Effect of potassium on the tolerance to PEG-induced water stress of two white clover varieties (Trifolium repens L.)

The response to water stress was studied on white clover grown hydroponically. Two varieties (Crau and Huia) were both subjected to a moderate and a more-severe stress, induced by polyethylene glycol (10 and 20% respectively), in the presence of a nutrient solution poor in potassium (K₁=0.005 mM), or abundantly supplied (K₂=5mM). Dawn water potential and nitrogen fixation (acetylene reduction activity) decreased with the increasing stress. Conversely, the stomatal resistance increased whenosmoticum was added. Crau had a lower stomatal resistance to the deficit, than did Huia. In relation with the K supply, treatment K₂ confirmed the superiority of Crau. Crau also showed greater nodule mass and number than Huia. The data show relationships between dawn water potential, stomatal resistance and nitrogen fixation activity.     

Plasticity of petioles of white clover (Trifolium repens) to blue light

Petiole response of white clover to variations in blue light (BL) was studied on the main axis and on primary and secondary branches. The objectives of the present work were to determine (1) the time course of petiole response to BL and (2) whether these responses were dependent on petiole location. Under BL, clover had shorter petioles, and the switch to conditions without BL increased the length of forthcoming petioles. The fitting of a logistic function was used to compare the effect of BL on final petiole length, maximum elongation rate and the duration of petiole elongation between axes and phytomers. Petiole response to BL was not dependent on its location within the plant (axis type or phytomer position along the axis). A reduction in BL induced a rapid increase in leaf elongation rate, despite a small decrease in the duration of petiole elongation. Moreover, petiole response was dependent on petiole stage of development: the increase in the maximum rate of petiole elongation was inversely proportional to the petiole stage of development at the time of the switch. We conclude that the effects of BL on petiole elongation were not dependent on its position within the plant, whereas internode elongation resulted from the integration of light environment at the plant level. The difference between the responses of orthotropic and plagiotropic organs of clover to BL is discussed in relation to their structural function and localisation in the canopy.     

Multivariate analysis of intraspecific responses to UV-B radiation in white clover (Trifolium repens L.)

White clover (Trifolium repens L.) is experiencing increased levels of ultraviolet‐B (UV‐B) radiation in temperate pastures due to the depletion of the stratospheric ozone layer. Based on 17 morphological, morphogenetic and physiological attributes, this study analysed the consequences of enhanced UV‐B on 26 white clover populations using principal components analysis (PCA). After 18 d of exposure to 13·3 kJ m ^{? 2} d ^{? 1} UV‐B in controlled environments, UV‐B significantly decreased above‐ground and below‐ground plant growth attributes, epidermal cell surface area and maximum quantum efficiency of photosystem II photochemistry (F_v/F_m). Aspects of cell division and cell expansion both were negatively affected by UV‐B. Stomatal density, specific leaf mass, root‐to‐shoot ratio and levels of UV‐B‐absorbing compounds increased in response to UV‐B. In the multivariate analysis, the main dimension of UV‐B sensitivity was characterized by changes in plant growth attributes. Alterations in partitioning within and between plant organs constituted a secondary tier of UV‐B responsiveness. Plant characteristics related to UV‐B tolerance included lower growth rate, smaller epidermal cell surface area and higher UV‐B‐induced levels of UV‐B‐absorbing compounds. The results suggest overall UV‐B tolerance for slower‐growing populations from less productive habitats with higher natural UV‐B irradiance.     

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.     

Nodulation of white clover (Trifolium repens) in the absence ofRhizobium

Summary Spontaneous nodules developed on the roots of white clover (Trifolium repens cv. Ladino) in the absence ofRhizobium. A small subpopulation of uninoculated clover plants (0.2%) exhibited white, single-to-multilobed elongated structures on their root systems when grown without fixed nitrogen. Clonal propagation using aseptic stolons confirmed the genetic stability of the observation. Few if any viable bacteria of unknown origin were recovered from surfacesterilized structures. Nodule contents were incapable of eliciting nodulation. Histological observations showed that these structures possessed all the characteristic features of indeterminate nodules, such as active meristem, cortex, endodermal layer, vascular strands, and a central zone with parenchyma cells. Infection threads, intercellular or intracellular bacteria were absent. Instead, numerous starch grains were observed in the central zone, a feature absent in normal nitrogen-fixing nodules. Our observation broadens the concept of spontaneous nodulation, believed to be restricted to alfalfa (Medicago sativa), to other legumes, and suggests a degree of generality among indeterminately nodulated legumes displaying natural heterozygosity.     

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     

Phosphorus supply and the growth of frequently defoliated white clover (Trifolium repens L.) in dry soil

A previous study found that increased phosphorus (P) supply to frequently defoliated white clover plants, growing in a low-P, dry soil, alleviated water stress symptoms and increased plant recovery on rewatering. In this study we determined how these stresses influence white clover growth. Measurements were made of the leaf canopy, stolon infrastructure and root system of the white clover plants growing in a low-P soil. Treatments included the factorial combination of four levels of P supply, two defoliation frequencies and two soil water treatments. White clover growth declined markedly when P-deficient plants were exposed to frequent defoliation and dry soil conditions. Leaf area was more affected than other parameters, in that the combination of stresses reduced leaf area to 2% of maximum observed for infrequently defoliated plants growing in high-P soil, with adequate water. Increased P supply generally increased the growth of all plant parts. Frequently defoliated plants growing in dry soil produced similar or greater leaf mass and leaf area as plants from similar treatments growing in wet soil, when the P supply increased to 50 mg P kg-1 soil. Higher P rates were able to negate the effect of dry soil on these frequently defoliated plants, as a result of larger water and P uptake. Also, the frequently defoliated plants with restricted root growth did not respond to a small increase in P supply (17 mg P kg-1 soil) for the leaf growth, irrespective of whether they were growing in wet or dry soil. Infrequently defoliated plants with greater root growth, compared to frequently defoliated plants, more than doubled their leaf mass with this P treatment.     

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.     

The effect of aluminium concentration on root hairs in white clover (Trifolium repens L.)

The effect of aluminium (Al) on root hair length and number is quantified using solution culture techniques with genotypes from white clover cultivar Tamar, that had previously been selected for long and short root hairs. The population differences were maintained in control (0 Al) treatments, with the long-haired population having hairs three times longer than the short-haired population. At an activity of 2.2 µM Al³⁺, root hair length decreased in both populations, the magnitude of the decrease being greater for the long-haired population. Root hair numbers decreased in a similar manner for both populations. At an activity of 4.4 µM Al³⁺ or higher, root hairs virtually disappeared and root growth was very stunted. The effect of Al on root hair development has not been previously quantified, however other workers have observed reduced root hair development in other species at activities of Al greater than 2.5 µM Al³⁺.     

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.     

Effect of nitrogen supply on frost resistance, nitrogen metabolism and carbohydrate content in white clover (Trifolium repens)

Effects of mineral nitrogen (2, 4, 6 and 8 m M NH₄NO₃) and nodulation with Rhizobium on frost hardiness in seedlings of white clover ( Trifolium repens ) have been studied. Seedlings of a population from Bodø (67°N lat.) were grown in Leonard jars under controlled conditions in a phytotron. For induction of frost hardening, plants were first exposed to 12 h photoperiod conditions for 2 weeks at 18°C, then for 2 weeks at 6°C and finally for 2 weeks at 0.5°C. Frost hardiness after treatments at 6 and 0.5°C was significantly enhanced by increasing nitrogen supply and was positively correlated with total nitrogen content of the stolons. Frost hardiness of nodulated plants correlated to the tissue nitrogen concentration. Content of soluble proteins in stolons decreased during hardening at 6°C but did not change during treatment at 0.5°C. There were minor changes in total amount of free amino acids during hardening. Both absolute and relative amounts of proline and arginine increased, and those of asparagine decreased during hardening. Absolute amounts of all free amino acids increased with increasing nitrogen supply, but the changes during hardening were similar in all treatments. There was a significant increase in the content of soluble carbohydrates during hardening. However, this increase was inversely related to nitrogen supply.     

Effects of temperature and photoperiod on frost resistance of white clover (Trifolium repens) ecotypes

Effects of photoperiod and temperature on frost resistance 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. Low-temperature-induced cold acclimation in all populations was significantly stimulated by short photoperiod and low temperature. The highest levels of frost resistance were found in the northernmost seedling populations and in clones from northern coastal locations.     

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.     

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.     

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.     

Leaf-atmosphere NH(3) exchange of white clover (Trifolium repens L.) in relation to mineral N nutrition and symbiotic N(2) fixation.

Plant-atmosphere NH(3) exchange was studied in white clover (Trifolium repens L. cv. Seminole) growing in nutrient solution containing 0 (N(2) based), 0.5 (low N) or 4.5 (high N) mM NO(3)(-). The aim was to show whether the NH(3) exchange potential is influenced by the proportion of N(2) fixation relative to NO(3)(-) supply. During the treatment, inhibition of N(2) fixation by NO(3)(-) was followed by in situ determination of total nitrogenase activity (TNA), and stomatal NH(3) compensation points (chi(NH(3))) were calculated on the basis of apoplastic NH4(+) concentration ([NH4(+)]) and pH. Whole-plant NH(3) exchange, transpiration and net CO(2) exchange were continuously recorded with a controlled cuvette system. Although shoot total N concentration increased with the level of mineral N application, tissue and apoplastic [NH4(+)] as well as chi(NH(3)) were equal in the three treatments. In NH(3)-free air, net NH(3) emission rates of <1 nmol m(-2) s(-1) were observed in both high-N and N(2)-based plants. When plants were supplied with air containing 40 nmol mol(-1) NH(3), the resulting net NH(3) uptake was higher in plants which acquired N exclusively from symbiotic N(2) fixation, compared to NO(3)(-) grown plants. The results indicate that symbiotic N(2) fixation and mineral N acquisition in white clover are balanced with respect to the NH4(+) pool leading to equal chi(NH(3)) in plants growing with or without NO(3)(-). At atmospheric NH(3) concentrations exceeding chi(NH(3)), the NH(3) uptake rate is controlled by the N demand of the plants.     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

Longevity of white clover (Trifolium repens) leaves, stolons and roots, and consequences for nitrogen dynamics under northern temperate climatic conditions

BACKGROUND AND AIMS: White clover (Trifolium repens) is, due to nitrogen (N) fixation, important to the N dynamics of several northern temperate agroecosystems. This study aimed at monitoring growth and death of major white clover plant organs to assess their potential contribution to within-season N input and risk of off-season N losses. METHODS: White clover ('Snowy') was studied in a plot and root window experiment in southeast Norway (60 degrees 42'N, 10 degrees 51'E). Leaves, stolons and roots were tagged for lifespan measurement in harvested and unharvested stands during two experimental years. The availability of soil inorganic N was measured by plant root simulator (PRS) probes. KEY RESULTS: The longevity of leaves and petioles ranged from 21 to 86 d (mean = 59 d), of main stolon sections from 111 to over 677 d (mean = 411 d) and of roots from 27 to 621 d (mean = 290 d). About 60 % of the leaves produced had turned over by the end of the growing season and another 30 % had died or disappeared by the subsequent spring. Harvesting reduced the longevity of stolons and increased plant fragmentation, but did not decrease leaf or root lifespan or increase soil N availability. From the plant organ turnover data, it was estimated that the gross N input to the soil-plant system from white clover in pure stand during two growing seasons corresponded to a 2.5-fold increase over the total N in harvestable shoots. CONCLUSIONS: The short lifespan and poor over-wintering of leaves showed their potential importance as a nitrogen source in the soil-plant ecosystem but also their potential contribution to the risk of off-season N losses.