Host range and overwintering sources of bean leaf roll and pea enation mosaic viruses in England

Bean leaf roll virus (BLRV) and pea enation mosaic virus (PEMV) were each transmitted by Acyrthosiphon pisum (Harris) to fifteen of thirty species of legumes in the glasshouse; eleven species were susceptible to both viruses. The only biennial or perennial species infected by BLRV were hop trefoil (Medicago lupulina L.), lucerne (M. sativa L.) and red clover (Trifolium pratense L.), but naturally infected sainfoin (Onobrychis viciifolia Scop.) and white clover (T. repens L.) were found. The only perennial species infected with PEMV in the glasshouse was kidney vetch (Anthyllis vulneraria L.). Eggs of A. pisum, which seems to be the main vector of BLRV and PEMV in England, were found in winter on several species of cultivated perennial legumes, most on lucerne, fewest on white clover. In spring, more viviparae of A. pisum were found on lucerne than on other perennial legumes, and many on lucerne, but few on red or white clover, were infective with BLRV. Lucerne is probably the main overwintering source of BLRV in areas where lucerne is common, but elsewhere red and white clovers are probably as important. No aphid collected from perennial legumes between 1965 and 1968 was infective with PEMV, but this virus can overwinter in common vetch (Vicia sativa L.). Lucerne infected with BLRV was usually symptomless or showed only transient mild yellowing of young leaves. Lucerne plants showing vein-yellowing, similar to that previously reported as a symptom of BLRV, were possibly infected with an aberrant strain of BLRV but more probably with BLRV and another aphid-transmitted agent. Inoculations from lucerne with vein-yellowing symptoms sometimes caused vein-yellowing, and sometimes typical BLRV-symptoms, in crimson clover (Trifolium incarnatum L.).     

Use of a 15N2 labelling technique to estimate exudation by white clover and transfer to companion ryegrass of symbiotically fixed N

Backgrounds and aims

N rhizodeposition by legumes leads to enrichment of N in soils and in companion plants. N rhizodeposition can be divided into two major components, root exudation and root senescence. Our aim was to quantify N root exudation in white clover (Trifolium repens L.) through an estimation of short-term N rhizodeposition and to assess its impact on N transfer to companion perennial ryegrass (Lolium perenne L.) grown in mixture with clover.

Method

¹⁵N₂ provided in the root atmosphere for 3 days was used to estimate transfer of symbiotically fixed nitrogen (SFN) to the growing medium by clover grown in pure stand and to ryegrass by clover grown in mixture for 2 months.

Results

The proportion of N rhizodeposited over the 3 days increased from 3.5 % of SFN in pure stand to 5.3 % in mixture. The ¹⁵N-enrichment of ammonium from the adhering substrate shows that a part of the rhizodeposited N was released in the form of ammonium. 4 % of the rhizodeposited N was taken up by ryegrass during the labelling period.

Conclusions

This study showed a significant contribution of root N exudation to the total N rhizodeposition of legumes and in the transfer of N to grasses.     

Nitrogen fixation and transfer in grass-clover leys under organic and conventional cropping systems

Background and aim

Symbiotic dinitrogen (N₂) fixation is the most important external N source in organic systems. Our objective was to compare symbiotic N₂ fixation of clover grown in organically and conventionally cropped grass-clover leys, while taking into account nutrient supply gradients.

Methods

We studied leys of a 30-year-old field experiment over 2 years in order to compare organic and conventional systems at two fertilization levels. Using ¹⁵N natural abundance methods, we determined the proportion of N derived from the atmosphere (PNdfa), the amount of Ndfa (ANdfa), and the transfer of clover N to grasses for both red clover (Trifolium pratense L.) and white clover (Trifolium repens L.).

Results

In all treatments and both years, PNdfa was high (83 to 91 %), indicating that the N₂ fixation process is not constrained, even not in the strongly nutrient deficient non-fertilized control treatment. Annual ANdfa in harvested clover biomass ranged from 6 to 16 g?N m^?2. At typical fertilizer input levels, lower sward yield in organic than those in conventional treatments had no effect on ANdfa because of organic treatments had greater clover proportions. In two-year-old leys, on average, 51 % of N taken up by grasses was transferred from clover.

Conclusion

Both, organically and conventionally cropped grass-clover leys profited from symbiotic N₂ fixation, with high PNdfa, and important transfer of clover N to grasses, provided sufficient potassium- and phosphorus-availability to sustain clover biomass production.     

Measurement of biological dinitrogen fixation in grassland: Comparison of the enriched 15N dilution and the natural 15N abundance methods at different nitrogen application rates and defoliation frequencies

A plant mixture of white clover (Trifolium repens L.), red clover (Trifolium pratense L.), and ryegrass (Lolium perenne L.) was established in the spring of 1991 under a cover-crop of barley. Treatments were two levels of nitrogen (400 and 20 kg N ha^-1) and two cutting intensities (3 and 6 cuts per season). Fixation of atmospheric derived nitrogen was estimated by two ¹⁵N dilution methods, one based on application of ¹⁵N to the soil, the other utilising small differences in natural abundance of ¹⁵N.Both methods showed that application of 400 kg N ha^-1 significantly reduced dinitrogen fixation, while cutting frequency had no effect. Atmospheric derived nitrogen constituted between 50 and 64% of harvested clover nitrogen in the high-N treatment, while between 73% and 96% of the harvested clover nitrogen was derived from the atmosphere in the low-N treatment. The amounts of fixed dinitrogen varied between 31–72 kg N ha^-1 and 118–161 kg N ha^-1 in the high-N and low-N treatment, respectively. The highest values for biological dinitrogen fixation were estimated by the enriched ¹⁵N dilution method.Estimates of transfer of atmospheric derived nitrogen from clover to grass obtained by the natural ¹⁵N abundance method were consistently higher than those obtained by the enriched ¹⁵N dilution method. Neither mineral nitrogen application nor defoliation frequency affected transfer of atmospheric derived nitrogen from clover to grass.Isotopic fractionation of ¹⁴N and ¹⁵N (B value) was estimated by comparing results for nitrogen fixation obtained by the enriched ¹⁵N dilution and the natural ¹⁵N abundance method, respectively. B was on average +1.20, which was in agreement with a B value determined by growing white clover in a nitrogen free media.     

N transfer in three-species grass-clover mixtures with chicory,ribwort plantain or caraway

Background and aims

There is substantial evidence that legume-derived Nitrogen (N) is transferred to neighboring non-legumes in grassland mixtures. However, there is sparse information about how deep rooted non-legume forage herbs (forbs) influence N transfer in multi-species grasslands.

Methodology

Red clover (Trifolium pretense L.) was grown together with perennial ryegrass (Lolium perenne L.) and one of three forb species: chicory (Cichorium intybus L.), ribwort plantain (Plantago lanceolata L.) or caraway (Carum carvi L.) in a field experiment. During the first year after the establishment, red clover leaves were labeled with ¹⁵N-urea to determine the N transfer from red clover to companion ryegrass and forbs.

Results

On an annual basis, up to 15 % of red clover N was transferred to the companion ryegrass and forbs, but predominantly to the grass. The forb species did not differ in their ability to take up clover N, but biomass production and soil N acquisition was higher in chicory and plantain than in caraway.

Conclusions

Grass relied to a great extent on clover N, whereas forbs relied on soil N. Soil ¹⁵N-enrichment indicated that N transfer occurred in the upper soil layers and that a dependence on clover-derived N did not necessarily give grass a growth advantage.

     

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.     

Cutting regime affects the amount and allocation of symbiotically fixed N in green manure leys

Cutting strategy effect on N₂ fixation and distribution of fixed N above and below ground in red clover (Trifolium pratense L.) and mixed red clover/perennial ryegrass (Lolium perenne L.) green manure leys was quantified in field experiments including in situ mezotrons and microplots. Symbiotically fixed N in clover, transfer of fixed N to grass in the mixed stands and the fate of ¹⁵N contained in mulch were estimated by isotope dilution. Below ground clover-derived N was estimated by leaf labelling. Total N₂ fixation was estimated by correcting fixed N in plant shoots with plant-derived N below ground and recycled N from mulch. The total N₂ fixation was larger in harvested and mulched stands (average 45 g?m^?2) than in the intact stands (32 g?m^?2). Of the fixed N, 53% (intact), 46% (harvested) and 60% (mulched) was found below ground. The average recycling of N in mulch was 21% and contributed 13.7% (pure clover) and 2.2% (mixed) of the clover N in the regrowth. Recycling of N did not decrease N₂ fixation in the mulched compared with harvested stands. The results indicate that cutting regime should be considered when estimating total amounts of N fixed by green manure leys.     

Long- and short-term effects of crop residues on aluminum toxicity,phosphorus availability and growth of pearl millet in an acid sandy soil

Pasture swards containing perennial ryegrass (Lolium perenne L.) alone or with one of five different white clover (Trifolium repens L.) cultivars were examined for production and transfer of fixed nitrogen (N) to grass under dairy cow grazing. Grass-only swards produced 21% less than mixed clover-grass swards during the second year after sowing. Production from grass-only plots under a mowing and clipping removal regime was 44% less than from grass-only plots under grazing. Much of this difference could be attributed to N transfer. In swards without clover, the ryegrass component also decreased in favour of other grasses.The average amount of fixed N in herbage from all clover cultivars was 269 kg N ha^–1 yr^–1. Above-ground transfer of fixed N to grasses (via cow excreta) was estimated at 60 kg N ha^–1 yr^–1. Below-ground transfer of fixed N to grasses was estimated at 70 kg N ha^–1 yr^–1 by ¹⁵N dilution and was similar for all clover cultivars. Thus, about 50% of grass N was met by transfer of fixed N from white clover during the measurement year. Short-term measurements using a ¹⁵N foliar-labelling method indicated that below-ground N transfer was largest during dry summer conditions.     

Establishment of reed canary grass with perennial legumes or barley and different fertilization treatments: effects on yield,botanical composition and nitrogen fixation

In two field experiments in northern Sweden, we investigated if intercropping reed canary grass (RCG; Phalaris arundinacea L.) with nitrogen‐fixing perennial legumes could reduce N‐fertilizer requirements and also if RCG ash or sewage sludge could be used as a supplement for mineral P and K. We compared biomass production, N uptake and N‐fixation of RCG in monoculture and mixtures of RCG with alsike clover (Trifolium hybridum L.), red clover (Trifolium pratense L.), goat's rue (Galega orientalis Lam.) and kura clover (Trifolium ambiguum M. Bieb.). In one experiment, RCG was also undersown in barley (Hordeum vulgare L.). Three fertilization treatments were applied: 100 kg N ha^?1, 50 kg N ha^?1 and 50 kg N ha^?1 + RCG ash/sewage sludge. We used a delayed harvest method: cutting the biomass in late autumn, leaving it on the field during the winter and harvesting in spring. The legume biomass of the mixtures at the inland experimental site was small and did not affect RCG growth negatively. At the coastal site, competition from higher amount of clover biomass affected RCG growth and spring yield negatively. N‐fixation in red clover and alsike clover mixtures in the first production year approximately covered half of recommended N‐fertilization rate. Goat's rue and kura clover did not establish well at the costal site, but at the inland site goat's rue formed a small but vital undergrowth. RCG undersown in barley gave lower yield, both in autumn and spring, than the other treatments. The high N treatment gave a higher spring yield at the inland site than the low N treatments, but there were no differences due to fertilization treatments at the coastal site. For spring harvest, there were no yield benefits of RCG/legume intercropping compared with RCG monoculture. However, intercropping might be more beneficial in a two‐harvest system.     

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

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

Rates of Hydrogen Ion Efflux by Nodulated Legumes Grown in Flowing Solution Culture with Continuous pH Monitoring and Adjustment

The net efflux of H⁺ from lucerne (Medicago saliva L.), redclover (Trifolium pratense L.) and white clover (Trifolium repensL.) growing in flowing solution culture and dependent upon symbioticfixation of atmospheric N, was measured over a 75 d experimentalperiod. Considerable and rapid increases in acidity of the nutrientsolution of up to 1.45 pH units were recorded when the pH wasriot held constant over a 30 h period. There was little differencein H⁺ efflux when solution pH was held constant at 4.75, 5.75or 6.75, but there was an immediate cessation when it was adjustedto 3.75. Differences in the daily net efflux of H⁺ closely followedthe pattern of daily differences in incoming radiation, andthere was also evidence of a diurnal pattern of H⁺ efflux. Althoughthere were initially distinct differences between the speciesin the calculated rate of net H⁺ efflux (µg H⁺ g^–1dry shoot d^–), by day 75 these had diminished. In allspecies, however, the maximum rate of efflux per unit of shootsoccurred during the earlier rapid phases of growth. The measuredefflux of H⁺ was well equated with the plant content of excesscations (as measured by ash alkalinity) and, on average, theratio of acidity produced to N assimilated (expressed as anequivalent) was 0-24. Medicago sativa L., Trifolium pratense L., Trifolium repens L., lucerne, red clover, white clover, acidification, cation/anion balance, flowing solution culture, H⁺ efflux, nitrogen fixation     

N2 fixation and nitrogen allocation to above and below ground plant parts in red clover-grasslands

Leys, used for grazing or production of forage to be conserved as silage or hay, are very important crops in northern areas. In order to measure the N₂ fixation in leys of varying ages and during different parts of the season, detailed measurements were taken of yield, N₂ fixation and the amounts of N remaining in the field after harvesting red clover (Trifolium pratense L.)-grass leys at a site in northern Sweden, where they are generally harvested twice per growing season. Entire plants, including stubble and roots, were sampled at the time of first and second harvest and, in addition, at the end of the growing season in three neighbouring fields, carrying a first, a second and a third year ley, respectively. N₂ fixation was measured by both ¹⁵N isotope dilution (ID) and ¹⁵N natural abundance (NA) methods. The proportion of clover dry matter (DM) in the stands increased from the first to the second harvest, but the grasses dominated throughout the entire season, especially below ground. The N concentrations, in both herbage and whole plants, were about twice as high in the clover as in the grasses. Seasonal variations in N concentrations were minor, and total N contents followed the same trends as DM. The clover acquired nearly all of its N from N₂ fixation: the proportion of N in clover herbage derived from N₂ fixation was often >0.8 throughout the season. The variations in the amounts of N₂ fixed during the course of the season corresponded well to the seasonal changes in clover biomass. Amounts of fixed N₂ allocated to clover herbage during the whole season were in the range 4 to 6 g N m⁻² in this unusually rainy year. Calculations of daily N allocation rates to herbage showed that N uptake rates were similar, and high, in grasses during May–June and July–August, while N₂ fixation rates in clover were about 10-fold as high in July–August as in May–June, reflecting the need for N in clover growth. The proportion of N remaining in clover stubble and roots after the first and second harvests was about 60 and 25%, respectively, while about 60% of the N in grasses remained in stubble and roots after both harvests. The considerable amounts of biomass and N that were left in field after harvesting red clover-grass leys are important for re-growth of the plants and provide substantial N fertilization for the next crop in the crop rotation.     

Root interactions between intercropped legumes and non-legumes—a competition study of red clover and red beet at different nitrogen levels

Aims

To investigate root competition in a legume/non-legume mixture, and how root growth of the legume is affected by the competition at increasing nitrogen (N) supply.

Methods

Red beet (Beta vulgaris L.) and red clover (Trifolium pratense L.) were grown in transparent rhizotron tubes either in mixture or as sole crop at N supplies of 0, 75 or 150 kg ha^-1. The root growth was evaluated by the root intensity on the rhizotron surface, root depth and plant uptake of ¹⁵N injected into the soil at the deeper part of the red clover root system.

Results

Competition with red beet decreased clover root intensity in deeper soil layers compared to clover grown as sole crop. The difference between clover in sole crop and in mixture was not evident at the highest N supply because the root growth of clover in sole crop appeared to be lowered at high N level. Increased N supply increased the dominance of red beet, but generally did not alter the root growth and distribution of the two species grown in mixture.

Conclusions

Clover root growth and rooting depth were inhibited by competition with red beet but the effect was not enhanced by increased N supply; hence the increased dominance of red beet at higher N level was likely due to its increased growth and competitiveness for other soil resources.     

Potential to improve transfer of N in intercropped systems by optimising host-endophyte combinations

Possibilities for improving N transfer from N2-fixing plants to non-N2-fixing plants by mycorrhiza have been investigated. Initially, the genetic variability with respect to N uptake was assessed by screening five varieties of chicory (Cichorium intybus L.), four of peas (Pisum sativum L.) and three of red clover (Trifolium pratense L.) in combination with eight isolates of arbuscular mycorrhizal fungi. The most promising plant - fungi combinations identified through the cultivar screening were used to optimise conditions for N transfer between intercropped N2-fixing plants (peas and clover) and non-N2-fixing chicory. In the first experiment, the recovery of fixed legume N was investigated using three cultivars, of chicory intercropped with pea variety, and inoculated with one of four mycorrhizal isolates. Roots of the N2-fixing pea and the non-N2-fixing chicory were separated by a root-free soil layer in a three-compartment container. A section of the legume roots was forced to grow into a separate compartment which received four split applications of ¹⁵N. The percentage of N in the chicory derived from transfer ranged between 3% and 50%. In a second experiment one chicory variety was intercropped with one red clover variety and inoculated with four mycorrhizal isolates respecetively. A harvest regime was chosen in which the shoots were harvested from intercropped plants at 3,4.5 and 6 months of age. At three months the percentage of N in the chicory derived from transfer ranged between 15% and 18% and at a plant age of 4.5 months from 46 to 77%. At six months the percentage of N in the chicory roots derived from transfer of legume N ranged from 20 to 34% and varied with fungal isolate. Our results show that there is potential for improving N transfer in intercropped plant systems through the methodological selection of suitable plant and mycorrhizal partners.     

Biomass yield and nitrogen fixation of legumes monocropped and intercropped with rye and rotation effects on a subsequent maize crop

The research is focused on an ecologically sound and highly productive cultivation system for fodder and/or biomass for thermal power generation on the basis of winter legumes and maize as subsequent summer crop, managed without additional nitrogen fertiliser. Therefore the yield of biomass and N-fixing capacity of a winter pea (Pisum sativum L.) and crimson clover (Trifolium incarnatum L.) monocropped and intercropped with rye (Secale cereale L.) were examined for five years in a field trial. In mid-June above-ground biomass of winter crops was removed and maize transplanted. The winter crops achieved maximum dry matter yield about three to five weeks before maturity. Mixed stands yielded more biomass than pure stands and exhibited greater yield stability. The relative advantage of intercropping, expressed as land equivalent ratio (LER), determined for intercropped winter pea/rye were 1.1 to 1.2 and for crimson clover/rye 1.3. At maturity, the amount of fixed nitrogen ranged between 178 kg N for crimson clover and 242 kg N ha^-1 for winter pea, respectively. At the end of anthesis (middle of June, harvesting stage for silage fodder) 75% and 88% of the total fixed nitrogen was achieved, for clover and pea, respectively. In intercropping the amount of fixed nitrogen was lower than in pure stands due to a lower seed density of the legume; however, the N-fixing efficiency was greater than in pure stands. N-release of the winter pea in a pure stand produced a maximum yield in maize (Zea mays L.) without additional N-fertiliser. An additional N mineral fertilisation of 75 to 150 kg N and 75 to 225 kg N was necessary to achieve maximum yields in maize following intercropped winter pea and crimson clover, respectively. Legumes in mixed stands with rye resulted in lower amounts of residual nitrogen after maize harvest. The beneficial effect of legumes on maize can be divided into N-effects and rotation effects. Both effects were positive regarding winter pea. The rotation effect of crimson clover in pure stands on maize was negative. Allelopathic effects and the high sensitivity of crimson clover to mineral nitrogen in the soil, released by residues of the preceding crop, winter rape (Brassica rapa L.), were discussed as the reason for this observation. The combination of the winter pea in pure stand and maize achieved the highest total biomass yield from winter and summer crops, unfertilised (156 dt ha^-1 dry). The combinations of intercropped legumes and maize produced biomass yields of 142 to 145 dt ha^-1. Because winter pea is highly susceptible to lodging, intercropping with low seed density of rye is recommended (3/4 winter pea, 1/4 rye). The rye crop prevents lodging by providing support and high rates of N-fixation are achieved with high seed density of pea. Intercropping with crimson clover and rye should be based on high seed densities of legumes, too because rye is highly competitive within those mixtures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Selective inhibitors of germination of legume seeds in activated sludge compost

Water extracts of the compost produced from activated sludge and coffee residue were found to be selectively inhibitory to seed germination of some legumes. Germination rate of white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) seeds were reduced to 2, 29 and 73% of the control, respectively, by water extracts of the compost (20 g l^–1). However, the extracts did not show any inhibition to seed germination of sorghum (Sorghum bicolor Moench), African millet (Eleusine coracana Gaertn.), and Komatsuna (Brassica rapa L.) at the same concentration. The inhibitors in the compost extracts were separated by ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC) and the inhibitory activities of seed germination were tested with white clover seeds. Five inhibitors were isolated and identified as 3,4-dichlorophenylacetic acid (3,4-DCP), 3,4-dichlorobenzoic acid (3,4-DCB), 3,4,5-trichlorophenylacetic acid, 3,4,5-trichlorobenzoic acid and mono-2-ethylhexylphthalate by ¹H-, ¹³C-NMR spectroscopy and mass spectrometry. The inhibitory activities of some authentic chemicals of the inhibitors and the related compounds were compared. The results indicated that the main inhibitor in the compost could be 3,4-DCB, which was contained at the concentration of 6.58 mg kg^–1 compost and showed the strongest inhibitory effect on seed germination of white clover among the tested compounds.     

Optimizing root architecture and increasing transporter gene expression are strategies to promote selenium uptake by high-se accumulating rice cultivar

Plant and Soil - To examine the legacy of pasture drill rows sown to various configurations of subterranean clover (Trifolium subterraneum L.), lucerne (Medicago sativa L.) and phalaris (Phalaris...     

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.     

Oxygen deficiency affects carbohydrate reserves in overwintering forage crops

Anaerobic conditions developing under an ice cover affect winter survival and spring regrowth of economically important perennial crops. The objective was to compare, during a prolonged period of low (<2%) O2 at low temperature, the concentration of carbohydrates of four plant species contrasting in their resistance to oxygen deficiency. Four perennial forage species, lucerne (Medicago sativa L.), red clover (Trifolium pratense L.), timothy (Phleum pratense L.), and cocksfoot (Dactylis glomerata L.) were subjected to a progressively developing oxygen deficiency stress by enclosing potted plants in gas-tight bags in late autumn for overwintering in an unheated greenhouse. Timothy was previously reported to be more resistant to oxygen deficiency than the three other species. Non-structural carbohydrates increased and remained at a higher concentration in timothy than in the other three species under low O2 concentration. Concentrations of sucrose, fructose, glucose, and fructans increased in response to oxygen deficiency in timothy, whereas the concentration of soluble sugars decreased under the same conditions in lucerne, red clover, and cocksfoot. The gene expression of glyceraldehyde-3-phosphate dehydrogenase increased in response to low oxygen concentration in oxygen deficiency-sensitive lucerne while it remained unchanged in the oxygen deficiency-resistant timothy. It is concluded that timothy maintains higher carbohydrate reserves under oxygen deficiency, a specific feature that could favour its winter survival and spring regrowth.     

Direct Somatic Embryoid Formation on Immature Embryos of Trifolium repens, T. pratense and Medicago sativa, and Rapid Clonal Propagation of T. repens

By direct somatic embryogenesis in vitro a clone of asepticplantlets can be raised from a single immature embryo of Trifoliumrepens (white clover) within about 6 weeks of pollination. Embryoidsare induced directly from intact zygotic embryonic tissue ona culture medium containing 0·025 or 0·05 mg 1^–1BAP and 1·0 g 1^–1 yeast extract. Similar directsomatic embryogenesis has also been achieved for Trifolium pratense(red clover) and Medicago sativa (lucerne). Applications ofembryo propagation by direct somatic embryogenesis are discussed,particularly in relation to multiple screening of host genotypesfor analysis of host/pathogen and legume/Rhizobium interactions. Trifolium repens L., Trifolium pratense L., Medicago sativa L., clover, lucerne, tissue culture, embryoid, somatic embryogenesis, legumes