Winter survival in red clover: experimental evidence for interactions among stresses.

BACKGROUND: There is a lack of knowledge on the combined effects of different stresses on plants, in particular different stresses that occur during winter in temperate climates. Perennial herbaceous plants in temperate regions are exposed to many different stresses during winter, but except for the fact that cold temperatures induce resistance to a number of them, very little is known about their interaction effects. Knowledge about stress interactions is needed in order to predict effects of climate change on both agricultural production and natural ecosystems, and to develop adaptation strategies, e.g., through plant breeding. Here, we conducted a series of experiments under controlled conditions to study the interactions between cold (low positive temperature), clover rot infection (caused by Sclerotinia trifoliorum) and freezing, in red clover (Trifolium pratense) accessions. We also compared our results with winter survival in field experiments and studied associations between stress and shoot growth. RESULTS: Exposure to low positive temperatures (cold acclimation) induced resistance to clover rot. There was a clear negative interaction effect between freezing stress and clover rot infection, resulting in up to 37% lower survival rate compared to what would have been expected from the additive effect of freezing and infection alone. Freezing tolerance could continue to improve during incubation under artificial snow cover at 3 °C in spite of darkness, and we observed compensatory shoot growth following freezing after prolonged incubation. At the accession level, resistance to clover rot was negatively correlated with growth in the field during the previous year at a Norwegian location. It was also negatively correlated with the shoot regrowth of control plants after incubation. Clover rot resistance tests under controlled conditions showed limited correlation with clover rot resistance observed in the field, suggesting that they may reveal variation in more specific resistance mechanisms. CONCLUSIONS: We here demonstrate, for the first time, a strong negative interaction between freezing and infection with a winter pathogen. We also characterize the effects of cold acclimation and incubation in darkness at different temperatures on winter stress tolerance, and present data that support the notion that annual cycles of growth and stress resistance are associated at the genetic level.

Allelopathic effect of the methanol extract of the weed species-red sorrel (Rumex acetosella L.) on the growth, phytohormone content and antioxidant activity of the cover crop - white clover (Trifolium repens L.).

Allelopathy is a biological process in which one organism releases biochemicals that affect the growth and development of other organisms. The current investigation sought to determine the allelopathic effect of Rumex acetosella on white clover (Trifolium repens) growth and development by using its shoot extract (lower IC50 value) as a foliar treatment. Here, different concentrations (25, 50, 100, and 200 g/L) of shoot extract from Rumex acetosella were used as treatments. With increasing concentrations of shoot extract, the plant growth parameters, chlorophyll and total protein content of Trifolium repens decreased. On the other hand, ROS, such as O2.- and H2O2, and antioxidant enzymes, including SOD, CAT, and POD, increased with increasing shoot extract concentration. A phytohormonal study indicated that increased treatment concentrations increased ABA and SA levels while JA levels were reduced. For the identification of allelochemicals, liquid‒liquid extraction, thin-layer chromatography, and open-column chromatography were conducted using R. acetosella shoot extracts, followed by a seed bioassay on the separated layer. A lower IC50 value was obtained through GC/MS analysis. gammaSitosterol was identified as the most abundant component. The shoot extract of Rumex acetosella has strong allelochemical properties that may significantly impede the growth and development of Trifolium repens. This approach could help to understand the competitive abilities of this weed species and in further research provide an alternate weed management strategy.

Lipid remodelling and the conversion of lipids into sugars associated with tolerance to cadmium toxicity during white clover seed germination.

Cadmium (Cd) is a leading environmental issue worldwide. The current study was conducted to investigate Cd tolerance of 10 commercial white clover (Trifolium repens) cultivars during seed germination and to further explore differences in lipid remodelling, glycometabolism, and the conversion of lipids into sugars contributing to Cd tolerance in the early phase of seedling establishment as well as the accumulation of Cd in seedlings and mature plants. The results show that Cd stress significantly reduced seed germination of 10 cultivars. Compared to Cd-sensitive Sulky, Cd-tolerant Pixie accelerated amylolysis to produce more glucose, fructose, and sucrose by maintaining higher amylase and sucrase activities under Cd stress. Pixie maintained higher contents of various lipids, higher DGDG/MGDG ratio, and lower unsaturation levels of lipids, which could be beneficial to membrane stability and integrity as well as signal transduction in cells after being subjected to Cd stress. In addition, Pixie upregulated expression levels of key genes (TrACX1, TrACX4, TrSDP6, and TrPCK1) involved in the conversion of lipids into sugars for early seedling establishment under Cd stress. These findings indicate that lipid remodelling, enhanced glycometabolism, and accelerated conversion of lipids into sugars are important adaptive strategies for white clover seed germination and subsequent seedling establishment under Cd stress. In addition, Pixie not only accumulated more Cd in seedlings and mature plants than Sulky but also had significantly better growth and phytoremediation efficiency under Cd stress. Pixie could be used as a suitable and critical germplasm for the rehabilitation and re-establishment of Cd-contaminated areas.

Chemical Basis for Determining the Allelopathic Potential of Invasive Plant Wall Barley (Hordeum murinum L. subsp. murinum).

The study investigated compounds present in the invasive grass Hordeum murinum L. subsp. murinum and tested the allelopathic potential of this plant against common meadow species Festuca rubra L. and Trifolium repens L. Gas chromatography-mass spectrometry (GC-MS) performed separately on the ears and stalks with leaves of wall barley revealed 32 compounds, including secondary metabolites, that may play an important role in allelopathy. Two compounds, N-butylbenzenesulfonamide (NBBS) and diphenylsulfone (DDS), were described for the first time for wall barley and the Poaceae family. The presence of 6,10,14-trimethylpentadecan-2-one (TMP) has also been documented. Aqueous extracts of H. murinum organs (ears and stalks with leaves) at concentrations of 2.5%, 5%, and 7.5% were used to evaluate its allelopathic potential. Compared to the control, all extracts inhibited germination and early growth stages of meadow species. The inhibitory effect was strongest at the highest concentration for both the underground and aboveground parts of the seedlings of the meadow species tested. Comparing the allelopathic effect, Trifolium repens proved to be more sensitive. In light of the results of the study, the removal of wall barley biomass appears to be important for the restoration of habitats where this species occurs due to its allelopathic potential.

Asynapsis and unreduced gamete formation in a Trifolium interspecific hybrid.

BACKGROUND: Unreduced gametes, a driving force in the widespread polyploidization and speciation of flowering plants, occur relatively frequently in interspecific or intergeneric hybrids. Studies of the mechanisms leading to 2n gamete formation, mainly in the wheat tribe Triticeae have shown that unreductional meiosis is often associated with chromosome asynapsis during the first meiotic division. The present study explored the mechanisms of meiotic nonreduction leading to functional unreduced gametes in an interspecific Trifolium (clover) hybrid with three sub-genomes from T. ambiguum and one sub-genome from T. occidentale. RESULTS: Unreductional meiosis leading to 2n gametes occurred when there was a high frequency of asynapsis during the first meiotic division. In this hybrid, approximately 39% of chromosomes were unpaired at metaphase I. Within the same cell at anaphase I, sister chromatids of univalents underwent precocious separation and formed laggard chromatids whereas paired chromosomes segregated without separation of sister chromatids as in normal meiosis. This asynchrony was frequently accompanied by incomplete or no movement of chromosomes toward the poles and restitution leading to unreduced chromosome constitutions. Reductional meiosis was restored in progeny where asynapsis frequencies were low. Two progeny plants with approximately 5 and 7% of unpaired chromosomes at metaphase I showed full restoration of reductional meiosis. CONCLUSIONS: The study revealed that formation of 2n gametes occurred when asynapsis (univalent) frequency at meiosis I was high, and that normal gamete production was restored in the next generation when asynapsis frequencies were low. Asynapsis-dependent 2n gamete formation, previously supported by evidence largely from wheat and its relatives and grasshopper, is also applicable to hybrids from the dicotyledonous plant genus Trifolium. The present results align well with those from these widely divergent organisms and strongly suggest common molecular mechanisms involved in unreduced gamete formation.

Major effect loci for plant size before onset of nitrogen fixation allow accurate prediction of yield in white clover.

KEY MESSAGE: Accurate genomic prediction of yield within and across generations was achieved by estimating the genetic merit of individual white clover genotypes based on extensive genetic replication using cloned material. White clover is an agriculturally important forage legume grown throughout temperate regions as a mixed clover-grass crop. It is typically cultivated with low nitrogen input, making yield dependent on nitrogen fixation by rhizobia in root nodules. Here, we investigate the effects of clover and rhizobium genetic variation by monitoring plant growth and quantifying dry matter yield of 704 combinations of 145 clover genotypes and 170 rhizobium inocula. We find no significant effect of rhizobium variation. In contrast, we can predict yield based on a few white clover markers strongly associated with plant size prior to nitrogen fixation, and the prediction accuracy for polycross offspring yield is remarkably high. Several of the markers are located near a homolog of Arabidopsis thaliana GIGANTUS 1, which regulates growth rate and biomass accumulation. Our work provides fundamental insight into the genetics of white clover yield and identifies specific candidate genes as breeding targets.

Transcriptome analysis reveals major transcriptional changes during regrowth after mowing of red clover (Trifolium pratense).

BACKGROUND: Red clover (Trifolium pratense) is globally used as a fodder plant due its high nutritional value and soil improving qualities. In response to mowing, red clover exhibits specific morphological traits to compensate the loss of biomass. The morphological reaction is well described, but the underlying molecular mechanisms and its role for plants grown in the field are unclear. RESULTS: Here, we characterize the global transcriptional response to mowing of red clover by comparing plants grown under greenhouse conditions with plants growing on agriculturally used fields. Unexpectedly, we found that biotic and abiotic stress related changes of plants grown in the field overlay their regrowth related transcriptional changes and characterized transcription related protein families involved in these processes. Further, we can show that gibberellins, among other phytohormones, also contribute to the developmental processes related to regrowth after biomass-loss. CONCLUSIONS: Our findings show that massive biomass loss triggers less transcriptional changes in field grown plants than their struggle with biotic and abiotic stresses and that gibberellins also play a role in the developmental program related to regrowth after mowing in red clover. Our results provide first insights into the physiological and developmental processes of mowing on red clover and may serve as a base for red clover yield improvement.

Laboratory and Field Evaluation of the Phytotoxic Activity of Sapindus mukorossi Gaertn Pulp Extract and Identification of a Phytotoxic Substance.

Interest in finding plant-based herbicides to supplement synthesized herbicides is increasing. Although the extract of Sapindus mukorossi Gaertn has been reported to have herbicidal activity, little is known about phytotoxic substances and their efficacy of weed control in the field. To identify phytotoxic substances, the bioassay-guided fractionation by column chromatography and high-speed counter-current chromatography (HSCCC) was carried out. The phytotoxic activity assay, performed by the agar medium method, showed that the 70% ethanol fraction exhibited strong root growth inhibition against Trifolium pratense with an 50% inhibitory concentration (IC50) value of 35.13 mg/L. An active compound was isolated from the 70% ethanol fraction and identified as hederagenin 3-o-ß-D-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (Compound A). Compound A had an IC50 value of 16.64 mg/L. Finally, a new formulation was prepared based on the 70% ethanol fraction, which exhibited good efficacy against broadleaf weeds in a carrot field. The fresh weight control efficacy was 78.7% by 45 days after treatment at the dose of 1500 g a. i./ha. Hence, the extract of S. mukorossi pulp could be a promising supplement to the synthesized herbicides. Furthermore, compound A from S. mukorossi may be responsible for its phytotoxic activity.

Trifolium subterraneum cover cropping for improving the nutritional status of a Mediterranean apricot orchard.

BACKGROUND: The utilization of Trifolium subterraneum L. cover crops may represent an innovative and efficient option in low-input and organic farming, especially in Mediterranean agroecosystems where low and irregular rainfall require frequent soil tillage and use of herbicides to reduce moisture losses and weed competitiveness. Since imbalances of soil macro- and micro-nutrients due to cover cropping establishment could be responsible for numerous problems in specialized orchards, such as disturbances in the normal tree growth and quality of fruits, the objective of this study was to investigate, the cumulative effects of a 3-years established T. subterraneum cover cropping, compared with a spontaneous flora and a conventional management (as a control), on the levels of mineral nutrients in the apricot leaves and fruits. RESULTS: Our findings indicated that T. subterraneum cover cropping tended to stimulate higher leaf macro- and micro-nutrients content than conventional management and flora spontaneous cover cropping. In addition, the presence of T. subterraneum cover cropping, especially with the incorporation of dead mulches into the soil, increased the content of potassium (K), nitrogen (N), calcium (Ca), iron (Fe) and manganese (Mn) in apricot fruits. CONCLUSION: Taking also into account the effects of T. subterraneum cover cropping on both the reduction of soil weed and enhancement of bacteria communities involved in the soil N-cycle, we may suggest its application in Mediterranean orchards as an eco-friendly alternative to synthetic herbicides for weed control and mineral N fertilizers, while enhancing the apricot tree nutritional status and fruit quality. © 2020 Society of Chemical Industry.

Revealing the full-length transcriptome of caucasian clover rhizome development.

BACKGROUND: Caucasian clover (Trifolium ambiguum M. Bieb.) is a strongly rhizomatous, low-crowned perennial leguminous and ground-covering grass. The species may be used as an ornamental plant and is resistant to cold, arid temperatures and grazing due to a well-developed underground rhizome system and a strong clonal reproduction capacity. However, the posttranscriptional mechanism of the development of the rhizome system in caucasian clover has not been comprehensively studied. Additionally, a reference genome for this species has not yet been published, which limits further exploration of many important biological processes in this plant. RESULT: We adopted PacBio sequencing and Illumina sequencing to identify differentially expressed genes (DEGs) in five tissues, including taproot (T1), horizontal rhizome (T2), swelling of taproot (T3), rhizome bud (T4) and rhizome bud tip (T5) tissues, in the caucasian clover rhizome. In total, we obtained 19.82 GB clean data and 80,654 nonredundant transcripts were analysed. Additionally, we identified 78,209 open reading frames (ORFs), 65,227 coding sequences (CDSs), 58,276 simple sequence repeats (SSRs), 6821 alternative splicing (AS) events, 2429 long noncoding RNAs (lncRNAs) and 4501 putative transcription factors (TFs) from 64 different families. Compared with other tissues, T5 exhibited more DEGs, and co-upregulated genes in T5 are mainly annotated as involved in phenylpropanoid biosynthesis. We also identified betaine aldehyde dehydrogenase (BADH) as a highly expressed gene-specific to T5. A weighted gene co-expression network analysis (WGCNA) of transcription factors and physiological indicators were combined to reveal 11 hub genes (MEgreen-GA3), three of which belong to the HB-KNOX family, that are up-regulated in T3. We analysed 276 DEGs involved in hormone signalling and transduction, and the largest number of genes are associated with the auxin (IAA) signalling pathway, with significant up-regulation in T2 and T5. CONCLUSIONS: This study contributes to our understanding of gene expression across five different tissues and provides preliminary insight into rhizome growth and development in caucasian clover.

Responses of microbial communities and metabolic activities in the rhizosphere during phytoremediation of Cd-contaminated soil.

Phytoremediation is an effective way to repair heavy metal contaminated soil and rhizosphere microorganisms play an important role in plant regulation. Nevertheless, little information is known about the variation of microbial metabolic activities and community structure in rhizosphere during phytoremediation. In this study, the rhizosphere soil microbial metabolic activities and community structure of Trifolium repensL. during Cd-contaminated soil phytoremediation, were analyzed by Biolog EcoPlate™ and high-throughput sequencing. The uptake in the roots of Trifolium repensL. grown in 5.68 and 24.23 mg/kg Cd contaminated soil was 33.51 and 84.69 mg/kg respectively, causing the acid-soluble Cd fractions decreased 7.3% and 5.4%. Phytoremediation significantly influenced microbial community and Trifolium repensL. planting significantly increased the rhizosphere microbial population, diversity, the relative abundance of plant growth promoting bacteria (Kaistobacter and Flavisolibacter), and the utilization of difficultly metabolized compounds. The correlation analysis among substrate utilization and microbial communities revealed that the relative abundance increased microorganisms possessed stronger carbon utilization capacity, which was beneficial to regulate the stability of plant-microbial system. Collectively, the results of this study provide fundamental insights into the microbial metabolic activities and community structure during heavy metal contaminated soil phytoremediation, which may aid in the bioregulation of phytoremediation.

Comparison of the Polyphenolic Profile of Medicago sativa L. and Trifolium pratense L. Sprouts in Different Germination Stages Using the UHPLC-Q Exactive Hybrid Quadrupole Orbitrap High-Resolution Mass Spectrometry.

Identification and quantification of polyphenols in plant material are of great interest since they make a significant contribution to its total bioactivity. In the present study, an UPLC-Orbitrap-MS/MS approach using the variable data acquisition mode (vDIA) was developed and applied for rapid separation, identification, and quantification of the main polyphenolic compounds in Medicago sativa L. and Trifolium pratense L. sprouts in different germination stages. Based on accurate MS data and fragment ions identification strategy, a total of 29 compounds were identified by comparing their accurate masses, fragment ions, retention times, and literatures. Additionally, a number of 30 compounds were quantified by comparing to the reference standards. Data were statistically analysed. For both plant species, the sprouts of the third germination day are valuable sources of bioactive compounds and could be used in phytotherapy and nutrition. Although Trifolium pratense L. (Red Clover) is considered to be a reference for natural remedies in relieving menopause disorders, alfalfa also showed a high level of biological active compounds with estrogenic activity.

Agronomic and qualitative characterization of multi-cut berseem clover (Trifolium alexandrinum L.) cultivars.

BACKGROUND: Berseem clover is the main forage crop grown in Mediterranean regions. There are plenty of cultivars that possess variability in their productivity and quality among the different cuts. Therefore, accurate agronomic and qualitative characterization is crucial for selecting the most promising cultivars for breeding and feeding purposes. In the present study, the agronomic characteristics, ruminal degradability and fermentation measures of five cuts of the five most prominent Egyptian beseem clover cultivars (Helaly, Serw, Giza6, Gemmeza1 and Sakha4) were evaluated. RESULTS: The Giza6 cultivar produced a significantly higher fresh yield and high whole plant dry matter content on the 3rd cut. Giza6 was among the superior cultivars in crude protein content and had the highest values of gas production at the 3rd cut, and ruminal degraded acid detergent fiber and propionate concentrations at the 2nd cut, at the same time as presenting the lowest ammonia concentrations at the 3rd and 5th cuts. Overall, the 3rd cut produced significantly higher fresh yield across all cultivars, except for Sakha4. The 3rd cut also produced a higher leaf to stem ratio compared to the 4th and 5th cuts. The 1st three cuts had higher degraded neutral detergent fiber than the 4th and 5th cuts. The 1st cut was characterized by low propionate concentration in the Helaly and Gemmeza1 cultivars compared to the other cuts. CONCLUSION: Expanded production of the high-yielding, high-quality cultivar Giza6 is recommended. When harvesting, three cuts is optimal for berseem clover cultivation for breeding and feeding programs. © 2020 Society of Chemical Industry.

Mutualists Stabilize the Coexistence of Congeneric Legumes.

Coexistence requires that stabilizing niche differences, which cause species to limit themselves more than others, outweigh relative fitness differences, which cause competitive exclusion. Interactions with shared mutualists, which can differentially affect host fitness and change in magnitude with host frequency, can satisfy these conditions for coexistence, yet empirical tests of mutualist effects on relative fitness and stabilizing niche differences are largely lacking within the framework of coexistence theory. Here, we show that N-fixing rhizobial mutualists mediate coexistence in four naturally co-occurring congeneric legume (Trifolium) species. Using experimental greenhouse communities, we quantified relative fitness and stabilizing niche differences for each species in the presence of rhizobia originating from conspecific or congeneric hosts. Rhizobia stabilized coexistence by increasing the self-limitation of Trifolium species grown with rhizobia isolated from conspecifics, thus allowing congeners to increase when rare. Greenhouse-measured invasion growth rates predicted natural, unmanipulated coexistence dynamics of Trifolium species over 2 years at our field sites. Our results demonstrate that interactions with shared mutualists can stabilize the coexistence of closely related species.

Metabolomics and physiological analyses reveal ß-sitosterol as an important plant growth regulator inducing tolerance to water stress in white clover.

MAIN CONCLUSION: ß-sitosterol influences amino acids, carbohydrates, organic acids, and other metabolite metabolism and homeostasis largely contributing to better tolerance to water stress in white clover. ß-sitosterol (BS) could act as an important plant growth regulator when plants are subjected to harsh environmental conditions. Objective of this study was to examine effects of BS on growth and water stress tolerance in white clover based on physiological responses and metabolomics. White clover was pretreated with or without BS and then subjected to water stress for 7 days in controlled growth chambers. Physiological analysis demonstrated that exogenous application of BS (120 µM) could significantly improve stress tolerance associated with better growth performance and photosynthesis, higher leaf relative water content, and less oxidative damage in white clover in response to water stress. Metabolic profiling identified 78 core metabolites involved in amino acids, organic acids, sugars, sugar alcohols, and other metabolites in leaves of white clover. For sugars and sugar alcohol metabolism, the BS treatment enhanced the accumulation of fructose, glucose, maltose, and myo-inositol contributing to better antioxidant capacity, growth maintenance, and osmotic adjustment in white clover under water stress. The application of BS was inclined to convert glutamic acid into proline, 5-oxoproline, and chlorophyll instead of going to pyruvate and alanine; the BS treatment did not significantly affect intermediates of tricarboxylic acid cycle (citrate, aconitate, and malate), but promoted the accumulation of other organic acids including lactic acid, glycolic acid, glyceric acid, shikimic acid, galacturonic acid, and quinic acid in white clover subjected to water stress. In addition, cysteine, an important antioxidant metabolite, was also significantly improved by BS in white clover under water stress. These altered amino acids and organic acids metabolism could play important roles in growth maintenance and modulation of osmotic and redox balance against water stress in white clover. Current findings provide a new insight into BS-induced metabolic homeostasis related to growth and water stress tolerance in plants.

Globular structures in roots accumulate phosphorus to extremely high concentrations following phosphorus addition.

Crops with improved uptake of fertilizer phosphorus (P) would reduce P losses and confer environmental benefits. We examined how P-sufficient 6-week-old soil-grown Trifolium subterraneum plants, and 2-week-old seedlings in solution culture, accumulated P in roots after inorganic P (Pi) addition. In contrast to our expectation that vacuoles would accumulate excess P, after 7 days, X-ray microanalysis showed that vacuolar [P] remained low (<12 mmol kg-1 ). However, in the plants after P addition, some cortex cells contained globular structures extraordinarily rich in P (often >3,000 mmol kg-1 ), potassium, magnesium, and sodium. Similar structures were evident in seedlings, both before and after P addition, with their [P] increasing threefold after P addition. Nuclear magnetic resonance (NMR) spectroscopy showed seedling roots accumulated Pi following P addition, and transmission electron microscopy (TEM) revealed large plastids. For seedlings, we demonstrated that roots differentially expressed genes after P addition using RNAseq mapped to the T. subterraneum reference genome assembly and transcriptome profiles. Among the most up-regulated genes after 4 hr was TSub_g9430.t1, which is similar to plastid envelope Pi transporters (PHT4;1, PHT4;4): expression of vacuolar Pi-transporter homologs did not change. We suggest that subcellular P accumulation in globular structures, which may include plastids, aids cytosolic Pi homeostasis under high-P availability.

4xTrifolium ambiguum and 2xT. occidentale hybridise despite wide geographic separation and polyploidisation: implications for clover breeding.

KEY MESSAGE: The widely divergent species 4xTrifolium ambiguum and 2xT.occidentale are inter-fertile long after speciation (including polyploidisation) has occurred. Tri-species hybrids (T. repens × T. ambiguum × T. occidentale) have the potential to achieve introgression of stress resistant traits from both wild species into white clover. Trifolium ambiguum and T. occidentale are geographically, adaptionally and phenotypically contrasting species in the white clover section (Trifoliastrum) of the genus. T. ambiguum occurs as a high-altitude polyploid series (2x, 4x, 6x) in W Asia and NE Europe. T. occidentale is a diploid coastal species, occurring at sea level in W Europe. This study investigated hybridisation between 4xT. ambiguum and 2xT. occidentale and considered the significance of the hybrids for introgression breeding of white clover. Partially fertile F1 hybrids between 4xT. ambiguum and 2x and 4xT. occidentale were generated by embryo rescue. Hybrid plant morphology and fertility varied widely and hybrids generally expressed traits from both species. Advanced generation (F2-F5) 4x hybrids were highly fertile and constitute a new synthetic allotetraploid species. FISH analyses of 4x hybrids showed multivalent chromosome configurations with homoeologous associations between T. ambiguum and T. occidentale chromosomes. Crosses of the hybrids with T. repens produced fertile tri-species progeny. These very divergent species remain inter-fertile long after speciation (including polyploidisation) has occurred. Tri-species hybrids have the potential to achieve introgression of stress resistance traits from both wild species into white clover.

Isoflavone production in hairy root cultures and plantlets of Trifolium pratense.

OBJECTIVES: The aim of this study was to develop a Trifolium pratense hairy root (HR) production protocol and select HR lines with high isoflavone yield following elicitor treatments. RESULTS: We obtained 13 independent HR lines, producing approximately three times more isoflavonoids than seedlings (3.3 mg/g dry weight) and in which 27 isoflavonoids were detected. Each HR line had its own isoflavonoid profile. These lines produced as major components daidzein, genistein, formononetin and biochanin A. Sucrose, salicylic acid (SA), yeast extract (YE) and flagellin 22 (flg22) were tested as elicitors. Using SA 140 mg/L, allowed the maximum isoflavonoid production in plantlets (11.9 mg/g dry weight) but reduced root growth, possibly as a result of its toxicity. The highest isoflavone production in HR (27.9 mg/g dry weight) was obtained using sucrose 60 g/L, for 3.5 days. CONCLUSION: This work reports the high production of various isoflavonoids with T. pratense elicited HR cultures.

Dairy soil bacterial responses to nitrogen application in simulated Italian ryegrass and white clover pasture.

Through clearing and use of fertilizer and legumes, areas of southwestern Australia's unique coastal sand plains can support relatively low-cost dairies. However, the ancient, highly weathered nature of the soils in this region makes the dairies susceptible to a range of threats, including nutrient leaching and erosion. Despite this, Western Australian dairy cows typically produce up to 5,500 L of milk per head annually supported by inorganic nitrogen (N) fertilizer (commonly 50:50 urea and ammonium sulfate) at rates up to <320 kg of N/ha per year. Where hotspots exist (up to 2,000 kg of N/ha per year), total N exceeds pasture requirements. We investigated plant and soil bacteria responses to N fertilizer rates consistent with Australian legislated production practices on dairy farms for pure and mixed swards of white clover (Trifolium repens) and Italian ryegrass (Lolium multiflorum) in a long-term pasture experiment in controlled glasshouse conditions. Although the soil bacterial community structure at phylum level was similar for white clover and Italian ryegrass, relative abundances of specific subgroups of bacteria differed among plant species according to the N fertilizer regimen. Marked increases in relative abundance of some bacterial phyla and subphyla indicated potential inhibition of N cycling, especially for N hotspots in soil. Ammonium concentration in soil was less correlated with dominance of some N-cycling bacterial phyla than was nitrate concentration. Changes in bacterial community structure related to altered nutrient cycling highlight the potential for considering this area of research in policy assessment frameworks related to nutrient loads in dairy soils, especially for N.

Cigarette butts have adverse effects on initial growth of perennial ryegrass (gramineae: Lolium perenne L.) and white clover (leguminosae: Trifolium repens L.).

Cigarette filters (butts) are currently the most abundant form of anthropogenic litter on the planet, yet we know very little about their environmental impacts on terrestrial ecosystems, including plant germination and primary production. When discarded, filters contain a myriad of chemicals resulting from smoking tobacco and some still contain unsmoked remnants. A greenhouse experiment was used to assess the impacts of discarded filters of regular or menthol cigarette, either from unsmoked, smoked, or smoked cigarettes with remnant tobacco, on the growth and development of Lolium perenne (perennial ryegrass) and Trifolium repens (white clover). After 21 days, shoot length and germination success were significantly reduced by exposure to any type of cigarette filter for the grass and clover. Although total grass biomass was not measurably affected, the root biomass and root:shoot ratio were less in the clover when exposed to filters from smoked regular cigarettes and those with remnant tobacco. Cigarette filters caused an increase in chlorophyll-a in clover shoots and an increase in chlorophyll-b in grass shoots. Accordingly, whilst the chlorophyll a:b ratio was increased in the clover exposed to cigarette filters, it was decreased in grass. This study indicates the potential for littered cigarette filters to reduce growth and alter short-term primary productivity of terrestrial plants.