Effect of sowing methods and sowing rate in organic seed production of timothy (Phleum pratense L.), meadow fescue (Festuca pratensis Huds.) and red clover (Trifolium pratense L.)

Different sowing methods and sowing rates were evaluated in organic seed production of timothy (two trials), meadow fescue (two trials) and red clover (one trial) in Southeast Norway, during 2010–2013. The plan included: (1) broadcast sowing of grass/clover, cover crop sown at 12 cm row distance; (2) sowing of cover and seed crop in crossed rows, both at 12 cm row distance; and (3) sowing of cover crop and seed crop in every other row. The three sowing rates were 5, 10 and 15 kg ha–1 in timothy and meadow fescue and 3, 6 and 9 kg ha–1 in red clover. On average for sowing rates and all trials with timothy, meadow fescue and red clover, first year’s seed yields were 5–6%, 20–25% and 19–25% higher on plots sown with cover crop and seed crop in every other row than on plots where seed crop had been broadcast or sown perpendicularly to the cover crop. The different sowing methods had no effect on weed coverage or weed contamination in the cleaned seed. Increasing sowing rate usually had a negative influence on seed yield, while weed coverage/contamination was not significantly affected. It is concluded that organic seed crops should be established with cover crop and seed crop in every other row at a low sowing rate. However, in an organic production system, even this favorable method will not always be sufficient to meet the requirement for seed crop purity.     

Application of isozyme data to the management of the United States national Brassica oleracea L. genetic resources collection

Summary Management of a genetic resources collection is more effective if a curator can accurately identify genotypes and accessions as well as assess intraspecific genetic relationships and the genetic structure of species. Consequently, a study was conducted to determine whether data from starch gel electrophoresis of a specific set of isozymes from plants of Brassica oleracea (cole crops) would be useful in answering four questions the answers to which are essential for effective curatorial activities: Can individual plants be identified? Can specific accessions be identified? What are the genetic relationships among botanical varieties within B. oleracea? What is the genetic structure of the species B. oleracea? Six loci from 4 enzyme systems (LAP, PGD, PGI, and PGM) were analyzed. Individuals and accessions could not usefully be identified using these isozymes, but genetic relationships within and genetic structure of the species were easily determined, resulting in specific recommendations for improving collection management. As expected, highly selected commercial lines exhibited less diversity than average, and so may be of limited value when trying to maximize diversity with a minimum number of accessions. In contrast, landraces and weakly selected lines are more diverse than average, and thus are useful in maximizing per accession diversity. Not only was 93% of the genetic variability in B. oleracea found among accessions and a mere 7% among varieties, but also a cluster analysis showed that accessions of a single botanical variety are often more similar to those of a different variety than to each other. These results suggest that in order to create a genetic resources collection of B. oleracea that faithfully represents the diversity in the species, a curator should assemble a broad array of accessions originating from diverse agroecological niches, having different levels of improvement, and representing all botanical varieties.Abbreviations LAP Leucine amino peptidase - PGD 6-Phosphogluconate dehydrogenase - PGI Phosphoglucoisomerase - PGM Phosphoglucomutase - RAPD Random amplified polymorphic DNA - RFLP Restriction fragment length polymorphism - UPGMA Unweighted pair-group method using arithmetic averages     

Floral procyanidins of the forage legume red clover (Trifolium pratense L.)

The chemical characteristics of the purified procyanidin polymers of the flowers of the forage legume red clover (Trifolium pratense L.) were studied by (13)C NMR, acid-catalyzed degradation with benzyl mercaptan, and electrospray ionization mass spectrometry (ESI-MS). The (13)C NMR showed that the fraction consisted of predominantly procyanidin polymers. The thiolysis reaction products indicated a mean degree of polymerization (mDP) of 9.3 with epicatechin (81%) as the abundant flavan-3-ol extension unit and the terminating units dominated by catechin (95%). ESI-MS showed a range of oligomeric procyanidin ions (DP of 2-11). The white clover floral prodelphinidins consist of terminal units with nearly equal proportions of epigallocatechin (52%) and gallocatechin (48%) and extender units showing epigallocatechin (56%) and gallocatechin (39%). The dramatic difference in the stereochemistry of the terminal and extender units observed for the red clover floral procyanidins contrasts with the mixture of cis and trans stereochemistry observed for white clover floral prodelphinidins.     

Genetic characterization of guava (Psidium guajava L.) germplasm in the United States using microsatellite markers

Genetic diversity of 35 Psidium guajava L. accessions and three related species (P. guineense Sw., P. sartorianum (O. Berg) Nied. and P. friedrichsthalianum (O. Berg) Nied.) maintained at the U.S. Department of Agriculture (USDA), National Plants Germplasm System, Hilo, HI, was characterized using 20 simple sequence repeat (SSR) markers. Diversity analysis detected a total of 178 alleles ranging from 4 to 16 alleles per locus. The observed mean heterozygosity (0.2) and inbreeding coefficient (0.8) indicated a high level of inbreeding among the accessions tested. Multi-locus DNA fingerprints based on the 20 SSR loci unambiguously differentiated all accessions and revealed the absence of duplicated samples. Ordination and cluster analyses suggested that the genetic relationships between majorities of the accessions could be explained by geographic origin, mainly including tropical America, Southeast Asia and Hawaii. A Bayesian cluster analysis partitioned the accessions into two groups and the partition was largely compatible with the result of ordination analyses. Distance-based cluster analyses further indicated that accessions from same geographical region or breeding programs grouped together in spite of the inter-regional exchange of germplasm. Accessions from Southeast Asia were dominantly white fleshed, whereas accessions from tropical America and Hawaii exhibited diverse flesh colors. The results also indicated that accessions from the same region were likely derived from a small number of common ancestors or progenitors. All 20 SSRs were transferable to P. guineense, P. sartorianum and P. friedrichsthalianum, indicating a close relationship between the cultigens and wild relatives. Application of SSR markers in the USDA/Agricultural Research Service germplasm collection provides new insight into the diversity of the guava germplasm, which will be valuable in future breeding endeavors and the conservation of guava genetic resources.     

Diversity of common bean (Phaseolus vulgaris L.) germplasm from Portugal

Common bean (Phaseolus vulgarisL.) is a traditional crop in Portugal, where farmers growvarieties selected and maintained by themselves. A collection of 88landraces of common bean was evaluated for 17 quantitative andqualitative traits and the biochemical marker phaseolin to displaythe degree of variation of this germplasm. Agronomic data weresubjected to cluster analysis and several groups were identified,with three groups clustering most of the landraces. Regardingphaseolin variation the C and T banding patterns are the mostfrequent ones, so the origin of the Portuguese beans is thus probablythe Andean region of South America. These results give informationabout the origin, diversity and breeding value of the Portuguesegermplasm, that could be useful to widen the genetic base ofcurrently cultivated bean varieties in Europe.     

Collection and characterization of naturalized populations of white clover (Trifolium repens L.) in Argentina

A collection formed from 53 accessions of white clover stolonswas carried out in naturalized populations from Argentina betweenparallels 26° and 37° South latitudes and 58° to 62°West longitude. Almost half of them (43%) wereobtained from grazed sites. Collected samples were grown in the fieldat Pergamino Experimental Station and compared with a local cultivar.Attributes scored or measured were petiole length, width and lengthof central leaflet, relation width/length, height, growing habit,leaf mark, stolon thickness and density, days to flowering,cyanogenesis, seed yield and winter herbage yield. There weresignificant correlations among seven pairs of characters. Allpopulations were cyanogenic within a range from 40 to 100%.Populations from grazed sites had prostrate habit and lower canopyheight than those from non grazed sites. The collection was clusteredin seven groups. Differences among groups were attributed toagroecological differences in sites of collection. Results obtainedencouraged in Argentina further collection missions for naturalizedpopulations of white clover.     

Cover crop and plant density in seed production of white clover (Trifolium repens L.)

Seed crops of white clover (Trifolium repens L.) are usually established with a cover crop. Provided sufficient light, white clover may compensate for low plant density by stoloniferous growth. Our objectives were (1) to compare spring barley or spring wheat used as cover crops for white clover and (2) to find the optimal seeding rate/row distance for white clover. Seven field trials were conducted in Southeast Norway from 2000 to 2003. Barley was seeded at 360 and 240 seeds m^?2 and wheat at 525 and 350 seeds m^?2. White clover was seeded perpendicularly to the cover crop at 400 seeds m^?2/13 cm row distance or 200 seeds m^?2/26 cm. Results showed that light penetration in spring and early summer was better in wheat than in barley. On average for seven trials, this resulted in 11% higher seed yield after establishment in wheat than in barley. The 33% reduction in cover crop seeding rate had no effect on white clover seed yield for any of the cover crops. Reducing the seeding rate/doubling the row distance of white clover had no effect on seed yield but resulted in slightly earlier maturation of the seed crop.     

Genetic diversity in melon (Cucumis melo L.): Anevaluation of African germplasm

The genetic diversity among 126 exotic (108) andreference array (RA) melon (Cucumismelo L.) accessions (18) was assessed byvariation at 49 random amplified polymorphic DNA marker bands(putative loci) using 29 10-mer primers. Africanaccessions of unknown melon market classes were compared to the RAaccessions from a broad range of C.melo subsp. melo groups(Cantalupensis, Conomon, Inodorus and Flexuosus). Althoughdifferences in groupings occurred after multidimensional scaling andcluster analysis, both analyses placed African accessions into twogroups, which were separate from RA groupings. One African group of33 accessions containing accessions from Zimbabwe (5),Zambia (24), Mali (1), one of two Senegalaccessions and two of three South African accessions examined. Thesecond group, which consisted of 67 accessions containing collectionsfrom Egypt (40), Tunisia (6), Libya(13), Morocco (1), Algeria (2),Ethiopia (1), Niger (1), Sierra Leone(1), S. Africa (1), Zambia (1) andZimbabwe (1). Depending on the multivariate analysistechnique employed, accessions from Kenya, Senegal and Ghana formedeither unique groupings or were grouped with accessions(Cantalupensis) from the RA. Both analyses indicate thatthe genetic differences inherent between the African gene pools isassociated with the geographic proximity of African countries(northern vs. central-southern Africa) in thegermplasm array examined. Moreover, these data indicate that thegenetic diversity of U.S. and European commercial RA germplasm(Cantalupensis and Inodorus) could be enhanced by theintroduction of genetic variation from African accessions, and thatit would be advantageous to acquire more accessions from thisgeographically and ecologically varied region to ensure the retentionof existing genetic diversity.     

Kinetics of inorganic and organic P release from red clover (Trifolium pratense L.) and ryegrass (Lolium multiflorum L.) upon frost or drying

An incubation study with fresh, frozen, and dried clover and rye-grass shoots gathered in autumn was performed in the laboratory (43 days). The aim was to study inorganic and organic phosphorus (P) release from plant material during decomposition without soil. Plant materials (2?cm size) were mixed with small glass beads and placed in large syringes for consecutive water extractions on 7 occasions. Leachates were analysed for inorganic and total P. At the first leaching event, 8% of total crop P was released from fresh, 24% from frozen and 27% from dried plant material. During decomposition, both inorganic and organic P was released following first order kinetics, with grass releasing mostly inorganic P and clover mostly organic P. After 43 days, 42%–50% of total crop P from fresh, 49%–51% from frozen and 57%–69% from dried material was released, with significant differences between treatments. Using the results in calculations on field scale showed that P released from overwintering crops under cold climate conditions can amount to several kg P ha^?1. Thus, senescence of overwintering aboveground biomass can be a significant source for P leaching from soils.     

Evaluation of Dry Peas (Pisum sativum L.) Varieties for Seedling Vigor Indices in Eastern Montana

Genetic and environmental factors lead to a variation in yield and protein content of dry pea (Pisum sativum L.) seeds. The quality of seed, particularly seed vigor, also influences the establishment of crop and thus final grain yield. The area and production for dry peas are increasing in the Northern Great Plains but knowledge is lacking on how the pea lines/varieties differ in the seed vigor at seven leaf stage. This field and greenhouse study evaluated the eight dry pea lines/varieties for seedling vigor indices and correlated them with grain yield and protein concentrations. Significant differences were observed among the lines/varieties for nodule number plant^?1 in greenhouse, and grain yield in field conditions. The highest number of nodules plant^?1 was obtained with the line MT632, which were at par with lines MT457, and MT190. The highest Vigor Index I was achieved with line MT632 associated with their more shoot lengths as compared to other lines/varieties. The highest Vigor Index II was obtained by variety Majoret and line MT632. The variety DS Admiral yielded 5205 kg ha^?1, which was 17.4 and 33.3% higher than lines MT460 and MT190, respectively. The highest seed protein content was obtained with variety Majoret (23.4%) having highest Vigor Index II and seed yield (4940 kg ha^?1) at par with variety DS Admiral. The lowest seed protein was found with variety DS Admiral (20.3%). The line named MT190 showed lowest yield potential along with the lower protein contents also. Studies show a positive and significant correlation between biomass and Vigor Index I only. Plant nitrogen uptake was positively and significantly correlated with biomass and Vigor Index I in greenhouse only. The results also indicated that seed vigor indices did not reveal any significant correlations with dry peas yield and protein content, so more efforts are needed to evaluate varieties for higher yield and protein content during initial stages of growth in order to maximize their acreage and productivity.     

Effects of chitin amendment of soil on microorganisms,nematodes, and growth of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.)

Effects of soil amendment with crabshell chitin on the growth of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.), and on populations of soil bacteria, fungi, and plant-parasitic and free-living nematodes were investigated in a pot trial. Five soil samples were collected from Te Puke (Paengaroa Shallow Sand, a Typic Hapludand) and five from Hamilton (Bruntwood silt loam, an Aquic Hapludand), New Zealand. Subsamples of each soil were either amended with chitin or unamended and planted with white clover and ryegrass. The ryegrass shoot weight in amended soil was greater (P<0.01), most probably due to N mineralised from chitin. A significantly lower (P<0.01) root: shoot ratio of ryegrass in the amended soil also suggested improved N availability, and therefore less root mass was needed to support a given shoot mass. A reduction in nodulation was observed in 12-day-old white clover seedlings (P<0.05) and also in 6-week-old seedlings (P<0.01). The shoot weight of white clover was significantly lower (P<0.05) in amended soil, possibly due to phytotoxic effects of chitin. Chitin increased (P<0.01) the populations of bacteria and fungi by 13-fold and 2.5-fold, respectively. The cyst nematode of white clover, Heterodera trifolii, was significantly reduced in chitin-amended soil, possibly due to increased levels of chitinase produced by rhizosphere microorganisms. Two other plant-parasitic nematodes, Pratylenchus spp. and Tylenchus spp., were also reduced in ryegrass roots and in soil as a result of the chitin amendment. However, the total number of free-living nematodes increased 5.4-fold in amended soil.     

Molecular Characterization of the USDA White Clover (Trifolium repens L.) Core Collection by RAPD Markers

White clover is one of the most important forage legume species worldwide, playing an important role in Southern Brazil temperate cultivated pastures. This work was aimed to characterize the genetic variability of the USDA white clover core collection formed by 78 accessions representing 50 countries, together with two very well known cultivars (Huia and Ladino Regal), using RAPD (Random Amplified Polymorphic DNA) markers to produce genetic fingerprints. There were used DNA bulks formed by the extraction and mixture of 20 random individuals from each accession. Twenty four primers were used, which revealed from 3 to 29 bands, forming a total of 371 polymorphic bans and only one monomorphic, ranging from 50 to 3098 bp. The results showed a genetic similarity among the accessions, ranging from 0.18 to 0.58 (Jaccard’s index), with an average of 0.24, allowing the identification of each individual accession using just three primers. The results also showed a large genetic variability within the white clover core collection, probably due to its reproduction mode and ploidy level, which could be used in plant breeding program.     

Repellency of insecticides and the effect of thiacloprid on bumble bee colony development in red clover (Trifolium pratense L.) seed crops

The study intended to compare repellency of three insecticides on bumble bees and honey bees in Norwegian red clover (Trifolium pratense L.) seed crops, and to examine effects of thiacloprid on bumble bee colony development in the field. The repellency study was carried out in a large-scale field trial in SE Norway in 2013. On average for observations during the first week after spraying, 17 and 40% less honey bees (P?=?.03) and 26 and 20% less bumble bees (P?=?.36) were observed on plots sprayed with the pyrethroids lambda-cyhalothrin and alpha-cypermethrin, respectively, than on unsprayed control plots. No pollinator repellency was found on plots sprayed with the neonicotinoid thiacloprid. Compared with unsprayed control the seed yield increases were 22% on plots sprayed with thiacloprid vs. 12–13% on plots sprayed with pyrethroids (P?=?.10). Follow-up studies in 2014–2016 focused on the effect of thiacloprid on bumble bee colony development in commercially reared nests of Bombus terrestris placed into red clover seed crops at the start of flowering. Unsprayed control crops were compared with crops sprayed either at the bud stage or when 18–44% of flower heads were in full bloom. Chemical analyses of adult bumble bees showed that thiacloprid was taken up in bees when crops were sprayed during flowering, but not detected when crops were sprayed at the bud stage. The bumble bees in late-sprayed crops also developed weaker colonies than in unsprayed crops. Dead bees with a high internal concentration of thiacloprid were found in one crop sprayed during the night at 35% flowering. This shows that thiacloprid is not bee-safe if sprayed after anthesis and that spraying has to be conducted at the bud stage to reduce its contamination of nectar and pollen.     

Improving of seed quality of black seed (Nigella sativa L.) by increasing seed phosphorus content in a calcareous soil

Phosphorus (P) sufficiency during seed formation and development can affect the quality of seeds production. For increasing P content of black seed (Nigella sativa) in a calcareous soil, series of experiments were conducted in completely randomized factorial design with 4 replications at the Faculty of Agriculture, Ferdowsi University of Mashhad, Iran, in 2012 and 2013. The combinations of vermicompost (V), sulfur (S), and Thiobacillus bacteria (T) were mixed with a calcareous soil fertilized with 0, 30 and 60 kg P ha^?1 in pots and incubated for 63 days. At the end of incubation period, black seeds were sown in the pots and plants were grown to maturity. Results showed that S+T and V treatments were significantly increased soil available P, emergence, plant P content, seed yield and vigor of the seeds production. There were positive relationship between soil and plant P concentration with the quality of seeds production.     

Physiological analysis of growth and nitrogen metabolism of intercropped pasture species subterranean clover (Trifolium subterraneum L.) and cocksfoot (Dactylis glomerata L.) supplemented with different forms of inorganic nitrogen

Growth and physiological parameters of intercropped subterranean clover (Trifolium subterraneum L.) and cocksfoot (Dactylis glomerata L.) were investigated under controlled and field conditions. Nitrogen nutrition was accomplished in symbiotic and heterotrophic forms in both the species studied under controlled concentration of mineral elements in the media and by in oculation with appropriate rhizobial strain. Intercropped variants were also grown in sand culture as follows: subterranean clover – 100%, cocksfoot – 100%, subterranean clover:cocksfoot– 75:25(%), subterranean clover: cocksfoot– 50:50(%), and subterranean clover:cocksfoot– 25:75(%). In hydroponic cultures, subterranean clover to cocksfoot proportion was always 50:50(%). Subterranean clover reacted positively with growth up to 1.25 mM nitrate concentration in the medium with stimulation of number of nodules formed and their nitrogenase activity. When separately grown, both subclover and cocksfoot reacted with higher plant biomass formation under higher nitrate nutrition. When intercropped, the process of growth stimulation in subclover was more prominent than in cocksfoot.     

Influence of soil applied boron on yield of berseem (Trifolium alexandrium L.) and soil boron fractions in calcareous soils

A greenhouse experiment was conducted in North-west India to study the effect of soil applied boron on yield of berseem (Trifolium alexandrium L.) and soil boron fractions in boron deficient calcareous soils. Three soils with varying calcium carbonate content viz. 0.75% (Soil I), 2.6% (Soil II), and 5.7% (Soil III) were collected from different sites of Ludhiana, Bathinda, and Shri Muktsar Sahib districts, Punjab, India. The treatments consisted of six levels of soil applied boron viz. 0.5, 0.75, 1.0, 1.25, 1.5, and 2.0 mg B kg^?1 along with control. The green fodder yield and dry matter yield increased significantly at 0.75 mg B kg^?1 soil treatment level in the first cutting, while these were significant at 1.0 mg B kg^?1 soil treatment level in all soils at second, third, and fourth cuttings. Among all three calcareous soils, Soil I with lower calcium carbonate was the best soil in respect of mean yield in comparison to Soil II and Soil III. Combined effect of boron level and soils had significant effect on yield of berseem. There was a significant increase in mean dry root biomass at 1.0 mg B kg^?1 soil level over control and then remained non-significant with further high levels of soil applied boron. The mean dry root biomass decreased significantly for the soils having 0.75%, 2.6%, and 5.7% calcium carbonate levels. Readily soluble fraction is considered to be easily available fraction of B for plant uptake and consisted of 0.47–0.62% in Soil I, 0.31–0.43% in Soil II, and 0.24–0.34% in Soil III of the total boron. Among all B fractions, mean readily soluble, specifically adsorbed, and oxide-bound fractions got increased significantly with increase in B levels. Readily soluble and organically bound B fractions were more in Soil I as compared to Soil II and Soil III. Specifically adsorbed boron, oxide bound fraction, residual and total boron were more in Soil III in comparison to Soil I and Soil II. Among all fractions, residual fraction accounted for the major portion of the total B. It comprised of 92.71–93.90% in Soil I, 94.51–95.40% in Soil II, and 94.91–95.25% in Soil III of the total boron.     

Optimum rate of nitrogen application and seed rate for deteriorated wheat (Triticum aestivum L.)

In order to study the effect of seed quality (SQ), seed rate (SR), and nitrogen rate (NR) on yield and yield components of wheat, an experiment was conducted as a factorial-split plot based on the randomized complete block design with four replications in Golestan Province, Iran. Treatments included the combination of SR and NR (recommended rate, 15 and 30% higher than the recommended rate) as the main plots, and SQ (control, 15, 30 and 45?h of accelerated aging) as the subplots. The interaction of NR?×?SR was significant on 1000-seed weight, seed yield, plant height, the number of seeds, biological yield, and harvest index. There was a significant reduction in all traits with an increase in seed aging. Seed yield had a significant and positive correlation with yield components. Using higher density and applying higher nitrogen fertilizer than those of recommended could reduce the negative effects of seed aging in wheat.     

Microbial growth and nitrogen immobilization in the root zone of barley (Hordeum vulgare L.), Italian ryegrass (Lolium multiflorum Lam.), and white clover (Trifolium repens L.)

Summary The effect of roots on microbial growth and N immobilization was investigated in a pot experiment with barley, Italian ryegrass, and white clover. We used a silty subsoil with a low soil organic matter content (0.16%C and 0.012%N), which allowed us to measure N immobilization as an increase in total soil organic N (planted versus unplanted). At sampling, the soil was easily removed from intact roots by gentle washing, with a negligible loss of root material. Plant growth and extra mineral N (in planted soil only) gave increased total counts (fluorescence microscopy) and viable counts (plate dilution) of bacteria, a higher proportion of larger cells, and increased viable counts as a percentage of total counts. Under monocots, 12–17% of the added fertilizer N was recovered as soil organic N. Although this N immobilization was attributed to microbial assimilation, less than 1/4 was actually recovered as microbial biomass N, as measured with the chloroform fumigation/N-extraction method or calculated from total bacterial counts. The white clover accumulated substantial amounts of N due to N₂ fixation. However, microbial N immobilization represented only 3% of the total N accumulation, showing that the microorganisms obtained a smaller share of biologically fixed N₂ than of the N applied as fertilizer. Extra additions of mineral N (monocots) enhanced microbial N assimilation, partly due to increased plant growth. The results also strongly indicated, however, that the microbial growth under monocots was N-limited in the latter part of the experiment and that fertilizer N had a direct effect on microbial growth. In the early phase of plant growth, N immobilization ranged from 33 to 58 mg N g^-1 root C. This level of immobilization required a release of organic C into the soil representing a minimum of 60–100% of that found in intact roots.     

The use of nitrogen-15 natural abundance in white clover (Trifolium repens L.) to determine nitrogen fixation under different management practices

 The ¹⁵N natural abundance (δ¹⁵N) of white clover (Trifolium repens L.) grown in pasture under different management practices was determined. Plants were split into leaflets, petioles and stolons and the ¹⁵N signature of each tissue was measured. The δ¹⁵N of leaflet tissue from plants of two non-N₂-fixing species (Lolium perenne L. and Ranunculus repens L.), growing in close proximity to the sampled T. repens, was also measured. By using T. repens plants grown in the absence of mineral N to provide reference material, the proportion of N derived from N₂ fixation (%Ndfa) in pasture plants was calculated. Within a plot, variation was present in the δ¹⁵N between the tissues of T. repens. Variation was also present between the same tissues under different management practices. The %Ndfa in the leaf material of T. repens varied from 34% to 100% between the plots. The use of different reference species did not affect the estimate of %Ndfa. Received: 14 December 1998