (line NNU). Growth, nodulation, N₂ fixation and NO₃ ^– uptake were measured over 7 weeks in flowing solution culture (Experiment 1) by plants dependent for N acquisition on either (i) NO₃ ^– uptake, (ii) NO₃ ^– uptake +N₂ fixation, or (iii) N₂ fixation only. Effects of plant N status on the short-term uptake and translocation of ¹⁵NH₄ ⁺ and ¹⁵NO₃ ^– were also investigated (Experiment 2). Nitrate uptake per plant by –fix/+NO₃ ^– line LNU was 50% of uptake by line NNU over 35 days, and there were significant differences in specific uptake rates of NO₃ ^– between the lines over the first 24 days. The `low NO<sub>3</sub> <sup>–</sup> uptake' phenotype was indistinct under +fix/+NO<sub>3</sub> <sup>–</sup> treatment. Nitrate lowered specific rates of nitrogen fixation by line NNU but had no effect on line LNU. Only low N status line LNU plants had lower short-term rates of NH<sub>4</sub> <sup>+</sup> and NO<sub>3</sub> <sup>–</sup> uptake than line NNU. It is concluded that the `low NO₃ ^– uptake' phenotype of line LNU is inconsistently expressed. Circumstantial evidence points to increased NO₃ ^– efflux and decreased xylem translocation of NO₃ ^– as possible explanations for the lower NO₃ ^– uptake by line LNU.     

Carbon distribution and nitrogen partitioning in a soil-plant system with barley (Hordeum vulgare L.), ryegrass (Lolium perenne) and rape (Brassica napus L.) grown in a14CO2-atmosphere

To examine the influence of plant-microorganism interactions on soil-N transformations (e.g. net mineralization, net immobilization) a pot experiment was conducted in a¹⁴C-labelled atmosphere by using different (two annuals, one perennial) plants species. It was assumed that variation in below-ground, microorganism-available C would influence N transformations in soil. Plant species were fertilized (low rate) with¹⁵N-labelled nitrogen and grown, during days 13 and 62 after germination, in a growth chamber with a¹⁴C-labelled atmosphere. Nitrification was inhibited by using nitrapyrin (N-Serve). During the chamber period, shoots were harvested, and associated roots and soil were collected on two sampling occasionm, e.g. after 4 and 7 weeks in the growth chamber.The distribution of net (%) assimilated¹⁴C was significantly affected by both plant and time factors, and there was a significant plant × time interaction. There were significant differences between plants in all plant-soil compartments examined as well as in the degree of the plant × time interaction.Differences in the¹⁴C distribution between plants were due to both interspecific and developmental variation. In general, when comparing¹⁵N and¹⁴C quantities between species, many of the differences found between plants can be explained by the differences determined in the weight of shoot or root parts. Despite the fact that amounts of C released were greater in ryegrass than in the other plant-treatments no unequivocal evidence was found to show that the effects of plant-microorganism interactions on soil-N mineralization were greater under ryegrass. Possible mechanisms accounting for the partitioning of N found among plant biomass, soil biomass and soil residues are discussed.     

Translocation of phosphorus from nodal roots in two contrasting genotypes of white clover (Trifolium repens)

Patterns of translocation of recently-assimilated phosphorus (P) exported from'young' source roots (located 3–4 nodes from the stolon apex) and 'old' source roots (located near the base of the stolon) on the primary stolon of clonal plants of the forage legume white clover ( Trifolium repens L.) were determined using ³²P. Plants of a small-leaved genotype and of a large-leaved genotype were grown in sand culture at two notionally limiting or near-limiting rates of P supply and one non-limiting rate of supply. The small-leaved genotype showed little response in growth rate to the full range of P treatments whereas growth of the large-leaved genotype at the non-limiting rate of P supply was 2. 4 times greater than at the two low rates of P supply. Source roots of both genotypes exported only 26–30% of the P they acquired to the shoot within 24 h when P supply was limited whereas at the high-P rate 54% of recently-assimilated P was exported. Patterns of translocation of exported P to specific sinks differed little between the genotypes and the P treatments; branches were the main sink, accounting for nearly 80% of the estimated amounts of P (μg day⁻¹) exported from young and old roots combined. Translocation patterns from individual roots were determined largely by the modular structure of plants and by the location of the root relative to the major sinks, and were therefore consistent with the same source-sink principles which govern carbohydrate translocation in clonally-growing species. There were strong suggestions that storage of P in stolons and roots played a much greater role in the growth of the small-leaved plants than of the large-leaved plants.     

Heritability of,and relationships between phosphorus and nitrogen concentration in shoot,stolon and root of white clover (Trifolium repens L.)

Ninety eight white clover genotypes were cloned and grown in pots at two levels of phosphorus (P) supply in soil. After harvest the nitrogen (N) and P content of shoot (leaf, petiole and unrooted stolon), stolon and root tissue was determined. Broad sense heritabilities for %N, %P, and proportion of total N or P in each tissue type were calculated. Heritabilities ranged from 0.22 to 0.68. They were generally higher for %P than %N; and higher in shoot and stolon tissue than root tissue for %P, %N, and proportion of N or P. Level of P in which plants were grown had little effect on heritability values. Genotypes from bred cultivars differed from those collected from hill country pastures for plant size, and partitioning of N and P to shoot, stolon and root. Relationships between plant characters were examined to determine the consequences of selection.