Breeding of nodulated red clover (Trifolium pratense) for high yield

The symbiotic effectiveness of four cultivars of red clover were compared and breeding programmes undertaken to increase nitrogen fixation and yield of agar- and pot-grown plants. Programme I used the moderately effective Rhizobium trifolii strain 0403 and Programme II the highly effective strain 5. Aggregate scores of plant size (leaf area) were chosen as the criterion of selection. All cultivars, inoculated with strain 0403, differed in time of initial nodulation and in dry matter yield and three differed in leaf area. None differed in nodule number or N-content. Dry matter and leaf area were highly correlated. Diallel crosses among highly effective selections in Programme 1 gave progeny yields that exceeded those of crosses between modal selections by averages of 6% in the first generation, 5% in the second generation and 23% in the third generation. Yields of the high cross category exceeded those of the original cultivar by an average of 9% in the second generation and by 25–101% in the third generation. Effects on leaf area were similar. Highly effective progeny tended to nodulate earlier and have more nodules than the remainder but differences were very small. Crosses between cultivars were generally more effective than those within cultivars, indicating heterotic effects. Similar results for yield were obtained in Programme II. The average increase in yield of crosses of third-generation material between highly effective selections compared with cv. S123 was 63%. The modal crosses were intermediate. In each Programme and generation there were large differences between parents within each cross category.     

Manganese tolerance in subterranean clover (Trifolium subterraneum L.) genotypes grown with ammonium nitrate or symbiotic nitrogen

Summary A wide range of clover accessions were screened for reaction to manganese (Mn) in solution culture. Growth was supported with ammonium nitrate (NH₄NO₃) or symbiotic nitrogen to assess Mn effects on symbiosis and the suitability of NH₄NO₃ dependent growth for assessing Mn tolerance in clover. Reduction of dry matter at Mn 45 ppm varied 0–70%, at Mn 90 ppm, 38–92%, the extent depending on genotype. Tolerant clovers tended to restrict the movement of Mn from roots to shoots. Several previously untested lines were the most tolerant while some commercial lines possessed poor tolerance. Ranks of tolerance for the two nitrogen (N) sources at Mn 45 ppm were correlated suggesting no dominant, discriminatory effects of N source on Mn tolerance; but inclusion of symbiotic effectiveness in a multiple correlation improved the relation between relative tolerances of genotypes under different N sources. Mn affected some aspects of symbiosis. Total nodule nitrogenase activity mainly reflected effects of Mn on nodule number but nitrogenase activity per nodule also contributed. To establish relative tolerances of subterranean clover to Mn growth with NH₄NO₃ is suitable and useful when symbiotic effectiveness is unknown.     

Characterization of novel microsatellite loci for red clover (Trifolium pratense L.) from enriched genomic libraries

Twenty‐seven polymorphic microsatellite markers were isolated from red clover (Trifolium pratense). Allelic variability and cross‐species amplification were assessed on 24 red clover and eight white clover (Trifolium repens) genotypes. The number of alleles detected in red clover ranged from two to 25. Observed and expected heterozygosities were high with average values of 0.71 and 0.88, respectively. Five of the 27 loci were also successfully amplified from white clover, where two to 13 alleles were detected. These highly polymorphic microsatellite loci provide powerful tools for population genetic studies as well as for marker‐assisted selection in this important forage legume species.     

The influence of long-term tillage systems on symbiotic N2 fixation of pea (Pisum sativum L.) and red clover (Trifolium pratense L.)

Pea as a grain legume and red clover as a forage legume in the seeding year were cultivated in two long-term differentiated tillage systems on a loess soil in Germany. A continuous conventional tillage system (plow; CT) and a continuous minimum tillage system (rotary harrow; MT) were established in 1970. With pea and red clover dry matter accumulation and N parameters (N accumulation, Ndfa, N-harvest-index, N balance) were investigated in 1998 and 1999. Differences in the N₂ fixation of pea due to the tillage system could clearly be shown whereas grain yields and total N accumulation were equal in both tillage systems and years. In both years a significantly (P < 0.05) higher Ndfa in the MT system was found at least in the final harvest (maturity of pea): 1998/1999, 0.42/0.54 in CT, 0.62/0.75 in MT. The differences in N₂ fixation of pea may be explained by the delayed soil N supply in MT at the beginning of the vegetative period. Simplified N balances of pea were -18 and –25 kg N ha^–1 in CT and –5 and +1 kg N ha^–1 in MT for 1998 and 1999, respectively. Red clover showed no significant differences in the DM and N accumulation between both tillage systems but a year dependent effect caused by different stubble and root yields between the years was apparent. With red clover slightly, but also significantly (P < 0.05) increased Ndfa values were found in the MT system compared to the CT system with 0.55/0.62 in CT (1998/1999) and 0.64/0.71 in MT. However, the difference in Ndfa between the tillage systems (9 percentage points) was much smaller with red clover than with pea (20 and 21 percentage points in 1998 and 1999, respectively). Soil N uptake of red clover using the longer growing season reflected the more adjusted N supply in both long-term differentiated tillage systems, whereas pea in using only a short-term vegetative period reacted stronger to the lower N mineralization in the MT system in springtime.     

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.     

Number of Rhizobia and delayed inoculation influence nodulation of clovers

The relationship between numbers of rhizobia and nodulation response of legumes is of considerable practical importance. Experiments were done under controlled conditions to determine the influence of numbers of Rhizobium leguminosarum biovar. trifolii on nodulation of arrowleaf clover (Trifolium vesiculosum Savi.) and crimson clover (T. incarnatum L.). Numbers of rhizobia in excess of 1000 per seed did not substantially increase earliness of nodulation or total number of nodules formed on the taproot. Nodules, however, were formed nearer the top of the taproot as numbers of rhizobia increased to 100,000 per seed. Delayed inoculation experiments indicated that nodulation sites for these clovers only remained susceptible to infection for less than 1 day. Delaying inoculation for 4 days resulted in only a 1 to 2 day delay in nodulation for arrowleaf and crimson clovers respectively and no delay for subterranean clover (T. subterraneum L.). Apparently, larger seedlings nodulated faster.     

Characterization of the anomalous infection and nodulation of subterranean clover roots by Rhizobium leguminosarum 1020

Anomalous nodulation of Trifolium subterraneum (subterranean clover) roots by Rhizobium leguminosarum 1020 was examined as a model of modified host-specificity in a Rhizobium-legume symbiosis. Consistent with previous reports, these nodules (i) appeared most often at sites of secondary root emergence, (ii) were ineffective in nitrogen fixation and (iii) were as numerous as nodules formed by an effective Rhizobium trifolii strain. R. leguminosarum 1020, grown on agar plates or in the clover root environment, did not bind the white clover lectin, trifoliin A. This strain did not attach in high numbers, and did not induce shepherd's crooks or infection threads, in subterranean clover root hairs. However, R. leguminosarum 1020 did cause branching, moderate curling and other deformations of root hairs. The bacteria probably entered the clover root through breaks in the epidermis at sites of lateral root emergence. The anomalous nodulation was inhibited by nitrate. Only trace amounts of leghaemoglobin were detected in the nodules by Western blot analysis. The nodules were of the meristematic type and initially contained well-developed infection, bacteroid and senescent zones. Infection threads were readily found in the infection zone of the nodule. However, the bacteroid-containing tissue senesced more rapidly than in the effective symbiosis between subterranean clover and R. trifolii 0403. This anomalous nodulation of subterranean clover by R. leguminosarum 1020 suggests a naturally-occurring alternative route of infection that allows Rhizobium to enlarge its host range.     

Calcium and Nodulation in Subterranean Clover (Trifolium subterraneum L.)

From a study of the effects of Ca ions on the nodulation of subterranean clover in flowing culture solutions it is concluded that root infection or nodule initiation has a higher Ca requirement than either nodule development or host plant growth in the presence of fixed nitrogen.     

In vitro selection for 2,4-D tolerance in red clover

Summary In vitro, selection is a viable method of selecting herbicide-tolerant crops. This research was to evaluate in vitro selection techniques for enhancing 2,4-D [(2,4-dichlorophenoxy) acetic acid] tolerance in red clover (Trifolium pratense L.). In vivo and in vitro responses to 2,4-D of eight diverse red clover populations were correlated (r=0.77), justifying in vitro selection for 2,4-D tolerance. Suspension cultures of a red clover genotype capable of regeneration were plated onto agar-based nutrient media supplemented with 0.18 mM 2,4-D for selection experiments. After two cycles of selection, 16 2,4-D tolerant callus lines were identified based on visual growth assessment. These lines were evaluated for 2,4-D tolerance (based on 2,4-D content), using a 2,4-D bioassay procedure which consisted of placing selected callus tissue pieces on top of oat (Avena sativa L.) coleoptile or internode sections. The relative amount of 2,4-D in the callus tissue was estimated by the amount of oat section elongation after 24 h. Two of the more tolerant callus lines had 61% and 83% less 2,4-D in their tissues than the susceptible control tissue. These studies indicated that in vitro selection can enhance the levels of 2,4-D tolerance in red clover callus tissue.Florida Agricultural Experiment Station Journal Series No. 8943     

Genetic variation in tissue cultures of red clover

Summary Design II matings were made among randomly selected clones of Arlington red clover (Trifolium pratense L.). Progeny were evaluated in vitro on two regeneration media for callus growth and differentiation. Additive genetic variance was a significant source of variability for nearly all traits evaluated, including somatic embryogenesis. In vitro traits, such as rapid callus growth, colony vascularization, root initiation, chlorophyll production and embryogenesis were highly heritable and should respond to breeding and selection. Dominance genetic variance was significant for only a few in vitro characters. Maternal and cytoplasmic factors were significant primarily in the early subcultures. Highly significant additive genetic correlation of performance on two regeneration media was found. A population selected on one of the regeneration media for such characteristics as improved plantlet regeneration, rapid callus growth, long term colony viability or the frequency of root initiation should show correlated improvement on the other medium. No significant differences for embryogenesis were attributable to differences in the regeneration media used. Furthermore, no interaction of additive genetic effects with regeneration media were observed. These data indicate that improvement in the frequency of plantlet regeneration from callus of red clover could effectively be achieved by breeding and selection for embryogenic types.The research reported in this paper (No. 80-3-152) is in connection with a project of the Kentucky Agric. Exp. Stn. and the paper is published with the approval of the director. Part of a thesis submitted by the senior author in partial fulfillment of the requirements for the M.S. degree     

Diversity study on Sclerotinia trifoliorum Erikks., the causal agent of clover rot in red clover crops (Trifolium pratense L.)

Since the 16th century, red clover has been an important crop in Europe. Since the 1940s, the European areal of red clover has been severely reduced, due to the availability of chemical fertilizers and the growing interest in maize. Nowadays there is a growing interest in red clover again, although some setbacks still remain. An important setback is the low persistence of red clover crops. Clover rot, caused by the ascomycete fungus Sclerotinia trifoliorum Erikss., is a major disease in Europe and reduces the persistence of red clover crops severely. The fungus infects clover plants through ascospores in the autumn, the disease develops during the winter and early spring and can kill many plants in this period. In early spring, black sclerotia, serving as surviving bodies, are formed on infected plants. Sclerotia can survive up to 7 years in the soil (Ohberg, 2006). The development of clover rot is highly dependent on the weather conditions: a humid fall, necessary for the germination of the ascospores and an overall warm winter with short periods of frost are favourable for the disease. Cold and dry winters slow the mycelial growth down too much and prevent the disease from spreading. Clover rot is difficult to control and completely resistant red clover varieties have yet to be developed. Because of the great annual variation in disease severity, plant breeders cannot use natural infection as an effective means to screen for resistant material. Breeding for resistant cultivars is being slowed down by the lack of a bio-test usable in breeding programs. When applying artificial infections, it is necessary to have an idea of the diversity of the pathogen. A diverse population will require resistance screening with multiple isolates. The objective of this research is to investigate the genetic diversity among isolates from the pathogen S. trifoliorum from various European countries. We assessed diversity using a species identification test based on the sequence of the beta-tubulin gene, vegetative compatibility grouping and AFLP.     

Determining the effectiveness of resistance to subterranean clover mottle sobemovirus in different genotypes of subterranean clover in the field using the grazing animal as virus vector

The effectiveness of resistance to subterranean clover mottle sobemovirus (SCMoV) previously identified in different genotypes of subterranean clover (Trifolium subterraneum) inoculated with infective sap in the glasshouse, was tested in two field experiments which used the grazing animal as virus vector. Replicated plots each consisting of paired test rows of 20 different genotypes were used. Clover plants infected with SCMoV were transplanted in between the paired test rows and these acted as sources of the virus for spread by grazing sheep. Although used in different years at different sites with different virus isolates, the field exposure methodology employed produced consistent results. The genotypes each behaved similarly in both experiments as regards the relative extents of SCMoV infection that developed, levels ranging from 0–98%. The previously identified resistance in six ‘highly resistant’ and three ‘partially resistant’ cultivars was effective under field conditions. However, the ‘partial resistance’ in three others was overcome, cvs Green Range and Mt Barker developing levels of infection approaching those in ‘susceptible’ cultivars, while an intermediate infection level developed in cv. Karridale. The three cultivars in which partial resistance was not effective all belonged to ssp. subterraneum. In subterranean clover breeding programmes, field screening using the grazing animal as a vector is advisable to determine whether SCMoV resistance found by sap inoculation is still effective under field conditions.     

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.     

Effects of pH,Ca and Al on the exudation from clover seedlings of compounds that induce the expression of nodulation genes inRhizobium trifolii

The expression of nodulation genes inR. trifolii is induced by flavone compounds present in clover root exudates. In the present experiments a bioassay with an indicator strain ofR. trifolii, which contained thelacZ gene fromEscherichia coli fused to theR. trifolii nodA gene, was used to measure the level ofnod gene expression inR. trifolii. Compounds that stimulatednodA gene expression were shown to be present in exudates of white clover (Trifolium repens L.) and nine cultivars of subterranan clover (T. subterraneum L.) seedling sgrown at a range of pH between pH 3.0 and pH 8.0. Thenod gene-induction activity of exudates was, however, reduced when seedlings of all clover species were grown at pH>7.0 and at pH<4.0 and pH<5.0 for white clover and subterranean clover respectively. No major differences were apparent in the activity of exudates from seedlings of the various cultivars of subterranean clover.Nod gene-induction activity of exudates was shown to increase markedly with seedling age. The presence of Ca at concentrations up to 10 mM in seedling culture solutions also resulted in marked increases in thenod gene-induction activity of seedling exudates. Increases in activity due to the presence of Ca were most apparent at low pH where between 5 and 10-fold increases were observed for white clover and subterranean clover respectively. Conversely, the presence of Al at concentrations up to 60 M in seedling culture solutions had no effect on thenod gene-induction activity of seedling exudates.The observations that both low pH and Ca concentrations affected thenod gene-induction activity of seedling exudates suggested that the net presence of stimulatory flavones in root exudates was an important contributing factor to the acid-sensitive step in nodule formation.     

Host genetic control of symbiosis in soybean (Glycine max L.)

Genes controlling nitrogen-fixing symbioses of legumes with specialized bacteria known as rhizobia are presumably the products of many millions of years of evolution. Different adaptative solutions evolved in response to the challenge of survival in highly divergent complexes of symbionts. Whereas efficiency of nitrogen fixation appears to be controlled by quantitative inheritance, genes controlling nodulation are qualitatively inherited. Genes controlling nodulation include those for non-nodulation, those that restrict certain microsymbionts, and those conditioning hypernodulation, or supernodulation. Some genes are naturally occurring polymorphisms, while others were induced or were the result of spontaneous mutations. The geographic patterns of particular alleles indicate the role of coevolution in determining symbiont specificites and compatibilities. For example, the Rj4 allele occurs with higher frequency (over 50%) among the soybean (G. max) from Southeast Asia. DNA homology studies of strains of Bradyrhizobium that nodulate soybean indicated two groups so distinct as to warrant classification as two species. Strains producing rhizobitoxine-induced chlorosis occur only in Group II, now classified as B. elkanii. Unlike B. japonicum, B. elkanii strains are characterized by (1) the ability to nodulate the rj1 genotype, (2) the formation of nodule-like structures on peanut, (3) a relatively high degree of ex planta nitrogenase activity, (4) distinct extracellular polysaccharide composition, (5) distinct fatty acid composition, (6) distinct antibiotic resistance profiles, and (7) low DNA homology with B. japonicum. Analysis with soybean lines near isogenic for the Rj4 versus rj4 alleles indicated that the Rj4 allele excludes a high proportion of B. elkanii strains and certain strains of B. japonicum such as strain USDA62 and three serogroup 123 strains. These groups, relatively inefficient in nitrogen fixation with soybean, tend to predominate in soybean nodules from many US soils. The Rj4 allele, the most common allelic form in the wild species, has a positive value for the host plants in protecting them from nodulation by rhizobia poorly adapted for symbiosis.     

nodT,a positively-acting cultivar specificity determinant controlling nodulation of Trifolium subterraneum by Rhizobium leguminosarum biovar trifolii

Rhizobium leguminosarum biovar trifolii strain TA1 nodulates a range of Trifolium plants including red, white and subterranean clovers. Nitrogen-fixing nodules are promptly initiated on the tap roots of these plants at the site of inoculation. In contrast to these associations, strain TA1 has a Nod^- phenotype on a particular cultivar of subterranean clover called Woogenellup (A.H. Gibson, Aust J Agric Sci 19: (1968) 907–918) where it induces rare, poorly developed, slow-to-appear and ineffective lateral root nodules. By comparing the nodulation gene region of strain TA1 with that of another R. leguminosarum bv. trifolii strain ANU843, which is capable of efficiently nodulating cv. Woogenellup, we have shown that the nodT gene (B.P. Surin et al., Mol Microbiol 4: (1990) 245–252) is essential for nodulation on cv. Woogenellup. The nodT gene is naturally absent in strain TA1. A cosmid clone spanning the entire nodulation gene region of strain TA1 was capable of conferring nodulation ability to R.l. bv. trifolii strains deleted for nodulation genes, but only on cultivars of subterranean clovers nodulated by strain TA1. This shows that cultivar recognition events are, in part, determined by genes in the nodulation region of strain TA1. Complementation studies also indicated that strain TA1 contains negatively-acting genes located on the Sym plasmid and elsewhere, which specifically block nodulation of cv. Woogenellup.     

Effects of phosphorus application and mycorrhizal inoculation on root characteristics of subterranean clover and ryegrass in relation to phosphorus uptake

The effects of phosphorus (P) application and mycorrhizal inoculation on the root characteristics of subterranean clover and ryegrass were examined. Phosphorus application increased total root length, root surface area and root volume of both plant species. In contrast, mycorrhizal infection only affected the root characteristics of subterranean clover. Ryegrass took up more P than non-mycorrhizal subterranean clover at all levels of application. However, mycorrhizal infection only increased P uptake by subterranean clover and there was no difference in P uptake between ryegrass and mycorrhizal subterranean clover at low levels of P application. When the P uptake was expressed on the basis of any of the root characteristics, subterranean clover were superior to ryegrass suggesting that the greater uptake of P by ryegrass is not due to a higher efficiency in absorption of P from soil solution, but rather to a large root system.     

QTL analysis of seed yield components in red clover (Trifolium pratense L.)

Cultivars of red clover (Trifolium pratense L.), an important forage crop in temperate regions, are often characterised by an unsatisfactory level of seed yield, leading to high production costs. This complex trait is influenced by many components and negatively correlated with other important traits, such as forage yield or persistence. Therefore, seed yield has proven to be difficult to improve. Thus, the objectives of this study were to assess association among seed yield components and to provide the basis for identifying molecular markers linked to QTLs for seed yield components to assist breeding for improved red clover cultivars. A total of 42 SSR and 216 AFLP loci were used to construct a molecular linkage map with a total map length of 444.2 cM and an average distance between loci of 1.7 cM. A total of 38 QTLs were identified for eight seed yield components. The traits seed number per plant, seed yield per head, seed number per head, head number per plant and percent seed set were highly correlated with seed yield per plant, and QTLs for several of these traits were often detected in the same genome region. Head number per plant may present a particularly useful character for the improvement of seed yield since it can easily be determined before seed maturity. In addition, two genome regions containing four or five QTLs for different seed yield components, respectively, were identified representing candidate regions for further characterisation of QTLs. This study revealed several key components which may facilitate further improvement of seed yield. The QTLs identified represent an important first step towards marker-assisted breeding in red clover.     

Modification of symbiotic interaction of pea (Pisum sativum L.) andRhizobium leguminosarum by induced mutations

Summary In pea (Pisum sativum L.), mutants could be induced, modified in the symbiotic interaction withRhizobium leguminosarum. Among 250 M₂-families, two nodulation resistant mutants (K₅ and K₉) were obtained. In mutant K₅ the nodulation resistance was monogenic recessive and not Rhizobium strain specific. Out of 220 M₂-families one mutant nod₃ was found which could form nodules at high nitrate concentrations (15 mM KNO₃). This mutant nodulated abundantly with severalRhizobium strains, both in the absence and presence of nitrate. Probably as the result of a pleiotropic effect, its root morphology was also changed. Among 1800 M₂-families, five nitrate reductase deficient mutants were obtained and one of them (mutant E₁) was used to study the inhibitory effect of nitrate on nodulation and nitrogen fixation.The results of the present investigation show that pea mutants which are modified in their symbiosis withRhizobium leguminosarum, can readily be obtained. The significance of such mutants for fundamental studies of the legume-Rhizobium symbiosis and for applications in plant breeding is discussed.     

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.