Trifolium Species Research Articles

Localized hypermutation and associated gene losses in legume chloroplast genomes

Point mutations result from errors made during DNA replication or repair, so they are usually expected to be homogeneous across all regions of a genome. However, we have found a region of chloroplast DNA in plants related to sweetpea (Lathyrus) whose local point mutation rate is at least 20 times higher than elsewhere in the same molecule. There are very few precedents for such heterogeneity in any genome, and we suspect that the hypermutable region may be subject to an unusual process such as repeated DNA breakage and repair. The region is 1.5 kb long and coincides with a gene, ycf4, whose rate of evolution has increased dramatically. The product of ycf4, a photosystem I assembly protein, is more divergent within the single genus Lathyrus than between cyanobacteria and other angiosperms. Moreover, ycf4 has been lost from the chloroplast genome in Lathyrus odoratus and separately in three other groups of legumes. Each of the four consecutive genes ycf4-psaI-accD-rps16 has been lost in at least one member of the legume "inverted repeat loss" clade, despite the rarity of chloroplast gene losses in angiosperms. We established that accD has relocated to the nucleus in Trifolium species, but were unable to find nuclear copies of ycf4 or psaI in Lathyrus. Our results suggest that, as well as accelerating sequence evolution, localized hypermutation has contributed to the phenomenon of gene loss or relocation to the nucleus.

Sex pheromone of the red clover casebearer moth, Coleophora deauratella, an invasive pest of clover in Canada

AbstractThe red clover casebearer, Coleophora deauratella Lienig & Zeller (Lepidoptera: Coleophoridae), is an invasive pest of Trifolium species (Fabaceae) in Canada. We identified candidate sex pheromone components from female pheromone gland extracts using coupled gas chromatographic–electroantennographic analysis detection. Three compounds elicited an electrophysiological response from antennae and were identified as: (Z)‐7‐dodecenyl acetate, (Z)‐5‐dodecenyl acetate, and (Z)‐7‐dodecen‐1‐ol. Field tests of the candidate pheromone components revealed that males were attracted to a binary mixture of (Z)‐7‐dodecenyl acetate and (Z)‐5‐dodecenyl acetate. Male moth trap capture was greatest in traps baited with lures containing 100:10 or 100:20 ratios of these pheromone components, respectively. Trap capture was reduced when (Z)‐5‐dodecenyl acetate was present below 10 or above 20% of (Z)‐7‐dodecenyl acetate. Equal numbers of male moths were captured in traps baited with 10, 100, and 1 000 μg of the attractive binary mixture. These findings allow for the development of a pheromone‐based monitoring system for this invasive pest of clover in Canada.

Complete genome sequence of Rhizobium leguminosarum bv. trifolii strain WSM1325, an effective microsymbiont of annual Mediterranean clovers.

Rhizobium leguminosarum bv trifolii is a soil-inhabiting bacterium that has the capacity to be an effective nitrogen fixing microsymbiont of a diverse range of annual Trifolium (clover) species. Strain WSM1325 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from root nodules collected in 1993 from the Greek Island of Serifos. WSM1325 is produced commercially in Australia as an inoculant for a broad range of annual clovers of Mediterranean origin due to its superior attributes of saprophytic competence, nitrogen fixation and acid-tolerance. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a microsymbiont of annual clovers. We reveal that its genome size is 7,418,122 bp encoding 7,232 protein-coding genes and 61 RNA-only encoding genes. This multipartite genome contains 6 distinct replicons; a chromosome of size 4,767,043 bp and 5 plasmids of size 828,924 bp, 660,973 bp, 516,088 bp, 350,312 bp and 294,782 bp.

Ecology, chorology and commonness of the Trifolium taxa in Greece

AbstractThe taxa of the genus Trifolium L. are considered valuable genetic plant resources worldwide. The study of their chorological and floristic characteristics is of great importance. This paper aims at evaluating the taxa of the genus found in the flora of Greece in terms of their ecological, chorological and commonness characteristics. A floristic catalogue was produced based on published, but not genus‐specific, floristic data collected at various parts of the country and field surveys carried out by the authors in years 2004 and 2005. Information on the localities of the taxa, either found in the field surveys or scattered into floristic reports, was gathered. The present analysis was based on several research results and bibliographic information. It was found that the Greek flora contains 113 taxa (95 species, 17 subspecies, one hybrid) out of a total of approximately 255 species reported worldwide; six of them being Greek endemics. The taxa are distributed into two subgenera of the genus; subgenus Chronosemium including 14 species and subgenus Trifolium that includes 98 taxa (without counting one hybrid) distributed in five out of eight sections: Trifolium (56), Vesicaria (18), Trifoliastrum (15), Trichocephalum (7) and Paramesus (2). The majority of the taxa are mediterranean elements (38%), annuals (76%), therophytes (76%), and of scapose growth type (78%). It was also found that the centre of distribution of Trifolium is located in Anatolia (Turkey) and Greece, so pointing out this part of east Mediterranean as an important “gene‐pool” region. The most common Trifolium species in Greece are T. repens, T. campestre, T. fragiferum, T. physodes and T. arvense (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Complete genome sequence of Rhizobium leguminosarum bv trifolii strain WSM2304, an effective microsymbiont of the South American clover Trifolium polymorphum.

Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse range of annual and perennial Trifolium (clover) species. Strain WSM2304 is an aerobic, motile, non-spore forming, Gram-negative rod, isolated from Trifolium polymorphum in Uruguay in 1998. This microsymbiont predominated in the perennial grasslands of Glencoe Research Station, in Uruguay, to competitively nodulate its host, and fix atmospheric nitrogen. Here we describe the basic features of WSM2304, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a nitrogen fixing microsymbiont of a clover species from the American center of origin. We reveal that its genome size is 6,872,702 bp encoding 6,643 protein-coding genes and 62 RNA only encoding genes. This multipartite genome was found to contain 5 distinct replicons; a chromosome of size 4,537,948 bp and four circular plasmids of size 1,266,105 bp, 501,946 bp, 308,747 bp and 257,956 bp.

Competition for nodule occupancy between introduced and native strains of Rhizobium leguminosarum biovar trifolii

The aim of this work was to evaluate the competitive ability between Rhizobium leguminosarum bv trifolii strain U204 used as commercial inoculants in Uruguay for Trifolium repens L. and Trifolium pratense L. and two native strains isolated from inoculated pastures of T. pratense. T126 is an efficient nitrogen fixer and a melanin producer strain; T70 is inefficient and a melanin non-producer strain; and U204 is very efficient in both hosts but is a melanin non-producer strain. Competitiveness between the strains was determined in experiments in pots and in growth pouches under controlled conditions. In the last experiment, we evaluated pH of plant nutrient solution and inoculum ratios. Plant dry weight was determined, and the identification of nodule bacteria was done using melanin production and DNA fingerprinting (GTG5-PCR). The U204 symbiotic efficiency was not affected by the co-inoculation with the others two native strains. The T70 strain was a poor competitor when was co-inoculated with one of the effective strains in both experiments. Our results confirmed a “selective nodulation” because an effective symbiosis occurred preferentially over an ineffective one in Trifolium species. The native effective strain competed with U204 for nodule formation in both clovers species, but the nodule occupancy depended on the inoculum ratio. The pH of nutritive solution did not affect competition ability of the studied strains. It may be possible to isolate efficient, competitive, and genetically different native rhizobial strains to be used as inoculant strains for clover pastures in Uruguay. Both (GTG)5-PCR and melanin production were useful methods to identify nodulating bacteria in competition studies.

Phenotypic, genotypic, and symbiotic diversities in strains nodulating clover in different soils in Spain.

Trifolium species are the most common legumes present in wild Spanish soils; however, there are no studies to date on the diversity of rhizobia nodulating clover in Spain. Twenty strains from different Spanish soils with acidic, neutral, and basic pH were selected to study their genotypic, phenotypic, and symbiotic features. The results showed that the isolates were genotypically diverse, displaying 12 different DNA fingerprint patterns and also 14 different plasmid profiles. Although they have 16S rRNA gene sequences that are nearly identical to that of the type strain of Rhizobium leguminosarum, their recA and atpD gene sequences were phylogenetically divergent from those of R. leguminosarum reference strains, and phenotypic divergence as well as different host ranges were also found. Although most of them nodulated both Trifolium and Phaseolus, only 5 strains were also able to nodulate Pisum. The results of the effectiveness analysis showed a high variability in the symbiotic characteristics of our strains and suggested that Pisum is the more restrictive host of this group. Interestingly, some of the Trifolium isolates showed an ability to promote growth of Pisum in the absence of nodulation.

Responses of genotypes from species of Trifolium, Ornithopus, Biserrula and Hedysarum to a highly virulent race of Phytophthora clandestina and new sources of resistance

AbstractThirty‐six genotypes, including 15 cultivars and 10 breeding lines of Trifolium subterraneum, a single genotype of each of seven other species of Trifolium (viz. Trifolium dasyurum, Trifolium glanduliferum, Trifolium incarnatum, Trifolium michelanium, Trifolium purpureum, Trifolium spumosum and Trifolium vesiculosum), Biserrula pelecinus, Hedysarum coronarium, Ornithopus compressus and Ornithopus sativus were screened under controlled environmental conditions for resistance to root disease caused by the most pathogenic race of Phytophthora clandestina occurring in Australia, namely race 177. This is the first time any of these genera/species other than T.subterraneum has ever been screened for its response to P. clandestina. The root disease caused by P.clandestina is the first report of susceptibility to this pathogen for the seven other species of Trifolium and also for B.pelecinus, H.coronarium and O.sativus. Within T.subterraneum, a very high level of resistance was identified in cvs Denmark, Junee and Meteora [scores ≤1.5 (0–5 scale where 0 = no disease) across two separate screening tests] and in the breeding lines SL027 and SM023 (scores ≤1.3 across two separate screening tests). Six of the seven other species of Trifolium (viz. T.dasyurum, T.glanduliferum, T.incarnatum, T.michelanium, T.purpureum and T.spumosum) showed a high level of resistance (scores ≤0.8 across two separate screening tests), while T.vesiculosum showed a disease score of ≤1.2 across both screening tests. O.compressus showed no disease in either test, and O.sativus showed a disease score of ≤0.7 across both screening tests. H.coronarium was susceptible with a disease score of ≤2.8 across two separate screening tests, while B.pelecinus was highly susceptible with disease scores of 3.5 and 4.6 in these tests. The high levels of resistance identified against P.clandestina are useful sources of resistance that can be exploited commercially, either directly to minimise damage from this disease or as parents in breeding programs to develop cultivars within the genera/species tested with improved resistance to this highly pathogenic race of P.clandestina.

Using elevated CO 2 to increase the biomass of a Sorghum vulgare × Sorghum vulgare var. sudanense hybrid and Trifolium pratense L. and to trigger hyperaccumulation of cesium

The most important challenge to use phytoremediation is how to improve its efficiency by increasing the accumulation of metals in plants, or by improving key plant biological traits that should enhance metal uptake. In this paper, we used open-top chambers to investigate the effects of elevated CO 2 (860 μL L −1) on biomass and Cs uptake by a Sorghum vulgare × Sorghum vulgare var. sudanense hybrid and Trifolium pratense L. growing on soils spiked with various levels of cesium (0, 300, 1500 and 3000 mg Cs kg −1). The results showed that elevated CO 2 not only increased aboveground biomass of the Sorghum and Trifolium species by 32–111%, and by 8–11%, respectively, compared to the ambient CO 2 treatment, but also caused more accumulation of Cs by Sorghum species (up to 73%) than Trifolium species (up to 43%). It was speculated that the increase in biomass and the improvement in Cs accumulation ability at elevated CO 2 could be related to lowered soil pH values, and changes in number and kind of microorganisms in the rhizospheres of the two tested species. This is the first report of a link among elevated CO 2, increased biomass and hyperaccumulation of Cs by Sorghum and Trifolium species.

The green manure value of seven clover species grown as annual crops on low and high fertility temperate soils

Annual and perennial clover species may differ in green manure value. Seven clover (Trifolium) species were grown as annual crops on low fertility (Breton) and high fertility (Edmonton) soils in Alberta. Four annual clovers [balansa (T. michelianum Savi), berseem (T. alexandrinum L.), crimson (T. incarnatum L.), and Persian (T. resupinatum L.)], three perennial clovers [alsike (T. hybridum L.), red (T. pratense L.), and white Dutch (T. repens L.)] and a non-legume reference crop [fall rye (Secale cereale L.)] were ploughed-down as green manure in autumn, and followed by barley (Hordeum vulgare L.). Annual clovers had greater biomass yields than perennial clovers, and berseem clover had the highest yield. At Breton, mean biomass N content was greater for perennial clovers (2.9 g N kg-1) than annual clovers (1.9 g N kg-1). Clover biomass at Breton yielded an average of 77 kg N ha-1, with N derived from the atmosphere averaging 88% by N difference method and 75% by 15N natural abundance method. At Edmonton, the green manures had few effects on soil nitrate and subsequent barley yields. At Breton, all clover green manures except balansa increased barley yields, and grain yields were greater following perennial clovers than annual clovers in one year. Annual clovers will provide forage biomass and add N in areas where rainfall is adequate, and they may be preferable under zero tillage. However there is no advantage of annual clovers, relative to perennial clovers, in terms of N supply.Key words: Green manure, clover, Trifolium species, nitrogen fixation

Impacts of seedling herbivory on plant competition and implications for species coexistence.

Although the causes and consequences of seedling herbivory for plant community composition are well understood, the mechanisms by which herbivores influence plant species recruitment to the established phase remain less clear. The aim was to examine how variation in the intensity of seedling herbivory interacts with growth-defence trade-offs and herbivore feeding preferences to affect plant community development. Using 14-d-old seedlings of Trifolium pratense and T. repens, relative growth and susceptibility to herbivory by the snail Helix aspersa was quantified to elucidate putative growth-defence trade-offs for these species. Then mixed assemblages of 14-d-old Trifolium seedlings were exposed to herbivory by zero, two, five or ten snails and determined how variation in the intensity of herbivory affected competitive interactions into the mature phase (as measured by total plant biomass at 120 d old). In the absence of herbivory, communities were dominated by T. pratense; a result expected on the basis that it yielded larger and presumably more competitive seedlings. However, when seedlings were exposed to herbivory, the balance of competition shifted. At low levels of herbivory (two snails), both Trifolium species contributed equally to total plant biomass. More intense herbivory (five snails) resulted in almost total mortality of T. pratense and dominance of the mature community by T. repens. The most intense herbivory (ten snails) effectively removed all seedlings from the experimental community. The study illustrates a mechanism whereby spatio-temporal fluctuations in seedling herbivory, when coupled with species-specific variation in competitive ability and sensitivity to herbivore attack, can differentially influence plant recruitment into the mature phase. This mechanism may be a key element in our attempts to understand plant species coexistence, since fluctuations in plant recruitment are fundamental to the many theories that view coexistence as a consequence of a spatio-temporal lottery for dominance over regeneration micro-sites.

N2 fixation in three perennial Trifolium species in experimental grasslands of varied plant species richness and composition

This study is the first to investigate quantitative effects of plant community composition and diversity on N2 fixation in legumes. N2 fixation in three perennial Trifolium species grown in field plots with varied number of neighbouring species was evaluated with the 15N natural abundance method (two field sites, several growing seasons, no N addition) and the isotope dilution method (one site, one growing season, 5 g N m−2). The proportion of plant N derived from N2 fixation, pNdfa, was generally high, but the N addition decreased pNdfa, especially in species-poor communities. Also following N addition, the presence of grasses in species-rich communities increased pNdfa in T. hybridum and T. repens L., while legume abundance had the opposite effect. In T. repens, competition for light from grasses appeared to limit growth and thereby the amount of N2 fixed at the plant level, expressed as mg N2 fixed per sown seed. We conclude that the occurrence of diversity effects seems to be largely context dependent, with soil N availability being a major determinant, and that species composition and functional traits are more important than species richness regarding how neighbouring plant species influence N2 fixation in legumes.

Ultrasound‐assisted solid‐phase extraction coupled with photodiode‐array and fluorescence detection for chemotaxonomy of isoflavone phytoestrogens in Trifolium L. (Clover) species

Detailed chemotaxonomic studies were undertaken to establish the qualitative profile and real amounts of the pharmacologically active isoflavone aglycones genistein, daidzein, formononetin, and biochanin A in aerial parts of thirteen Trifolium L. (clover) species, native to Poland. A newly elaborated micropreparative technique--SPE--on BakerBond octadecyl, cyclohexyl, and phenyl cartridges was used in combination with ultrasound-assisted extraction for isolation of isoflavone aglycones from hydrolyzed samples. The effectiveness of all three SPE sorbents in the purification of plant extracts was compared and very high recoveries (>96%) were documented for four isoflavones. Classical photodiode-array and very sensitive fluorescence detection, coupled with reversed-phase high-performance liquid chromatography (RP-HPLC), were employed to obtain the most reliable qualitative and quantitative results. Chemotaxonomic differences combined with flower color variability were demonstrated within thirteen clover species. Concentration levels of particular isoflavones in ten Trifolium species possessing flowers with white, pink, or purple-red corolla ranged from approximately 3 to approximately 3300 microg/g dry weight, while in three yellow flowering clovers (T. aureum, T. dubium, and T. campestre) isoflavone compounds have not been detected at all. RSD values, determined for intra- and inter-day precision of the quantitative results, were not higher than 6.2% and 7.1%, respectively.

Fatty acids and other lipid composition of five Trifolium species with antioxidant activity

The contents of fatty acids and other lipids of Trifolium balansae Boiss, Trifolium stellatum Lin., Trifolium nigrescens Viv. subsp. petrisavi (Clem) Holmboe, Trifolium constantinopolitanum Ser., and Trifolium resupinatum L. var. resupinatum L. (Leguminosae) were determined by gas chromatography-mass spectrometry (GC-MS). The whole plant hexane extracts of five Trifolium species contained eight fatty acids consisting of linolenic (31.1%) and palmitic (18.9%) acids as the most abundant unsaturated and saturated fatty acids, respectively. The total unsaturation for the oils of five Trifolium species was 30.6–42.2%. In addition to fatty acids, some alkanes and phytol were also obtained. The antioxidant activity and contents of fatty acids and lipids of five Trifolium species T. balansae, T. stellatum, T. nigrescens subsp. petrisavi, T. constantinopolitanum, and T. resupinatum var. resupinatum are presented for the first time in this study.

Breaking Seed Dormancy of Some Annual Medicago and Trifolium Species by Different Treatments

The germination abilities of 14 annual legume species (six Medicago and eight Trifolium) were tested under three dormancy breaking treatments: In the first treatment, mechanical scarification plus sodium hypochlorite (MSS); in the second, chemical scarification with sulphuric acid (CSA); in the third, incubation in water bath at 90 °C (IWB) were used for breaking the dormancy. All Medicago species except for Medicago polymorpha showed improvement in germination rates using MSS. The seeds of M. polymorpha showed higher germination rates with CSA or IWB than MSS. For Trifolium species, results indicated that there was a great variability among species with regard to the treatments. Seeds of species showed a broad range of dormancy requirements for germination. The CSA treatment significantly increased the germination rates of Trifolium lappaceum, T. scabrum and T. strictum. On the other hand, the germination rates of T. spumosum, T. spadieceum and T. angustifolium were significantly improved by MSS. The seeds of T. badium showed higher germination rates with IWB than the other applications.

The influence of NaCl salinity and hypoxia on aspects of growth in Trifolium species

The effects of salinity and hypoxia on growth, nutritive value, and ion relations were evaluated in 38 species of Trifolium and 3 check legume species (Trifolium fragiferum, Trifolium michelianum, and Medicago sativa) under glasshouse conditions, with the aim of identifying species that may be suitable for saline and/or waterlogged conditions. In the first set of experiments, plants were grown hydroponically at four NaCl concentrations (0, 40, 80, and 160 mm NaCl) and harvested after exposure to these treatments for 4 weeks. NaCl concentrations up to 160 mm reduced dry matter production in most species; however, there were differences in salt tolerance among species, with T. argutum, T. diffusum, T. hybridum, and T. ornithopodioides performing well under the saline conditions (dry matter production was reduced by less than 20%). Concentrations of Na+ and Cl− in the shoots increased with increasing salinity levels, and species again differed in their capacity to limit the uptake of these ions. Dry matter digestibility at 0 mm ranged from 49.8% (T. palaestinum) to 74.0% (T. vesiculosum) and decreased with increasing NaCl concentrations. A second set of experiments evaluated the tolerance of Trifolium species to hypoxic conditions in the glasshouse. Shoot growth, and to a lesser extent root growth, were reduced in all Trifolium species when plants were exposed to stagnant, non-aerated conditions for 28 days, but T. michelianum, T. resupinatum, T. squamosum, T. nigrescens, T. ornithopodioides, T. salmoneum, and T. fragiferum were the least affected species. All species acclimated to the oxygen-depleted conditions by increasing the gas-filled porosity in the roots. This study has provided information that will assist in the identification of forage species for saline and/or waterlogged areas.

Trifolium L. – A review on its phytochemical and pharmacological profile

Plants from the genus Trifolium have been used in traditional medicine by many cultures. In Turkish folk medicine, for example, some Trifolium species are used for their expectorant, analgesic, antiseptic properties and also to treat rheumatic aches. Some species are also grown as pasture crops for animals in the Mediterranean. The high quercetin concentration and soyasaponin occurrence make the seeds of some Trifolium species a potential source of health beneficial phytochemicals for use in human nutrition. However, Trifolium pratense has also gained popularity due to research into its use for the treatment for menopausal symptoms. This paper provides an overview of the phytochemical and pharmacological profile of Trifolium species.

Genetic similarity among Trifolium species based on isozyme banding pattern

The study deals with similarity among 25 species of the genus Trifolium represented by 134 accessions. Clustering of the species based on isozyme banding pattern of five enzymes revealed that T. repens was distinctly different from other species. T. repens and T. retusum formed independent clusters. The group of species comprising of T. pratense, T. cherleri, T. spumosum, T. subterraneum, T. resupinatum, T. alexandrinum, T. echinatum, T. constantinopolitanum and T. tembense exhibited considerable similarity to the second cluster. This group joined another group of five species, i.e. T. nigrescens, T. glomeratum, T. apertum, T. alpestre and T. hybridum with nearly 50% similarity. T. purpureum, T. hirtum, T. campestre, T. incarnatum, and T. argutum grouped separately. There was no marked difference for banding pattern among T. alexandrinum genotypes. T. alexandrinum showed close affinity with T. subterraneum and T. resupinatum.T. lappaceum, T. diffusum, T. campestre, T. incarnatum and T. argutum showed only 44.8% similarity with other Trifolium species. Grouping together of accessions belonging to individual species indicated that incompatibility among species under study had restricted interspecific hybridization. Species belonging to subgenus Lotoidea clustered with species of subgenus Trifolium. Chonosemium species T. campestre formed one cluster with two Trifolium species T. hirtum and T. incarnatum. T. nigrescens was placed quite apart from the T. repens.

Leaf epidermal characteristics of Trifolium L. species from Serbia and Montenegro

A micromorphological analysis of leaf epidermal tissue of 18 Trifolium species was carried out using light and scanning electron microscopy, in order to obtain qualitative and quantitative data on epidermal characteristics and to evaluate their taxonomic significance. The shape of epidermal cells and their cell walls, epidermal wax depositions, presence of papillae, type and number of stomata, type, distribution and number of non-glandular and glandular trichomes were analyzed. The results showed that species that belong to the same section had some common leaf epidermal characteristics. Combinations of those characters proved to be useful in taxonomy at the level of the sections, but not for identification of single species.

Volatile composition of three Trifolium species

Volatile composition of three Trifolium species