Responses to UV-B radiation in Trifolium repens L. – physiological links to plant productivity and water availability

This study used comparisons across nine populations of Trifolium repens (white clover) in conjunction with drought to examine physiological responses to ultraviolet‐B radiation (UV‐B). Plants were exposed for 12 weeks to supplementation with 13.3 kJ m^?2 d^?1 UV‐B, accompanied by 4 weeks of drought under controlled environmental conditions. UV‐B increased the levels of UV‐B‐absorbing compounds and of flavonol glycosides and this effect was synergistically enhanced by water stress. These changes were more pronounced for the ortho‐dihydroxylated quercetin, rather than the monohydroxylated kaempferol glycosides. UV‐B increased leaf water potential (ψ_L) by 16% under drought and proline levels by 23% under well‐watered conditions. The intraspecific comparisons showed that higher UV‐B‐induced levels of UV‐B‐absorbing compounds, of quercetin glycosides and of ψ_L were linked to lower plant productivity and to higher UV‐B tolerance under well‐watered conditions. These findings suggest that: (1) slow‐growing T. repens ecotypes adapted to other stresses have higher capacity for physiological acclimation to UV‐B; and (2) that these attributes also contribute to decreased UV‐B sensitivity under drought.     

Response of Trifolium repens to UV-B radiation: morphological links to plant productivity and water availability

This study used nine populations of Trifolium repens L. (white clover) to investigate possible relationships between plant morphological attributes and responses to ultraviolet-B (UV-B) radiation. Plants were exposed to 0 or 13.3 kJ·m(-2) ·day(-1) UV-B for 12 weeks. Drought was applied in parallel to these treatments during the last 4 weeks of the experiment to test whether limited moisture availability would alter morphological UV-B responses. UV-B affected plant morphology under well-watered conditions, reducing leaf size by 15%, leaf number by 5% and stolon elongation by 19%. The number of leaf primordia in the apical bud was decreased by 4% under UV-B, and by 12% under drought. In drought-exposed plants, leaf size was reduced by 50%, leaf number by 30% and stolon elongation by 60%. In addition, drought reduced specific leaf area (SLA) by 33% and increased leaf percentage dry mass (PDM) by 40%. UV-B-induced reduction in plant biomass in the T. repens populations was associated with higher plant productivity and this was further linked to larger leaf size as well as to lower PDM. In conclusion, the findings suggest that morphological attributes conferring fast potential growth under productive conditions carry a cost in the form of lower biomass accumulation under UV-B.     

Photoinhibition of white clover seed germination at low water potential

Photosensitivity of germination of white clover ( Trifolium repens L. cv. Podkowa) seeds was studied under water deficit (low water potential) conditions at 25°C. The seeds showed negative photoblastism, which was most pronounced at -0.03 MPa polyethylene glycol solution. Inhibition was observed at two different wavelength bands with maxima at 660 nm (R) and around 730 nm (FR). Red light acted identically to white light (maximum inhibition ca 50%). The effect of far-red illumination was less inhibitory (20–30%). The photoresponse required long illuminations (3 h exposures); saturation level was at 0.1 W m⁻², independently of the light quality. White clover seed germination showed no reversibility of the effects of R and FR light. Prolonged illumination with R and FR increased the inhibition, and intermittent illumination had a higher effect than a continuous one. It was concluded that the photoinhibition of germination of seeds of Trifolium repens involves a reaction dependent on the rate of phytochrome interconversion, a property that is characteristic for the high irradiance reaction.     

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.     

A comparison of ovule abortion in self-incompatible and self-fertile lines of white clover (Trifolium repens L.)

Plants of white clover (Trifolium repens L.) cv. Olwen and three genotypes of each of three self-fertile inbred lines of white clover were grown from seed in a bee-proof glasshouse. When in flower, a series of crosses within and between the self-incompatible (SI) and self-fertile (SF) lines was made and ovule growth and seed production measured 7 and 28 days after pollination (DAP) respectively. At harvest, the SF plants had a lower proportion of ovules aborting and more seeds per floret than the SI plants, though the mean seed weight of the SI plants was greater. Ovule abortion was observed in both the SF and SI plants though the number of aborted ovules was greater in the SI plants. The stage when abortion occurred also differed. All of the ovule abortion in the SI plants occurred within a 7-day period after pollination. In the SF plants, some abortion was observed in this period but also between 7 and 28 DAP, when the seed was harvested. This suggested that abortion, up to 7 DAP, is due primarily to genetic factors whilst subsequent abortion, between 7 and 28 DAP, is due to non-genetic factors.     

Morphological compatibility of white clover and perennial ryegrass cultivars grown under two nitrate levels in flowing solution culture

The effects of nitrate (NO3-) supply on shoot morphology, vertical distribution of shoot and root biomass and total nitrogen (N) acquisition by two perennial ryegrass (Lolium perenne L.) cultivars (AberElan and Preference) and two white clover (Trifolium repens L.) cultivars (Grasslands Huia and AberHerald) were studied in flowing nutrient culture. Cultivars were grown from seed as monocultures and the clovers inoculated with Rhizobium. The 6-week measurement period began on day 34 (grasses) and day 56 (clovers) when the NO3- supply was adjusted to either 2 mmol m-3 (low nitrogen, LN) or 50 mmol m-3 (high nitrogen, HN). These treatments were subsequently maintained automatically. Plants were harvested at intervals to measure their morphology and N content. Cultivars of both species differed significantly in several aspects of their response to NO3- supply. In the grasses, the LN treatment increased the root : shoot ratio of AberElan but did not affect the distribution of root length in the root profile. In contrast, this treatment changed the root distribution of Preference compared with HN, resulting in a larger proportion of root length being distributed further down the root profile. The morphology of white clover Grasslands Huia was for the most part unaffected by the level of NO3- supply. In contrast, AberHerald exhibited different growth strategies, with LN plants increasing their stolon weight per unit length at the expense of leaf production, leaf area and stolon length, whereas HN plants showed reduced stolon thickness, greater leaf area production and stolon length per plant. Cultivars with different morphological/physiological strategies in response to NO3- supply may be of value in the construction of 'compatible mixtures' aimed at reducing oscillations in sward clover content by extending the range of conditions that allow balanced coexistence of species to occur.     

Physiological responses to water stress following a conditioning period in berseem clover

Berseem clover (Trifolium alexandrinum L.) is an important crop in semi-arid regions; its herbage and seed yields are often reduced by water stress. Our objectives were (i) to determine the effect of water stress, applied after a conditioning period, on water relations, proline accumulation and plant dry weight, and (ii) to investigate if some physiological responses differed in varieties of berseem. Five cultivars (Axi, Bigbee, Lilibeo, Sacromonte and Saniros) were grown in a controlled environment, and subjected to four irrigation treatments (T1, T2, T3 and T4 referring to plants irrigated to field capacity every 1, 2, 3 or 4 d, respectively) during a conditioning period (12 d). T1 treatment indicated the well-watered control, whereas T2, T3 and T4 treatments represented the conditioned plants. Leaf water potential (Ψ), osmotic potential (Ψπ), relative water content (RWC), gravimetric soil water content (GSWC) and leaf proline concentration were recorded during the conditioning period and a subsequent water deficit period (3 d) applied at early flowering growth stage. The conditioned plants subjected to subsequent water deficit maintained higher values of Ψ, Ψπ, RWC and GSWC, and lower values of leaf proline concentration. Reductions in parameter values were inversely related to the water stress severity that plants had previously experienced. At the end of the experiment, T1 showed 42%, 58% and 31% lower values for Ψ, Ψπ and RWC, respectively, than those of T4. Conditioned plants were also shorter and accumulated less leaf, stem and total dry weight. The conditioning treatments did not affect the relation between Ψ and Ψπ since conditioned plants show similar values of Ψπ as the control at the same Ψ value. Thus, drought acclimation in berseem clover contributed to water stress tolerance by the maintenance of tissue hydration. The berseem cultivars examined showed differences in plant growth parameters, but they were very similar for physiological responses to water deficit. The main genetic difference was recorded for turgor maintenance capacity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Incidence and severity of pest and disease damage to white clover foliage at 16 sites in England and Wales

From 1985 to 1987, leaves of white clover ( Trifolium repens ) cv. Grasslands Huia were examined for damage by pests and fungal diseases on seven occasions at up to 16 sites in England and Wales. Pest damage was recorded at all sites on all sampling dates. Over all sampling dates, the mean number of leaves damaged by slugs ranged from 23% to 67%, and that by weevils ( Sitona spp.) ranged from 3% to 62%. Also, up to 30% of leaves were damaged by other, unidentified pests. At individual sites, total pest damage frequently exceeded 90% of leaves. The area of leaf damaged by pests ranged from 2 - 12%. Fungal diseases were recorded in July and September but not in May, and were more prevalent in September 1985 than in September 1986 or 1987. Black blotch ( Cymadothea trifolii ) was the most frequently recorded disease, and the mean number of leaves damaged ranged from 4% to 21%. The mean area of leaves covered by lesions was 1–2%. Infections by viruses were assessed on two occasions, using indicator plants and electron microscopy, and only a very low incidence of arabis mosaic virus and red clover necrotic mosaic virus was recorded.     

Prolific direct plant regeneration from cotyledons of white clover

Summary A facile procedure has been developed to regenerate white clover (Trifolium repens L.) plants, rapidly and directly from cotyledon explants of 3 day old seedlings. Scanning electron microscopy and histological sectioning demonstrated that shoot meristems developed from individual epidermal cells on the adaxial surface of the cotyledonary stalk, proximal to the site of excision. Initial cell divisions occurred after 2 days of culture and regenerated plants were transferred to soil within 6–8 weeks. Regenerated plants were normal, flowered and set seed. The highest shoot regeneration frequency (an average of 20 shoots per cotyledon) was obtained using an MS based medium containing 1.0 mg 1^-1 6-benzylaminopurine and 0.05 mg 1^-1 -napthaleneacetic acid. A similar regeneration frequency was obtained from cotyledon explants taken from eight different white clover cultivars.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - MS Murashige and Skoog medium     

Seasonal plasticity is more important than population variability in effects on white clover architecture and productivity

Background and AimsThe drivers of white clover (Trifolium repens) architecture and productivity are still imperfectly understood. Our aim here was to investigate the impact of genetic background, neighbourhood and season on different architectural traits, clover and total biomass yield, as well as the relationship between those traits and yield.MethodsWe grew eight white clover populations in pure stands and in mixed stands with contrasting mixture partners. Over four consecutive regrowth periods within 1 year, we measured trait sizes and determined clover and total yield amounts.Key ResultsThe size of the architectural traits differed between populations and changed in response to neighbourhood and season. Population did not affect the sign or degree of those changes. Among the tested factors, season was by far the most important driver of white clover architecture, with the seasonal pattern notably differing between architectural traits. Clover and total yield were positively related to the architectural traits leaf area, petiole length, internode length and specific leaf area. Whereas the direction of the relationship was widely unaffected, its magnitude was clearly altered by neighbourhood and season.ConclusionsOur results show that seasonal effects are the key for a deeper understanding of the architecture of white clover individuals and to improve the productivity of white clover communities.     

Variation amongst survivor populations of white clover collected from sites across Europe: growth attributes and physiological responses to low temperature

Experiments were carried out at IGER, Aberystwyth, UK to investigate traits of direct relevance to the processes of overwintering and spring growth in white clover (Trifolium repens L.). The plant material used was derived from baseline populations of the cultivar AberHerald and survivor populations generated after 2-3 years' growth in Germany (Kiel), Sweden (Uppsala) and Switzerland (Zürich). The aims of the experiments were to measure the level of genetic shift that had occurred in certain traits due to selection in the survivor populations by comparing these with the baseline population. The adaptive significance of traits was assessed by determining the extent to which stabilizing selection had operated to reduce levels of intra-population variation. Significant differences were found in the responses of leaf production to two temperature treatments in the survivor populations from Germany and Sweden compared with the Swiss and baseline material. Plants of the former two populations produced much more leaf than the others at the higher temperature, but leaf production rates at the lower temperature did not differ. As this experiment used cloned genotypes in the two treatments, the result suggests that a higher degree of phenotypic plasticity for this trait had been selected for in the German and Swedish populations. These populations also showed greater rates of regrowth of leaves from terminal buds exposed to sub-zero temperatures, but there were no differences between populations in levels of freezing tolerance, or in stolon carbohydrate content. Genetic shift occurred in the degree of unsaturation of stolon lipids, with all three survivor populations possessing higher proportions of unsaturated fatty acids than the baseline. Stabilizing selection also operated on this trait in the survivor populations, suggesting that it is of adaptive significance in cool climates.     

A simple model of feedback regulation for nitrate uptake and N2 fixation in contrasting phenotypes of white clover

A simple three equation model is proposed for the feedback regulation of nitrate uptake and N2 fixation, based on the concentration of the organic N substrate pool within the plant and two parameters denoting the N substrate concentrations at which half-maximal inhibition occurs. This model simulated three contrasting phenotypes of white clover (Trifolium repens L.) inbred lines with (1) normal rates of nitrate uptake and N2 fixation (NNU); (2) low rates of nitrate uptake (LNU); and (3) very low rates of N2 fixation (VLF). The LNU phenotype was simulated by a decrease in the value of the inhibition parameter for nitrate uptake and the VLF phenotype was simulated by a decrease in the value of the N2 fixation inhibition parameter. The model was tested against nitrate uptake data obtained from white clover plants growing in flowing nutrient culture. There was an accurate prediction of the increase in nitrate uptake caused by N2 fixation activity of the NNU and LNU inbred lines being interrupted by a switch in gas phase from air to Ar : O2. The model was also tested against data for nitrate uptake, N2 fixation and %N from fixation for the three inbred clover lines grown in flowing nutrient culture at 0, 5 or 20 mmol m(-3) N(3-). Again there was accurate prediction of nitrate uptake, although simulated values for N2 fixation were more variable. The simple model has potential use as a sub-routine in larger models of legume growth under field conditions.     

Effect of four acid soils on root growth of white clover seedlings using a soil-on-agar procedure

White clover (Trifolium repens L.) is widely distributed in the Appalachian region, except on highly acid soils. We used a procedure where a thin layer of soil is placed on top of solidified water agar to characterize effects of acid soil on seedling root growth. Our objectives were to evaluate the soil-on-agar technique by using four soils (non-limed and limed) with diverse chemical characteristics and to relate root emergence to the chemical properties of the soils. We used three white clover cultivars, Grasslands Huia, Grasslands Tahora and Sacramento. Daily counts of root emergence from soil into agar were made for 12 d. Liming hastened white clover root emergence in three of the four soils. Days to 40% emergence were closely related (P < 0.01) to soil pH and to species of soil solution Al that are associated with Al toxicity in dicotyledonous plants. The r2 values for the regression of days to 40% root emergence on were 0.95, 0.96, 0.94 and 0.96, respectively. Apparently, the primary factor responsible for delayed root emergence in the soil-on-agar procedure was Al toxicity. Because of the close relationship between root emergence and activity of toxic species of soil solution Al, we propose that the soil-on-agar technique should be useful for characterizing the response of many small-seeded species to Al.     

Effects of temperature and photoperiod on vegetative growth of white clover (Trifolium repens) ecotypes

Effects of photoperiod and temperature on vegetative growth of seedling populations and clones of white clover ( Trifolium repens L.) originating from various latitudes (58°48'–69°54'N) and altitudes (up to 1100 m above sea level) were studied in a phytotron. Dry matter production, stolon elongation, petiole elongation and leaf lamina size were enhanced by long photoperiod. The requirement for long photoperiod increased with decreasing temperature. At 6°C the maximum growth was recorded under 24-h daylength. At 18°C already an increase in photoperiod from 12 to 15 h significantly enhanced growth, and maximum growth was obtained at 21-h photoperiod. The studied populations responded similarly to daylength, and the results did not indicate photoperiodic ecotypes in the material. The southernmost clones and populations generally had the highest dry matter production at all temperature treatments (6–18°C). Variation between clones within one location was, however, significant, and rapidly growing clones were found also in high-latitude locations. Dry matter production was poorly correlated with the morphological characters observed, but in some cases significant correlation with leaf lamina size was found.     

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     

Involvement of a high irradiance response in photoinhibition of white clover seed germination at low water potential

Seeds of white clover ( Trifolium repens L., cv. Podkowa) were germinated at water potential ψ=−0.3 MPa in darkness, at 25°C. A short exposure to blue light (B) inhibited germination in a manner similar to that described earlier for red (R) and far‐red (FR) light (Niedźwiedź‐Siegień and Lewak 1989). No reversibility of B, R and FR effects was observed. Saturation irradiance and energy was the lowest for R and the highest for B. The reciprocity of irradiance versus time of exposure was observed only for non‐saturating irradiances of B, R and FR.     

Photosynthesis and carbon export in white clover plants grown at various levels of phosphorus supply

Net photosynthesis, concurrent carbon export and starch, sucrose and inorganic phosphorus concentrations were measured in leaves of white clover ( Trifolium repens L. cv. Grasslands Huia) grown at four levels of phosphorus supply in the presence or absence of mineral nitrogen. The nitrogen treatments had no effect on growth, photosyntheis or carbon export. At the three higher levels of phosphorus supply, the amount of carbon exported was about 77% of net fixation. Photosynthesis and export per leaf decreased with phosphorus supply, primarily through the effect of phosphorus supply on leaf area. The rate of photosynthesis was reduced only at the lowest level of phosphorus supply.
Inorganic phosphorus rose with phosphorus supply but starch concentration was unaffected. Sucrose was reduced at the lowest level of phosphorus supply but not significantly affected at higher levels. The ratio between starch and sucrose concentration was also unaffected at the higher levels, but was increased at the lowest level of supply. There thus appeared to be direct effects of phosphorus supply on photosynthesis, partitioning of carbon to carbohydrates and, by implication, export, only at the lowest level of phosphorus supply. As leaf area and plant growth were affected over the whole range of phosphorus supply, factors other than photosynthesis per se must have determined the response of growth to phosphorus supply.