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.     

Infection of clover by plant growth promoting Pseudomonas fluorescens strain 267 and Rhizobium leguminosarum bv. trifolii studied by mTn5-gusA

Plant growth promoting Pseudomonas fluorescens strain 267, isolated from soil, produced pseudobactin A, 7-sulfonic acid derivatives of pseudobactin A and several B group vitamins. In coinoculation with Rhizobium leguminosarum bv. trifolii strain 24.1, strain 267 promoted clover growth and enhanced symbiotic nitrogen fixation under controlled conditions. To better understand the beneficial effect of P. fluorescens 267 on clover inoculated with rhizobia, the colonization of clover roots by mTn5-gusA marked bacteria was studied in single and mixed infections under controlled conditions. Histochemical assays combined with light and electron microscopy showed that P. fluorescens 267.4 (i) efficiently colonized clover root surface; (ii) was heterogeneously distributed along the roots without the preference to defined root zone; (iii) formed microcolonies on the surface of clover root epidermis; (iv) penetrated the first layer of the primary root cortex parenchyma and (v) colonized endophytically the inner root tissues of clover.     

Root exudates: a pathway for short-term N transfer from clover and ryegrass

The short-term transfer of nitrogen (N) from legumes to grasses was investigated in two laboratory studies. One study was done in pots where the roots of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.) were allowed to co-exist, and a second study was performed using a micro-lysimeter system designed to maintain nutrient flow from the clover to the grass, whilst removing direct contact between the root systems. The ¹⁵N-dilution technique was used to quantify the transfer of N between species. Levels of ammonia and amino acids were measured in root exudates. The amounts of N transferred were in the same order of magnitude in both the pot and micro-lysimeter experiments. In the micro-lysimeter experiment, 0.076 mg of N were transferred per plant from clover to ryegrass during the course of the experiment. Ammonium exudation was much higher than amino acid exudation. The most abundant amino acids in both clover and ryegrass root exudates were serine and glycine. However, there was no correlation between the free amino acid profile of root extracts and exudates for both plant species: Asparagine was the major amino acid in clover roots, while glutamine, glutamate and aspartate were the major amino acids in ryegrass roots. Comparison of exudates obtained from plants grown in non-sterile or axenic conditions provides evidence of plant origin of ammonium, serine and glycine.     

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.     

Evaluation of host and pathogen responses for screening soil amendments to control Sclerotium rolfsii

Three strains of Bradyrhizobium, 280A, 2209A and 32H1, that nodulated peanuts (Arachis hypogaea L.), were tested for their ability to grow and survive at elevated temperatures of up to 42°C in laboratory culture. Strain 32H1 was unable to grow at 37°C and was more sensitive to elevated temperatures than the other two strains. All three produced heat-shock proteins of molecular weights 17 kDa and 18 kDa. Two greenhouse experiments were conducted to determine the effect of high root temperature on nodulation, growth and nitrogen fixation of peanut. Two peanut varieties (Virginia cv NC7 and Spanish cv Pronto) were inoculated and exposed to root temperatures of 30°, 37° and 40°C. Nodulation and nitrogen fixation were strongly affected by root temperature but there was no variety × temperature interaction. At a constant 40°C root temperature no nodules were formed. Nodules were formed when roots were exposed to this temperature with diurnal cycling but no nitrogen fixation occurred. Highest plant dry weight, shoot nitrogen content and total nitrogen were observed at a constant root temperature of 30°C. Increasing root temperature to 37°C reduced average nitrogen content by 37% and total nitrogen by 49% but did not reduce nodulation. The symbiotic performance of the strains corresponded to their abilities to grow and survive at high temperature in culture.     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Effects of nitrogen supply and high temperature on the growth and physiology of the chickpea

Chickpeas were grown with or without nitrate nitrogen feeding, or nodulated with Rhizobium leguminosarum. High [40°C day, 25°C night (HT)] and moderate [25°C day, 177°C night (LT)] temperature regimes were employed during growth. Growth rates, photosynthetic capacity and enzymes of carbon and nitrogen metabolism were monitored to assess the acclimatory capacity of the chickpea. Initial growth rates were stimulated by high temperatures, particularly in nitrate-fed and nodulated plants. Older HT plants had fewer laterals, smaller leaves, and fewer flowers were produced than in LT plants. There was some indication of an acclimation of photosynthesis to high temperatures and this was independent of nitrogen supply. Rubisco activity was increased by high growth temperatures. However, HT plants also had higher transpiration rates and lower water use efficiency than LT plants both in respective growth conditions and when compared in a common condition. High temperatures reduced shoot nitrate reductase activity but had little effect on root activity, which was the same if not greater than activity in LT roots. The amino acid, asparagine, was found at high concentrations in all treatments. Concentrations were maintained throughout growth in HT plants but declined with age in LT plants.     

Influence of root temperature on growth of Pinus sylvestris,Fagus sylvatica,Tilia cordata and Quercus robur

One-year-old tree seedlings were incubated in a greenhouse from April to July, under natural daylight conditions, with their root systems at constant temperatures of 5, 10, 15, 20, 25, 30 and 35 °C and with the above ground parts kept at a constant air temperature of 18–20 °C. The course of height growth, total mass increment, root, shoot and leaf weight as well as leaf areas were measured. The results indicate that clear differences exist in the optimal root zone temperatures for various growth parameters in different tree species. Pinus sylvestris had a maximal height increment at about 5–10 °C and maximal total mass increment at 15 °C root temperature. In contrast, the optimum for Quercus robur was at 25 °C. Tilia cordata and Fagus sylvatica had their optima for most growth parameters at 20 °C. The root temperature apparently indirectly influenced photosynthesis (dry weight accumulation) and respiration loss. From the observed symptoms and indications in the literature it seems probable that a change in hormone levels is involved as the main factor in the described effects. Variation of root temperature had only an insignificant effect on bud burst and the time at which the shoots sprouted. Apparently species of northern origin seem to have lower root temperature optima than those of more southern origin. This is to be verified by investigation of other tree species.     

Effect of Temperature of the Culture Medium on Growth and Nitrogen Fixation of Inoculated Legumes and Rhizobia

Two pea (Pisum sativum L.) cultivars and a kidney bean (Phaseolus vulgaris L.) cultivars were grown in water cultures at different diurnal temperatures (15, 20, 24, 27, 30°C) or at 10°C night temperature combined with various day temperatures (20, 24, 27, 33 or 35°C) in the root medium. The inoculated plants were, more sensitive to the extreme temperatures than the plants supplied with combined nitrogen (KNO₃). The middle-European pea cv. Violetta was adapted to somewhat higher root temperatures than the northern one cv. Torsdag II, the latter showing better growth at lower temperatures, when the plants were inoculated with the same Finnish Rhizobinm strain (HA1). Especially at optimum day temperatures the nitrogen fixation and consequently the dry weights of the inoculated plants were greatly increased when the night temperature was lowered. The optimum temperature for the growth of free-living Rhizobium strains (HA1 and H43) for peus was found to be 25°C and that of a strain (P103) for beans somewhat higher. Effective nitrogen fixation by nodulated legumes without a supply of combined nitrogen is achieved only when the optimum temperature range for root function is very close to the optimum for the rhizobia.     

Temperature and seedling age affect susceptibility of perennial ryegrass seedlings to pathogenic fungi

Summary Effects of temperature and seedling age on survival of perennial ryegrass (Lolium perenne L.) seedlings grown on sand-wheat wholemeal cultures of different isolates ofFusarium spp. (9 isolates),Pythium spp. (9 isolates), andChaetomium spp. (1 isolate) are reported. Some isolates were virulent over the whole range of temperatures tested (7.5–27.5°C). The virulence of others depended on temperature. Most isolates were less virulent at intermediate temperatures (12.5–22.5°C) than at higher or lower temperatures. At 25°C ryegrass seedlings were susceptible to fungal attack for only a limited period after germination commenced. This period differed for different fungi, but for most isolates tested, seedlings were resistant after 2–3 days.     

Effects of genotype and temperature on pollen tube growth in perennial ryegrass (Lolium perenne L.)

Summary Low yield in seed crops of perennial ryegrass is related to low fertilization efficiency and low temperature during anthesis. To study the effect of genotype and temperature on pollen performance, we conducted greenhouse experiments at controlled temperatures. Individual florets of four genotypes that are known to differ in seed production were hand pollinated at four temperatures (14°, 18°, 22°, 26° C) both in vivo and via a semiin-vitro method involving excised florets on agar. Pollen germination and tube growth were determined with UV-fluorescence microscopy and scored in six classes at 2 h after pollination in vitro and after 0.5, 2 and 5 h in vivo. In vitro, both genotype and temperature had a significant effect on the performance of self-pollen. Pollen tube growth increased with temperature. In cross-pollinations, the pistil parent had a significant effect on pollen tube growth, and there was also a significant pistil-by-temperature interaction. In vivo, genotype and temperature significantly affected pollen performance. The genotype-by-temperature interaction was only significant 5 h after pollination. One genotype with low seed yield was pseudoself-compatible and was a relatively poor mother after cross-pollination. The effects of genotype and temperature on the growth of self-pollen might be exploited in a breeding programme.A.G. Stephenson was on a sabbattical leave at SVP in 1987     

Effect of white clover cultivar on apparent transfer of nitrogen from clover to grass and estimation of relative turnover rates of nitrogen in roots

The apparent transfer of N from clover to associated grass was evaluated over a four year period both on the basis of harvested herbage and by taking account of changes in N in stubble and root (to 10 cm depth) in swards with perennial ryegrass and three different white clover cultivars differing in leaf size. The large leaved Aran transferred 15% of its nitrogen while Huia transferred 24% and the small leaved Kent Wild White transferred 34%. When changes in stubble and root N were taken into account the percentage of N transferred was calculated to be 5% less than in harvested herbage only, as the small leaved types had proportionately more N in the roots and stolons, but the large leaved type was probably more competitive towards the grass.Loss of N from clover roots from July to October was compared to that from grass roots in a grass/white clover sward continuously stocked with steers using a method which incorporated tissue turnover and ¹⁵N dilution techniques. Less than 1 mg N m^-2 d^-1 was lost from the grass roots. In contrast 8 mg m^-2 d^-1 were estimated to be lost from clover roots while 12 mg N m^-2 d^-1 were assimilated.It is concluded that clover cultivar and competitive ability on grass have to be taken into account together with the relationship between N turnover in roots and N available for grass growth when modelling N transfer in grass/clover associations.     

Short-term survival of rhizobia on arrowleaf clover seed sown at different depths

Seed of arrowleaf clover (Trifolium vesiculosum Savi) were inoculated with a streptomycin resistant mutant ofRhizobium leguminosarum biovartrifolii and planted on the surface of a Norwood fine sandy loam and at 10 and 25 mm depths. Populations of rhizobia declined from an excess of 10,000 seed⁻¹ immediately after inoculation to less than 100 within three to four days after sowing on the soil surface when water was the peat inoculant adhesive. Gum arabic as the adhesive promoted the survival of rhizobia. Populations of rhizobia on surface sown seed declined much more rapidly than on seed buried in soil. Although, the soil was nearly air dry, rhizobia on buried seed survived at populations exceeding 1,000 seed⁻¹. The maximum soil temperatures ranged between 21 and 36°C over the sampling time and did not seem to have a major influence on short term survival of rhizobia. Delayed germination of seed due to the higher temperature would indirectly influence the number of viable rhizobia present at germination.     

Nitrogen limitations to field bean productivity: A comparison of combined nitrogen applications with Rhizobium inoculation

Summary Nitrogen limitations to the yield of a field crop ofVicia faba have been examined. Application of nitrogen totalling 560 kg/ha increased dry matter yield at flowering by 674 kg/ha (32%) and grain yield at final harvest by 1.6 tonnes/ha (24%). Attempts to reduce nitrogen limitations by replacing the native rhizobia with strains ofRhizobium leguminosarum selected for high rates of nitrogen fixation were unsuccessful but the introduction of poor rhizobia reduced grain yield. The reasons for this and the implications of the results for crop improvement are discussed.     

Effect of Temperature and Nitrogen Supply on the Growth of Perennial Ryegrass and White Clover. 2. A Comparison of Monocultures and Mixed Swards

White clover (Trifolium repens L.) and Perennial ryegrass (Loliumperenne L.) plants were grown, in Perlite, in simulated swardsas either monocultures or mixtures of equal plant numbers. Theywere supplied with a nutrient solution either high (220 µgg^–1) or low (40 µg g^–1) in ¹⁵N-labelled nitrateand grown to ceiling yield at either high (20°C day/15°Cnight) or low (10°C day/8°C night) temperature. Temperature had little effect on the maximum rates of grosscanopy photosynthesis which were similar in High-N grass andHigh-N and Low-N clover monocultures. However these maxima werereached more slowly in clover than grass, and more slowly atlow rather than high temperature. Nitrogen supply increasedphotosynthesis in grass but not in clover. Clover had higherN contents than grass in all four treatments, although in anygiven treatment its N content was lower, and contribution ofN₂-fixation relative to nitrate uptake higher, in mixture thanin monoculture. Conversely, grass had higher N contents in mixturethan monoculture, because more nitrate was available per plantand not because of transfer of biologically fixed N from clover. Under Low-N, clover outyielded grass in mixture, particularlyat high temperature. The grass plants in the Low-N mixtureshad higher N contents and higher SLA, LAR and shoot: root ratiosthan those in monoculture. It is proposed that competition forlight is the cause of the low relative yield and negative aggressivityof grass in these swards. Under High-N, grass outyielded cloverin monoculture and mixture, at both temperatures but particularlyat low temperature when grass had a high aggressivity. Nitrogenand yield component analyses shed no light on clover's apparentlylow competitive ability and evidence is drawn from the previouspaper to demonstrate that grass grew faster than clover onlyas spaced individuals during non-com petitive growth. The relativemerits of measures of competitive ability based on final harvestdata and physiological data taken over a growth period are discussed. Trifolium repens L., white clover, Lolium perenne, perennial ryegrass, competition, temperature, nitrogen     

Reduction of the nodulation barrier inMedicago laciniata by alteration of the root temperature

Medicago laciniata, an annual leguminous plant of Saharo-Sindian origin, is particularly refractory to root nodulation by most strains ofRhizobium meliloti. Using a series of such bacterial strains belonging to the 8 groups of Brockwell and Hely, and a variety of environmental conditions, it was noted that several normally non-nodulating strains (at 20°C) produced ineffective nodules at root temperatures of 24°C to 28°C. Nodulation at 20°C failed to occur in the presence of a wide variety of test compounds and physical conditions. No phytoalexins or anti-Rhizobium growth inhibitors were isolated from inoculated root tissue at any temperature. Temperature shift experiments indicated no infection of the root hairs at 20°C, and infection threads produced at the permissive root temperature failed to elongate after transfer to 20°C. However, if meristematic activity had been initiated in the inner root-cortical cells as a result of infection thread penetration at 28°C, no blockage of nodule maturation occurred upon subsequent transfer to 20°C root temperature. Nodules produced at 28°C were completely devoid of nitrogenase activity, although the apical (but not the distal) regions contained normal-appearing bacteriods, surrounded by enclosing membranes, and possessed a fully functional leghaemoglobin. A shortage of metabolic energy did not appear to be involved in the ineffective response. A hypothesis to explain the nodulation phenomenon observed was based on the observation in the roots of 2 factors present at 20°C but not at 28°C.     

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.     

Influence of dissimilarities in temporal and spatial N-uptake patterns on15N-based estimates of fixation and transfer of N in ryegrass-clover mixtures

The temporal N-uptake patterns of white clover (Trifolium repens L.) mixed with perennial ryegrass (Lolium perenne L.) and of red clover (Trifolium pratense L.) mixed with Italian ryegrass (Lolium multiflorum Lam.) were determined in successive harvests of herbage within the growth cycles of a ley established near Zürich (Switzerland). Rooting patterns were examined by injecting¹⁵N-fertilizer at soil depths ranging from 10 to 40 cm. The results were analyzed to determine the effect of variations in time and depth of N-uptake on the¹⁵N-based measurement of N from symbiosis (N_sym) and N from transfer (N_trans).Grasses in mixture appeared to have deeper rooting systems than grass monocultures, which led to an overestimation of N transfer from white clover to perennial ryegrass if¹⁵N was spread on the soil surface.White clover generally lagged behind grass in soil N- uptake. Soil N-uptake of red clover slowed down before that of the grass because % N_sym almost reached 100% during the second half of each growth cycle. However, the effect of these dissimilarities on the seasonal average of %N_sym did not exceed 2%.It is concluded that at the observed high levels of N₂ fixation, failure to account for the N-uptake patterns of the test and reference crops only slightly affected the estimates of % N_sym and % N_trans, and did not invalidate the observed differences between species.     

Use of white clover as an alternative to nitrogen fertiliser for dairy pastures in nitrate vulnerable zones in the UK: productivity, environmental impact and economic considerations

Perennial ryegrass and perennial ryegrass/white clover permanent dairy pastures are compared with respect to productivity, environmental impact and financial costs in nitrate vulnerable zones (NVZ) in the UK. With appropriate management, and utilisation of recommended perennial ryegrass and white clover cultivars, white clover is likely to stabilise at around 20% of total dry matter production in a mixed pasture. Plant dry matter production and milk production from a perennial ryegrass/white clover pasture are likely to be similar to that from a perennial ryegrass pasture receiving 200 kg N ha⁻¹ annum⁻¹ and around 70% of that obtained with perennial ryegrass supplied with 350–400 kg N ha⁻¹ annum⁻¹. Nitrate, phosphorus and methane losses from the system and decreases in biodiversity relative to a grazed indigenous sward are likely to be similar for a perennial ryegrass/white clover pasture and a perennial ryegrass pasture receiving 200 kg N ha⁻¹ annum⁻¹: nitrate leachate from both systems is likely to comply with European legislation. Greenhouse gas emissions resulting from nitrogen (N) fertiliser production would be avoided with the perennial ryegrass/white clover pasture. Within NVZ stocking rate restrictions, white clover can provide the N required by a pasture at a lower financial cost than that incurred by the application of N fertiliser.     

Interactions between nitrate uptake and N2 fixation in white clover

Summary White clover (Trifolium repens L.) plants grown in pots and supplied with the same concentration x days of¹⁵N labelled nitrate, but in contrasting patterns and doses had similar N concentrations but differed in the proportions devived from N₂ fixation and nitrate. N₂-fixation and nodule dry weight responded rapidly (2–3 days) to changes in nitrate availability. Plants exposed frequently to small doses of nitrate took up more nitrate (and hence relied less on N₂-fixation) and had greater dry weights and shoot: root ratios than those exposed to larger doses less often. In mixed ryegrass (Lolium perenne L.)/clover communities clover's ability to either successfully compete for nitrate or fix N₂ gave it consistently higher N concentrations than grass whether they were given high or low nitrate nutrient. This higher N concentration was accompanied by greater dry weights than grass in the low nitrate swards but not where high levels of nitrate were applied.