Within-population variation in localized and integrated responses of Trifolium repens to biotically patchy environments

Summary Genets of Trifolium repens (white clover) were collected from three patches of old permanent pasture dominated by Agrostis capillaris, Holcus lanatus or Lolium perenne. Plants derived from the genets were grown with plants of one grass species present on one side of each T. repens, and a different grass species on the other side, in all combinations of two of the three grasses. Different modules (a node with its associated internode, leaf, and axillary bud) on the same clover plant responded independently to the microenvironment provided by their own neighbouring grasses. In contrast, all apical meristems on the plant reacted similarly, showing a unified response and integrating the effects of the different microenvironments experienced by the whole clover plant. This is consistent with what is known both physiologically about the nutrition of meristems and modules, and ecologically about the exploratory growth habit of the species. Averaged over all associated grasses, there was no significant variation in the final dry weight of the different clover genets but these differed in their growth habit response to different grasses. In response to Agrostis as a neighbour, each meristem of T. repens rapidly produced many small modules. New modules were produced more slowly and were larger when Holcus or Lolium was the neighbour. The same pattern of differences occurred among clovers sampled from different backgrounds. Either genetic differences paralleled plastic responses, or plastic changes in phenotype that developed in response to different neighbours in the field persisted in the greenhouse. Plants taken from backgrounds of different grass species showed different responses to growing with those grass species. The differences were manifest primarily in a positive leading diagonal effect of Holcus or not-Holcus. They were the result primarily of differences in the dry weight per module and the probability of development of the axillary bud into a branch. This confirms earlier results, and implicates the central importance of branching as a means of local response to the microenvironment.     

The response of white clover (Trifolium repens L.) seedlings to spring root temperatures: The relative roles of the plant and the Rhizobium bacteria

Three experiments are reported which examine the relative roles of host and Rhizobium genotypes as factors limiting clover (Trifolium repens L.) growth at low soil temperatures.In the first experiment un-nodulated clover and perennial ryegrass (Lolium perenne L.) were grown with non-limiting nitrate at root temperatures of 8, 10 and 12°C. The ryegrass had substantially better relative growth rates (RGR) than the clover with the biggest difference occurring at 8°C. Alterations in growth rate with temperature were more marked in clover than in ryegrass but the latter still produced several times more dry matter than clover at each temperature.In the subsequent experiments clover nodulated with different strains of rhizobia was grown with and without non-limiting additions of nitrate at root temperatures of 9, 12 and 15°C. Plants receiving nitrate generally produced more dry matter than those dependent upon Rhizobium for nitrogen but differences in yield between these treatments did not alter with temperature. This suggests that limitations imposed by nitrogen fixation are similar at both high and low temperatures. Indeed, there was some evidence that nitrogen limitations were rather more pronounced at the highest temperature. The first experiment clearly demonstrated that the clover genotype makes particularly poor use of nitrate at low root temperatures when compared to its common companion perennial ryegrass.It can be concluded that improvements in spring growth of clover will rest largely with alterations to the plant genotype and its ability to use combined nitrogen for growth at lower temperatures rather than with changes in rhizobia or any symbiotic characters.     

Self fertilization and seed set in Trifolium repens L. by in situ and in vitro pollination

Summary The frequency of seed formation has been determined from self-pollination in situ (by hand) and in vitro for Trifolium repens. Selfing in situ was measured over a period of 3 years in which plants were held either at 35 °C for 24 h post-pollination (1984 and 1985) or held at ambient temperatures (1986). Mean yield of self-seed per 100 florets was 2.8 in 1984, 5.2 in 1985 and 2.2 in 1986. This was based on over 15,000 selfings per year with seven varieties and a total of 166 genotypes. In general, seed set following self-pollination was low; 53% of all genotypes set less than one seed per 100 florets selfed. Selfing of 340 excised florets in vitro with six genotypes gave a mean of 30.6 seeds per 100 florets. Temperature treatments (post-pollination) had no significant effect on seed yield in vitro. Treatment of florets in vitro for 24 h post-pollination with 0.1% CO₂ increased the yield of self seed with three genotypes but had no effect on a fourth genotype.     

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.     

Morphological responses to nutrient availability in four clonal herbs

This paper examines morphological plasticity of clonal plants of contrasting habitats and of contrasting architectures in response to nutrient supply. The hypotheses were tested that plants from rich habitats possess greater plasticity in response to variation in resource supply than species from poor habitats, and that rhizomatous species are less plastic in their response than stoloniferous species. Two sympodial rhizomatous herbs (Carex flacca, C. hirta) and two monopodial stoloniferous herbs (Trifolium fragiferum, T. repens) were subjected to four levels of nutrient supply in a garden experiment. One of the two species of each genus (C. hirta, T. repens) is from fertile and the other from infertile habitats. We measured 1) whole plant characters: total plant dry weight, number of modules (product of a single apical meristem) and number of ramets; 2) ramet characters: ramet leaf area and ramet height; and 3) spacer characters: branches per module, length per module and length per module internode.All measured characters in the Trifolium species significantly responded to treatment: the values for all measured characters increased with higher levels of fertilization. The differences in plant characters between fertilization levels were larger in Trifolium repens than in T. fragiferum in terms of whole plant characters, ramet characters and stolon internode length. The two Carex species did not differ in their responses to treatment in terms of most characters measured. In ramet characters and in some whole plant characters the species from fertile habitats were more plastic than those from infertile habitats. In spacer characters this pattern was not found. Foraging could not be demonstrated unequivocally.Morphological plasticity in the stoloniferous (Trifolium) species was much larger than in the rhizomatous (Carex) species. This seems in accordance with a foremost storage function of rhizomes, as against a foremost explorative function of stolons.     

Host selectivity and the mediation of competition by the root hemiparasite Rhinanthus minor

Summary Rhinanthus minor (Yellow-rattle) was grown in replacement series mixtures with Lolium perenne and Trifolium repens. The hemiparasitic interaction resulted in Relative Yield Totals (the sum of the yields in mixture relative to those in monoculture) considerably above 2. The hemiparasite caused a greater decrease in the yield of the legume and also performed better on the legume, indicating that T. repens was a better host for R. minor than L. perenne under the experimental conditions. When L. perenne and T. repens were grown in binary mixture with or without R. minor the hemiparasite affected considerably the competitive relationship between the two species by selectively parasitizing the legume. The effect of R. minor on competition between the two species was, however, dependent upon the nutrient status of the soil: the higher the level of soil nitrogen the fewer haustorial connections were made with T. repens and the less was the depression in its yield. In another series of experiments in which Festuca rubra, Holcus lanatus and L. perenne were grown in various binary mixtures with or without R. minor it was also shown that the yield of a preferred host was depressed to the advantage of a non-preferred host. It is suggested that the mediation of competition by the hemiparasite provides a mechanism by which it might affect the structure and diversity of plant communities.     

The effect of grasses on the quality of transmitted radiation and its influence on the growth of white clover Trifolium repens

Summary Plants of white clover Trifolium repens were grown under the canopies of three grass species, Lolium perenne, Agrostis tenuis and Holcus lanatus, and under simulated canopies of black polythene and controls were exposed to unfiltered natural radiation. The canopies were adjusted so that they transmitted equal intensities of Photosynthetically Active Radiation (P.A.R.). The ratio of red to far red radiation () was unchanged under the black polythene canopies but was reduced under canopies of Lolium and Agrostis and even more so under Holcus. The effect of canopy filtered radiation on the growth of clover was greatly to reduced internode length, mean number of nodes, the number of branched nodes and the number of rooted nodes and greatly to increase petiole length. The effect of canopies of Holcus was greater than that of the other grass species both in its effect on and on the responses of the clover plant to its shade.     

Pollination in vitro: effects on the growth of pollen tubes,seed set and gametophytic self-incompatibility in Trifolium pratense L. and T. repens L.

Summary Growth of pollen tubes and seed set were compared after hand pollination in situ and in vitro in two self-incompatible species, Trifolium pratense and Trifolium repens. Adhesion of pollen grains to the stigma was greater in vitro for both species. After cross-pollination, in vitro culture gave a significant increase in the cumulative growth of pollen tubes in pistils of T. pratense compared to in situ conditions. After selfing in T. repens, pollen tube growth was significantly increased by in vitro culture of florets. Seed set after crossing in situ and in vitro was similar for both species. Seed set after selfing in vitro was not increased in T. pratense. Several genotypes of T. repens were classified as very good, good and poor selfers based on their capacity for seed set following selfing in situ. In vitro pollination increased self seed formation by 1.7-, 18.0- and 31.0-fold for each class, respectively. Ovules located nearest to the style were fertilized more often after selfing than after crossing.     

Nitrous oxide emissions from grass swards during the eighth year of elevated atmospheric pCO2 (Swiss FACE)

Emissions of N₂O were measured during the growth season over a year from grass swards under ambient (360 μL L^?1) and elevated (600 μL L^?1) CO₂ partial pressures at the Free Air Carbon dioxide Enrichment (FACE) experiment, Eschikon, Switzerland. Measurements were made following high (56 g N m^?2 yr^?1) and low (14 g N m^?2 yr^?1) rates of fertilizer application, split over 5 re‐growth periods, to Lolium perenne, Trifolium repens and mixed Lolium/Trifolium swards. Elevated pCO₂ increased annual emissions of N₂O from the high fertilized Lolium and mixed Lolium/Trifolium swards resulting in increases in GWP (N₂O emissions) of 179 and 111 g CO₂ equivalents m^?2, respectively, compared with the GWP of ambient pCO₂ swards, but had no significant effect on annual emissions from Trifolium monoculture swards. The greater emissions from the high fertilized elevated pCO₂Lolium swards were attributed to greater below‐ground C allocation under elevated pCO₂ providing the energy for denitrification in the presence of excess mineral N. An annual emission of 959 mg N₂O‐N m^?2 yr^?1 (1.7% of fertilizer N applied) was measured from the high fertilized Lolium sward under elevated pCO₂. The magnitude of emissions varied throughout the year with 84% of the total emission from the elevated pCO₂Lolium swards measured during the first two re‐growths (April–June 2001). This was associated with higher rainfall and soil water contents at this time of year. Trends in emissions varied between the first two re‐growths (April–June 2001) and the third, fourth and fifth re‐growths (late June–October 2000), with available soil NO₃^? and rainfall explaining 70%, and soil water content explaining 72% of the variability in N₂O in these periods, respectively. Caution is therefore required when extrapolating from short‐term measurements to predict long‐term responses to global climate change. Our findings are of global significance as increases in atmospheric concentrations of CO₂ may, depending on sward composition and fertilizer management, increase greenhouse gas emissions of N₂O, thereby exacerbating the forcing effect of elevated CO₂ on global climate. Our results suggest that when applying high rates of N fertilizer to grassland systems, Trifolium repens swards, or a greater component of Trifolium in mixed swards, may minimize the negative effect of continued increasing atmospheric CO₂ concentrations on global warming.     

Plant competition and disease in genetically diverse wheat populations

Summary The direct and indirect effects of plant genetic diversity on epidemics and the influence of disease on plant competition were investigated using the wheat (Triticum aestivum)/stripe rust (Puccinia striiformis) system. Replacement series consisting of a susceptible and a resistant wheat genotype or two wheat genotypes susceptible to different races of stripe rust were grown in the presence and absence of the pathogen. Stripe rust severity, number of seed heads, seed yield, and seed weight were determined separately for each wheat genotype in the mixtures and the pure stands. The frequency of susceptible genotypes in a mixture explained up to 67% of the variation in disease severity. However, competitive interactions among plant genotypes sometimes appeared to alter susceptibility and obscured the relationship. In pure stands of single genotypes, disease severity explained between 52 and 58% of the variation in seed yield. In mixtures, coefficients of determination were only 10 and 31%, suggesting a strong influence of plant-plant interactions on seed yield. These results suggest that host-parasite coevolutionary models need to account for the strong effect that specific plant genotype combinations may have on disease severity and plant reproduction.Paper No. 9818 of the journal series of the Oregon Agricultural Experiment Station     

Nitrogen fixation by white clover when competing with grasses at moderately low temperatures

It was the aim of this study to determine the way in which low temperature modifies the effect of a competing grass on nitrogen fixation of a forage legume. White clover (Trifolium repens L.) was grown in monoculture or in different planting ratios with timothy (Phleum pratense L.) or perennial ryegress (Lolium perenne L.) in growth chambers at either 7.5/5°C (LoT) or 15/10°C (HiT) average day/night temperatures, and with 2.5 or 7.5 mM ¹⁵N-labelled nitrate in the nutrient solution.Competition with grass led to a marked increase in the proportion of clover nitrogen derived from symbiosis (% N_sym). This increase was slower at LoT where % N_sym was reduced considerably; it was closely related to the reduction in the amount of available nitrate as a result of its being utilized by the grass.Nitrogen concentration in white clover herbage and dry matter yield per clover plant were reduced, for the most part, when a competing grass was present. The amount of nitrogen fixed per plant of white clover decreased markedly with temperature. Low temperature consequently accentuated competition for nitrate. The capacity of white clover to compete successfully was limited by its slower growth and nitrogen accumulation.     

Effects of intercropping white cabbage with clovers on pest infestation and yield

During two consecutive years the effects of intercropping fresh market white cabbage with two species of clover on pest populations and yield were studied. White cabbage cv. Minicole was intercropped withTrifolium repens (white clover) andTrifolium subterraneum (subterranean clover) as compared to the monocrop. During the season observations were made on pest population developments, especially ofMamestra brassicae L. (cabbage moth),Brevicoryne brassicae L. (cabbage aphid),Delia brassicae L. (cabbage root fly), and evaluation of caterpillar feeding injury. At harvest the yield in quantity and quality was determined to be able to assess the gross financial result. Intercropping effects in terms of suppression of oviposition and larval populations of various pests were found. Although no pesticides were used and competition reduced the weight, the quality of the intercropped cabbages lead to a better financial result compared to the monocropped cabbage crop. The results are discussed in the perspective of the practical implications in the context of IPM.     

The effect of shoot and root genotype on phosphorus concentrations of shoots and roots

Genotypes of two morphologically different populations of white clover (Trifolium repens L.) were reciprocally and self-grafted. Ungrafted stolon tips were also grown as controls. Grafting per se had no significant effect on shoot size, root size, leaflet width or shoot and root % P. The scion genotype had a significant effect on shoot and root size, and leaflet width. Neither scion nor rootstock genotype had a significant effect on either shoot or root % P. However, there was a significant scion × rootstock × P level interaction for shoot % P. This along with other evidence suggests that conflicting results regarding effects of scion and rootstock on % P content of plants within species is probaby due to the interaction of scion and rootstock with environment.This work was undertaken at the Department of Agricultural Botany, University of Reading, Reading, England.This work was undertaken at the Department of Agricultural Botany, University of Reading, Reading, England.     

Selective grazing of acyanogenic white clover: Variation in behaviour among populations of the slug Deroceras reticulatum

Summary Collections of the slug Deroceras reticulatum were made from grassland sites containing contrasting frequencies of the cyanogenic morph of white clover, Trifolium repens. In choice chamber experiments, slugs obtained from sites with a low frequency of cyanogenic clover showed a significantly greater degree of selective eating of acyanogenic morphs than slugs taken from a site containing a high frequency of cyanogenic clover. Differences in selectivity between populations were caused both by differences in the rate of initiation of feeding on cyanogenic morphs, and by differences in the extent of damage once feeding had been initiated. The implications of these results for the cyanogenic polymorphism of T. repens are discussed.     

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.     

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.     

Nodulation of Trifolium repens with modified Bradyrhizobium and the nodulation of Parasponia with Rhizobium leguminosarum biovar trifolii

Thirty one strains of Rhizobium leguminosarum biovar trifolii isolated from the North and South American continents, New Guinea, USSR, Turkey and Australia, nodulated P. andersonii ineffectively when grown in plant growth tubes and in Leonard jars. Nodules were slow to form, sometimes taking over 100 days. Reisolates of R. leguminosarum biovar trifolii from P. andersonii nodulated Trifolium repens and their identity was confirmed using serological techniques. Dual occupation of nodules by Rhizobium and Bradyrhizobium in P. andersonii is reported. The reduced effectiveness of the Bradyrhizobium symbiosis depended on the relative numbers of Rhizobium occupants in this dual system. R. leguminosarum biovar trifolii and Bradyrhizobium strains from Parasponia were able to co-exist in nodules on P. andersonii and maintain similar populations in the rhizosphere and on culture media. Bradyrhizobium strains, separated from R. leguminosarum biovar trifolii, were able to initiate and form nodule-like structures on T. repens. Bradyrhizobium bacteria were identified as the sole occupants of the cells of the nodule-like structures on Trifolium repens using an immunogold labelling technique applied to ultrathin sectins. The re-isolates of Bradyrhizobium obtained from these nodule-like structures on T. repens were able to effectively nodulate P. andersonii.     

Competition between cyanogenic and acyanogenic morphs of Trifolium repens

Summary To investigate the cost of the dimorphic cyanogenic system in Trifolium repens L., genotypes of the cyanogenic (T_c) and acyanogenic (T_a) morphs were grown in mixtures over a range of ratios and in pure stands at two levels of total density (low in a first experiment, high in a second experiment). The principles of the competition analysis employed were those related to the inverse linear model response. The morphs were compared using two interaction indices, the substitution rate and the relative resource total (RRT). The relative fitness of the two morphs, i.e. biomass and number of flowers per plant, suggests that the T_a morph has a competitive advantage over the T_c morph.     

Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors

A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.     

Leaf shape variation and herbivore consumption and performance: a case study with Ipomoea hederacea and three generalists

The effect of leaf shape variation on plant-herbivore interactions has primarily been studied from the perspective of host seeking behavior. Yet for leaf shape to affect plant-herbivore coevolution, there must be reciprocal effects of leaf shape variation on herbivore consumption and performance. We investigated whether alternative leaf morphs affected the performance of three generalist insect herbivores by taking advantage of a genetic polymorphism and developmental plasticity in leaf shape in the Ivyleaf morning glory, Ipomoea hederacea. Across four experiments, we found variable support for an effect of leaf shape genotype on insects. For cabbage loopers (Trichoplusia ni) and corn earworms (Helicoverpa zea) we found opposing, non-significant trends: T. ni gained more biomass on lobed genotypes, while H. zea gained more biomass on heart-shaped genotypes. For army beetworms (Spodoptera exigua), the effects of leaf shape genotype differed depending on the age of the plants and photoperiod of growing conditions. Caterpillars feeding on tissue from older plants (95 days) grown under long day photoperiods had significantly greater consumption, dry biomass, and digestive efficiency on lobed genotypes. In contrast, there were no significant differences between heart-shaped and lobed genotypes for caterpillars feeding on tissue from younger plants (50 days) grown under short day photoperiods. For plants grown under short days, we found that S. exigua consumed significantly less leaf area when feeding on mature leaves than juvenile leaves, regardless of leaf shape genotype. Taken together, our results suggest that the effects of leaf shape variation on insect performance are likely to vary between insect species, growth conditions of the plant, and the developmental stage and age of leaves sampled. Handling editor: May Berenbaum.