The first genetic maps for subterranean clover (Trifolium subterraneum L.) and comparative genomics with T. pratense L. and Medicago truncatula Gaertn. to identify new molecular markers for breeding

This study reports on the construction of the first genetic maps of subterranean clover (Trifolium subterraneum L.), a diploid, inbreeding annual pasture legume, and alignment of its linkage groups with those of red clover (T. pratense L.) and Medicago truncatula Gaertn. Transferability of red and white clover (T. repens L.) simple sequence repeat (SSR) markers to subterranean clover was observed. A total of 343 SSR loci were mapped into eight subterranean clover linkage groups, with 6?C31 loci per linkage group and 27 loci with similar locations between two distinct F ₂ mapping populations. Phenotypic data obtained for flowering time, content of three isoflavonoids (formononetin, genistein and biochanin A), hardseededness, leaf markings, calyx pigmentation and hairiness of stems were analyzed, together with genotypic data. Genomic intervals influencing each trait were assigned to one to three chromosome regions, accounting for 5.5?C59.8% of the phenotypic variance. Syntenic relationships were observed among subterranean clover, red clover and Medicago truncatula genomes. Comparisons of loci shared between the three species indicated that at least two chromosomal regions have undergone duplications in the subterranean clover genome. Candidate genes for isoflavone content were identified using M. truncatula as a reference genome. Synteny-based segmentation observed in Brassicaceae chromosomes helped to account for the apparent segmental-based relationship between the clover genomes, particularly within the subterranean clover lines. The proposed segmental nature of clover genome could account for the extensive variation observed between the parental genotypes, while not preventing production of fertile intercrosses.     

Studies on the host-finding ability of the aphid parasitoid,Trioxys complanatus (Hym.: Braconidae) in lucerne and clover

The spotted alfalfa aphid (SAA),Therioaphis trifolii (Monell)f. maculata, was first recorded in Australia in 1977. It is a major pest of lucerne and other pasture medics but not of clover (Trifolium spp.). The parasitoidTrioxys complanatus Quilis was introduced into Australia in late 1977 as part of a biological control program for SAA and made a substantial contribution to the management of the pest. Since 1989, aphids which are individually indistinguishable from SAA have been causing substantial yield loss to clover pastures in western and south-eastern Australia. This aphid, now known in Australia as the spotted clover aphid (SCA), is genetically distinct from SAA and has a much wider host range. Populations of SCA in clover are rarely parasitised byT. complanatus. A series of experiments was undertaken to determine whether the greater susceptibility of SAA in lucerne than of SCA in clover to parasitisation byT. complanatus is due to the attraction of the parasitoid preferentially to lucerne or to the differential attractiveness of the aphids themselves. SAA and SCA were offered to the parasitoids in arenas of increasing complexity from single trifoliate leaves of lucerne and clover on agar through single potted plants in small cages to groups of potted plants in a large cage. Results showed that the parasitoids exhibited a preference for lucerne though, in the cage situation, they did find and parasitise SCA on clover. They did not differentiate between SAA and SCA on lucerne.     

Lipid metabolism in mixtures of red clover (Trifolium repens) and perennial ryegrass (Lolium perenne) in lab scale silages and in vitro rumen incubations

Most often, farmers consider red clover an unattractive forage because of its low ensilability. Nevertheless, several in vivo and in vitro experiments also showed advantages of red clover silages such as decreased rumen biohydrogenation of polyunsaturated fatty acids. This has been attributed to a possible protective role of protein-bound phenols, with polyphenol oxidase playing a key role in their formation. This enzyme is active in red clover, but not in other green forages, such as, for example, perennial ryegrass. Therefore, the aim was to study the lipid metabolism within red clover/ryegrass mixtures in lab scale silages and during in vitro rumen batch incubations. Ensilability of red clover increased with higher proportions of ryegrass in the silage mixture. However, the lipid-protecting mechanism of red clover does not seem to occur in the co-ensiled ryegrass as lipolysis of polar lipids linearly increased with increasing proportions of ryegrass (86.0%, 91.6%, 89.9%, 93.1% and 95.6% in 60-day-old silages with 100/0, 75/25, 50/50, 25/75 and 0/100 red clover/ryegrass, respectively). Rumen lipolysis and biohydrogenation of C18:3n-3 and C18:2n-6 were negatively related to red clover proportions in the silage mixtures. The lipid-protective mechanism in red clover silages is confirmed, but it seems not to be transferred to lipids in co-ensiled forages.     

Isolation and structural identification of the trihydroxamate siderophore vicibactin and its degradative products from Rhizobium leguminosarum ATCC 14479 bv. trifolii

The Rhizobia are a group of free-living soil bacteria known for their ability to symbiotically infect the roots of specific host plants as well as to produce siderophores in order to compete with other microorganisms for the limited availability of iron in the rhizosphere. In this study, Rhizobium leguminosarum ATCC 14479, which preferentially infects the red clover Trifolium pratense, was found to produce the trihydroxamate siderophore vicibactin (C₃₃H₅₅N₆O₁₅) under iron restricted conditions. In addition, two other iron-binding, siderophore-like compounds: C₂₀H₃₆N₄O₁₀, C₃₁H₅₅N₆O₁₅, were isolated and purified from the culture media. Due to the structural similarity of the latter compounds to vicibactin based on electrospray-mass spectrometry and nuclear magnetic resonance data, these heretofore unreported molecules are thought to be either modified or degraded products of vicibactin. Although vicibactin has previously been found to be commonly produced by other rhizobial strains, this is the first time it has been chemically characterized from a clover infecting strain of R. leguminosarum.     

Host Suitability of Twelve Leguminosae Species to Populations of Meloidogyne hapla and M. chitwoodi

Legumes of the genera Astragalus (milkvetch), Coronilla (crownvetch), Lathyrus (pea vine), Lotus (birdsfoot trefoil), Medicago (alfalfa), Melilotus (clover), Trifolium (clover), and Vicia (common vetch) were inoculated with a population of Melaidogyne chitwoodi from Utah or with one of three M. hapla populations from California, Utah, and Wyoming.Thirty-nine percent to 86% of alfalfa (M. scutellata) and 10% to 55% of red clover (T. pratense) plants survived inoculation with the nematode populations at a greenhouse temperature of 24 ± 3°C. All plants of the other legume species survived all nematode populations, except 4% of the white clover (T. repens) plants inoculated with the California M. hapla population. Entries were usually more susceptible to the M. hapla populations than to M. chitwoodi. Galling of host roots differed between nematode populations and species. Root-galling indices (1 = none, 6 = severely galled) ranged from 1 on pea vine inoculated with the California population of M. hapla to 6 on yellow sweet clover inoculated with the Wyoming population of M. hapla. The nematode reproductive factor (Rf = final nematode population/initial nematode population) ranged from 0 for all nematode populations on pea vine to 35 for the Wyoming population of M. hapla on alfalfa (M. sativa).     

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.     

A galactoglucomannan from the leaf and stem tissues of red clover (Trifolium pratense)

A galactoglucomannan has been isolated by fractionation of the alkali-soluble hemicelluloses of the leaf and stem tissues of red clover (Trifolium pratense L.). The hemicellulose contains galactose, glucose, and mannose residues in the molar ratios 0.25:1.0:1.1 and accounts for ca. 25% of the mannose residues present in the clover tissues. Structural studies showed that the hemicellulose has a main chain of β-(1→4)-linked D-glucopyranosyl and D-mannopyranosyl residues, to which are attached α-(1→6)-linked D-galactopyranosyl residues.     

Sugar and organic Acid constituents in white clover

Major ethanol-soluble carbohydrate and organic acid constituents of white clover (Trifolium repens) have been identified by use of high-performance liquid chromatography and gas chromatography. In leaves, petioles, roots, and nodules, pinitol (3-O-methyl chiro-inositol) is the predominant sugar, with sucrose present in lower concentration. In leaves and petioles there are significant levels of α- and β-methyl glucosides, linamarin, glucose, and fructose. In the nodules glucose is rarely present at detectable levels. The concentration of pinitol is generally greater than 25 millimolar in each tissue examined whereas the level of sucrose varies depending on the time of day. Sucrose is the major sugar significantly labeled during 1 hour administration of ¹⁴CO₂ and accounts for more than 99% of all the radioactivity detected in the nodules at early times. Between 3 and 7 hours after labeling, 6% of the radioactivity is found in the organic acids fraction and 5% in the basic fraction of nodules. Malonic acid does not appear to be present in unusually high concentrations in either leaves or nodules of white clover.     

Phenotype profiling of Rhizobium leguminosarum bv. trifolii clover nodule isolates reveal their both versatile and specialized metabolic capabilities

Rhizobium leguminosarum bv. trifolii (Rlt) are soil bacteria inducing nodules on clover, where they fix nitrogen. Genome organization analyses of 22 Rlt clover nodule isolates showed that they contained 3–6 plasmids and majority of them possessed large (>1 Mb), chromid-like replicon with exception of four Rlt strains. The Biolog phenotypic profiling comprising utilization of C, N, P, and S sources and tolerance to osmolytes and pH revealed metabolic versatility of the Rlt strains. Statistical analyses of our results showed a clear bias toward specific metabolic preferences, tolerance to unfavorable osmotic conditions, and increased nodulation activity of the strains having smaller amount of extrachromosomal DNA. The K5.4 and K4.15 lacking a large megaplasmid possessed substantially diverse metabolism and belonged to effective clover inoculants. In conclusion, besides overall metabolic versatility, some metabolic specialization may enable rhizobia to persist in variable environments and to compete successfully with other bacteria.     

Reproduction of Meloidogyne incognita on Winter Cover Crops Used in Cotton Production

Substantial reproduction of Meloidogyne incognita on winter cover crops may lead to damaging populations in a subsequent cotton (Gossypium hirsutum) crop. The amount of population increase during the winter depends on soil temperature and the host status of the cover crop. Our objectives were to quantify M. incognita race 3 reproduction on rye (Secale cereale) and several leguminous cover crops and to determine if these cover crops increase population densities of M. incognita and subsequent damage to cotton. The cover crops tested were ‘Bigbee’ berseem clover (Trifolium alexandrinum), ‘Paradana’ balansa clover (T. balansae), ‘AU Sunrise’ and ‘Dixie’ crimson clover (T. incarnatum), ‘Cherokee’ red clover (T. pratense), common and ‘AU Early Cover’ hairy vetch (Vicia villosa), ‘Cahaba White’ vetch (V. sativa), and ‘Wrens Abruzzi’ rye. In the greenhouse tests, egg production was greatest on berseem clover, Dixie crimson clover, AU Early Cover hairy vetch, and common hairy vetch; intermediate on Balansa clover and AU Sunrise crimson clover; and least on rye, Cahaba White vetch, and Cherokee red clover. In both 2002 and 2003 field tests, enough heat units were accumulated between 1 January and 20 May for the nematode to complete two generations. Both AU Early Cover and common hairy vetch led to greater root galling than fallow in the subsequent cotton crop; they also supported high reproduction of M. incognita in the greenhouse. Rye and Cahaba White vetch did not increase root galling on cotton and were relatively poor hosts for M. incognita. Only those legumes that increased populations of M. incognita reduced cotton yield. In the southern US, M. incognita can complete one to two generations on a susceptible winter cover crop, so cover crops that support high nematode reproduction may lead to damage and yield losses in the following cotton crop. Planting rye or Meloidogyne-resistant legumes as winter cover crops will lower the risk of increased nematode populations compared to most vetches and clovers.     

Attraction of clover head weevil, Hypera meles, to flower bud volatiles of several species of Trifolium

Steam distilled volatiles from flower buds of white clover, Trifolium repens, attracted significantly greater numbers of female clover head weevils, Hypera meles, than males, in laboratory bioassays. Persian clover, T. resupinatum, bud extract, which attracted significantly more females than either small hop clover, T. dubium or red clover, T. pratense, also displayed qualitative and quantitative differences in the gas chromatographic profile of its bud volatiles. The strong attraction of normal females to Persian clover in laboratory bioassays was abolished by antennectomy, suggesting that attraction is regulated by olfactory receptors located on weevil antennal clubs. Weevils demonstrated a similar attraction to Persian clover in the field, indicating the possibility of developing a lure made from the flower bud extract of this clover to trap weevils for control or population monitoring purposes.     

Nitrogen transfer from forage legumes to nine neighbouring plants in a multi-species grassland

Legumes play a crucial role in nitrogen supply to grass-legume mixtures for ruminant fodder. To quantify N transfer from legumes to neighbouring plants in multi-species grasslands we established a grass-legume-herb mixture on a loamy-sandy site in Denmark. White clover (Trifolium repens L.), red clover (Trifolium pratense L.) and lucerne (Medicago sativa L.) were leaf-labelled with ¹⁵N enriched urea during one growing season. N transfer to grasses (Lolium perenne L. and xfestulolium), white clover, red clover, lucerne, birdsfoot trefoil (Lotus corniculatus L.), chicory (Cichorium intybus L.), plantain (Plantago lanceolata L.), salad burnet (Sanguisorba minor L.) and caraway (Carum carvi L.) was assessed. Neighbouring plants contained greater amounts of N derived from white clover (4.8?g?m^-2) compared with red clover (2.2?g?m^-2) and lucerne (1.1?g?m^-2). Grasses having fibrous roots received greater amounts of N from legumes than dicotyledonous plants which generally have taproots. Slurry application mainly increased N transfer from legumes to grasses. During the growing season the three legumes transferred approximately 40?kg?N ha^-1 to neighbouring plants. Below-ground N transfer from legumes to neighbouring plants differed among nitrogen donors and nitrogen receivers and may depend on root characteristics and regrowth strategies of plant species in the multi-species grassland.     

Secondary Metabolites of Micromycetes in Plants of the Family Fabaceae,Genus <Emphasis Type="Italic">Trifolium</Emphasis>

The component composition and content of mycotoxins in perennial grasses of five species of the genus Trifolium, selected from natural biocenoses in different periods of vegetation, were studied. In the terrestrial parts of plants, the permanent presence of alternariol, cyclopiazonic acid, and emodin was shown. It was noted that differences in contamination are most clearly expressed in meadow clover Trifolium pratense L. and white clover Trifolium repens L., while alsike clover Trifolium hybridum L. occupies an intermediate position. It was found that zigzag clover Trifolium medium L. and mountain clover Trifolium montanum L., morphologically close to meadow clover, practically did not differ in the composition of toxic metabolites. The ways of formation of metabolic equilibrium in plants with participation of microscopic fungi are discussed.     

Genetic diversity and structure of Rhizobium leguminosarum populations associated with clover plants are influenced by local environmental variables

The identification and conservation of indigenous rhizobia associated with legume plants and their application as biofertilizers is becoming an agricultural worldwide priority. However, little is known about the genetic diversity and phylogeny of rhizobia in Romania. In the present study, the genetic diversity and population composition of Rhizobium leguminosarum symbiovar trifolii isolates from 12 clover plants populations located across two regions in Romania were analyzed. Red clover isolates were phenotypically evaluated and genotyped by sequencing 16S rRNA gene, 16S-23S intergenic spacer, three chromosomal genes (atpD, glnII and recA) and two plasmid genes (nifH and nodA). Multilocus sequence typing (MLST) analysis revealed that red clover plants are nodulated by a wide genetic diversity of R. leguminosarum symbiovar trifolii sequence types (STs), highly similar to the ones previously found in white clover. Rhizobial genetic variation was found mainly within the two clover populations for both chromosomal and plasmid types. Many STs appear to be unique for this region and the genetic composition of rhizobia differs significantly among the clover populations. Furthermore, our results showed that both soil pH and altitude contributed to plasmid sequence type composition while differences in chromosomal composition were affected by the altitude and were strongly correlated with distance.     

Use of available crop by-products as alternative bedding materials to wheat straw for rearing broilers

The provision of suitable bedding for rearing broilers is an ongoing challenge for poultry producers. The aim of the present study was to evaluate the effect of using different types of crop residues as alternative bedding materials to wheat straw on growth performance, carcass traits, health, and welfare of broilers; moreover, the effects on the bedding and air quality were analyzed. We randomly assigned 180 1-day-old chickens (Cobb 500) across six bedding groups (three replicates of 10 birds each). Chickens in all groups were reared under similar housing conditions. Chickens of one group were reared on a wheat straw bedding (control), whereas those of the remaining five groups were reared on clover straw, cornstalk chips (CS), sugarcane top chips, chopped palm spines (CPS), and corn ear husks. Cornstalk chip and chopped palm spines were associated with the lowest (P < 0.05) ammonia concentrations compared with the remaining beddings. The mean values of bedding moisture percentage (P < 0.05), caking score, and ammonia concentrations (P < 0.05) were decreased with 6-week-old broilers reared on cornstalk chips. At 6 weeks, wheat straw, clover straw, sugarcane top chips and corn ear husks resulted in higher (P < 0.0001) BW and average daily gain compared with CS and CPS. Broilers reared on clover straw bedding showed the best FCR during 4–6 weeks of age (P < 0.01), and those kept on clover straw and sugarcane top chips exhibited the best (P < 0.01) feed conversion ratio (1.61 and 1.64, respectively) during 0–6 weeks of age. The carcass dressing yield, airborne dust concentration, bedding pH, and bacterial counts did not differ among the different bedding materials. The proportions of the drumsticks, gizzards, and abdominal fat of 6-week-old broilers were affected by bedding type (P < 0.05); but the differences among the bedding treatments were small. The mean incidence of footpad dermatitis and breast blisters was significantly less using clover straw. Broilers reared on chopped palm spine had the highest (P < 0.05) heterophil/lymphocyte ratio when compared with their counterparts. Conclusively, using clover straw or cornstalks as an alternative bedding material may be a beneficial strategy and should be highly recommended for rearing broilers.     

Impact of the spatial scale of grass–legume mixtures on sheep grazing behaviour,preference and intake,and subsequent effects on pasture

The benefits of using white clover in pastures have been widely recognised for many years. However, clover is perceived as being unreliable because of its typically low content, which is spatially and temporally variable, in mixed pastures. One proposed solution to increase the proportion of clover in the diet of grazing animals and composition in the pasture is to spatially separate clover from grass within the same field. In a field experiment ryegrass and white clover were grown in fine mixtures, and in pure alternating strips of ryegrass and clover of 1.5 m, 3 m or 18 m width within a field. Pastures were grazed for two grazing periods of 9 and 12 weeks, and measurements of sward surface height (SSH), herbage mass and composition and clover morphology were taken. Grazing behaviour was also observed. Results showed that spatial separation in the long term, when compared with a fine mixture, increased clover availability (18% to 30% v. 9%, based on standing dry matter) and was not grazed to extinction. Ewes maintained their preference for clover throughout the experiment (selection coefficient 2 to 5), which resulted in a reduction in the SSH of clover in monocultures to <3 cm and significant changes to the morphology of clover (smaller leaves, shorter petioles and thicker stolon), at the expense of maximising their intake. Spatial separation in the short term may therefore allow grazing animals to select their preferred diet; however, in the long term in continuously grazed pasture, their preference for clover depletes its availability.     

Receptor Site on Clover and Alfalfa Roots for Rhizobium

Sites on white clover and alfalfa roots that bind Rhizobium trifolii and R. meliloti capsular polysaccharides, respectively, were examined by fluorescence microscopy. Fluorescein isothiocyanate-labeled capsular material from R. trifolii bound specifically to root hairs of clover but not alfalfa. Binding was most intense at the root hair tips. Treatment of clover roots with 2-deoxyglucose (2-dG) prevented binding of R. trifolii capsular material to the roots. The sugar 2-dG enhanced the elution of clover root protein, which could bind to and specifically agglutinate R. trifolii but not R. meliloti or R. japonicum. The mild elution procedure left the roots intact. Agglutination of R. trifolii and passive hemagglutination of rabbit erythrocytes coated with the capsular material of R. trifolii were specifically inhibited by 2-dG. These results suggest that clover roots contain proteins that cross-link complementary polysaccharides on the surface of clover root hairs and infective R. trifolii through 2-dG-sensitive binding sites. Alfalfa root hairs were shown to specifically bind to a surface polysaccharide from R. meliloti.