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Revegetation is a promising strategy for large-scale bauxite residue disposal and management, potentially influencing the geochemical stability of potentially toxic elements (PTEs) through rhizosphere processes. However, the geochemical behaviors of PTEs and the underlying mechanisms during bauxite residue revegetation remain unclear. This study examined the migration and transformation behaviors of PTEs and their underlying mechanisms in the bauxite residue-vegetation-leachate system under various revegetation strategies, including single and co-planting of perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.), over a 100-day microcosm experiment. The results showed significant decreases in pH, EC, Na, Al, and Cr levels in the leachate under various revegetation strategies, with slight increases in Cu, V, As, and Pb. Over time, the pH, EC, Na, Cr, Cu, V, Pb, and As levels in the leachate decreased, while those of Al, Fe, Mn, and Zn increased. The mean pH, EC, and concentrations of Na, Al, Fe, and Cr in the leachate of the revegetated treatments decreased by 6%-8%, 21%-33%, 2%-4%, 19%-27%, 7%-22%, and 15%-26%, respectively, while the mean concentrations of Mn, V, Zn, and As increased by 47%-134%, 26%-46%, 39%-47%, and 3%-10%, respectively, compared to the unamended treatment. Co-planting generally exhibited a greater impact on leachate components compared to single planting. Available contents of Al, Cr, and Pb decreased by 81%-83%, 57%-77%, and 55%-72%, respectively, while those of other PTEs increased in the revegetated bauxite residue. Co-planting significantly reduced the availability of PTEs compared to single planting. Except for Na and Mn, the bioaccumulation and transportation factors of PTEs in both vegetation species remained below 1 under various revegetation strategies. The migration and transformation behaviors of PTEs in the bauxite residue-vegetation-leachate system were mainly influenced by pH and nutrient levels. These findings provide new insights into the migration and transformation behaviors of PTEs during bauxite residue revegetation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Soil nutrients exhibit heterogeneity in their spatial distribution, presenting challenges to plant acquisition. Notably, phosphorus (P) heterogeneity is a characteristic feature of soil, necessitating the development of adaptive strategies by plants to cope with this phenomenon. To address this, fully crossed three-factor experiments were conducted using red clover within rhizoboxes. Positions of P in three conditions, included P even distribution (even P), P close distribution (close P), and P far distribution (far P). Concurrently, N addition was two amounts(0 and 20 mg kg - 1 ), both with and without AMF inoculation. The findings indicated a decrease in aboveground biomass attributable to uneven P distribution, whereas N and AMF demonstrated the potential to affect aboveground biomass. In a structural equation model, AMF primarily increased aboveground biomass by enhancing nodule number and specific leaf area (SLA). In contrast, N addition improved aboveground biomass through increased nodule number or direct effects. Subsequently, a random forest model indicated that under the far P treatment, fine root length emerged as the primary factor affecting aboveground biomass, followed by thickest root length. Conversely, in the even P treatment, the thickest root length was of paramount importance. In summary, when confronted with uneven P distribution, clover plants adopted various root foraging strategies. AMF played a pivotal role in elevating nodule number, and SLA., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The clover seed weevil, Tychius picirostris Fabricius (Coleoptera: Curculionidae), is a major pest in Oregon white clover seed crops. Reliance on synthetic pyrethroid insecticides and limited availability of diverse modes of action (MoAs) has increased insecticide resistance selection in regional T. picirostris populations, emphasizing the need to evaluate novel chemistries and rotational strategies for effective insecticide resistance management (IRM). The efficacy of 8 foliar insecticide formulations for managing T. picirostris adult and larval life stages was determined in small and large-plot field trials across 2 crop years. In both years, bifenthrin (Brigade 2EC), the grower's standard, showed negligible adult and larval suppression. Insecticide formulations with isocycloseram and cyantraniliprole active ingredients reduced adult and larval populations when applied at BBCH 59-60 (prebloom) and BBCH 65-66 (full bloom) growth stages, respectively. While differences in T. picirostris abundance were observed among insecticide treatments, seed yield differences were not detected in large-plot trials. Larval abundance was correlated with reduced seed yield, and an economic threshold of ≥3 larvae per 30 inflorescences was determined as a conservative larval threshold to justify foliar applications of diamide insecticides. Additional commercial white clover seed fields were surveyed to compare larval scouting techniques, including a standard Berlese funnel and a grower's do-it-yourself funnel. Both larval extraction techniques were correlated and provided similar estimates of larval abundance. These findings demonstrate new MoAs, optimal insecticide application timing, and larval monitoring methods that can be incorporated into an effective T. picirostris IRM program in white clover seed crops., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Industrialization-driven surface ozone (O 3 ) pollution significantly impairs plant growth. This study evaluates the effectiveness of exogenous protectants [3 mg L⁻ 1 abscisic acid (ABA), 400 mg L⁻ 1 ethylenediurea (EDU), and 80 mg L⁻ 1 spermidine (Spd)] on Trifolium repens subjected to O 3 stress in open-top chambers, focusing on plant growth and dynamics of culturable endophytic fungal communities. Results indicate that O 3 exposure adversely affects photosynthesis, reducing root biomass and altering root structure, which further impacts the ability of plant to absorb essential nutrients such as potassium (K), magnesium (Mg), and zinc (Zn). Conversely, the application of ABA, EDU, and Spd significantly enhanced total biomass and chlorophyll content in T. repens. Specifically, ABA and Spd significantly improved root length, root surface area, and root volume, while EDU effectively reduced leaves' malondialdehyde levels, indicating decreased oxidative stress. Moreover, ABA and Spd treatments significantly increased leaf endophytic fungal diversity, while root fungal abundance declined. The relative abundance of Alternaria in leaves was substantially reduced by these treatments, which correlated with enhanced chlorophyll content and photosynthesis. Concurrently, EDU and Spd treatments increased the abundance of Plectosphaerella, enhance the absorption of K, Ca, and Mg. In roots, ABA treatment increased the abundance of Paecilomyces, while Spd treatment enhanced the presence of Stemphylium, linked to improved nitrogen (N), phosphorus (P), and K uptake. These findings suggest that specific symbiotic fungi mitigate O 3 -induced stress by enhancing nutrient absorption, promoting growth. This study highlights the potential of exogenous protectants to enhance plant resilience against O 3 pollution through modulating interactions with endophytic fungal communities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Companion planting of white clover (Trifolium repens L.) with orchard grass (Dactylis glomerata L.), a famous hay grass, improves the forage quality of orchard grass. Microbiome profiling techniques can reveal the specific role of white clover companion planting with orchard grass. This study aimed to explore the microbiome distribution and gene functions of rhizosphere and non-rhizosphere soil via companion planting systems of white clover and orchard grass. From metagenomics sequencing analysis, we confirmed the significant role of white clover on soil environment modeling during companion planting with orchard grass. Twenty-eight biomarkers of rhizosphere soil organisms were identified during companion planting, including Proteobacteria, Betaproteobacteria, Flavobacteriia, and Caulobacterales. The number of gene functions of nitrogen and carbon fixation in companion planting was higher than that in single plants, indicating new functional flora for companion planting. We characterized specific rhizosphere effects, typical biomarker flora, and potential regulatory mechanisms for white clover-related companion planting by metagenomics analyses., (© 2024. The Author(s).)

Improving the cultivation mode and technology for traditional Chinese medicine has become important for its sustainable development. Monoculture enhances plant diseases, which decreases yield and quality. Intercropping is an effective measure to counterbalance that negative effect. In this study, we focused on Panax quinquefolium L. (ginseng) and four treatments were set up: the control without intercropping, P. quinquefolius + ryegrass (Lolium perenne L.), P. quinquefolius + red clover (Trifolium pratense L.), and P. quinquefolius + ryegrass + red clover. An LC-MS/MS system was used to detect the changes in the P. quinquefolius secondary metabolites, and high-throughput sequencing technology was used to determine the changes in the P. quinquefolius' rhizosphere soil microorganisms. Ginsenoside content, soil enzyme activities, and arbuscular mycorrhizal infection rate of P. quinquefolius were also measured using HPLC, ELISA kits, and microscopy, respectively. Co-intertia and Pearson's analysis were performed to explore the relationship between the metabolites and the P. quinquefolius microorganisms. Intercropping significantly increased the content of ginsenoside metabolites and recruited a large number of beneficial bacteria to the P. quinquefolius rhizosphere. The P. quinquefolius secondary metabolites were associated with the rhizosphere microbial community. For example, the dominant microorganisms, such as Acidobacteriota and Chloroflexi, played a key role in promoting the synthesis of ginsenoside Rd and (20R) ginsenoside Rg3 by P. quinquefolius. Intercropping led to changes in the P. quinquefolius secondary metabolites by driving and reshaping the rhizosphere microorganisms. These findings revealed the potential application of intercropping for improving the quality of P. quinquefolius., (© 2024 Scandinavian Plant Physiology Society.)

Species that repeatedly evolve phenotypic clines across environmental gradients have been highlighted as ideal systems for characterizing the genomic basis of local environmental adaptation. However, few studies have assessed the importance of observed phenotypic clines for local adaptation: conspicuous traits that vary clinally may not necessarily be the most critical in determining local fitness. The present study was designed to fill this gap, using a plant species characterized by repeatedly evolved adaptive phenotypic clines. White clover is naturally polymorphic for its chemical defence cyanogenesis (HCN release with tissue damage); climate-associated cyanogenesis clines have evolved throughout its native and introduced range worldwide. We performed landscape genomic analyses on 415 wild genotypes from 43 locations spanning much of the North American species range to assess the relative importance of cyanogenesis loci vs. other genomic factors in local climatic adaptation. We find clear evidence of local adaptation, with temperature-related climatic variables best describing genome-wide differentiation between sampling locations. The same climatic variables are also strongly correlated with cyanogenesis frequencies and gene copy number variations (CNVs) at cyanogenesis loci. However, landscape genomic analyses indicate no significant contribution of cyanogenesis loci to local adaptation. Instead, several genomic regions containing promising candidate genes for plant response to seasonal cues are identified - some of which are shared with previously identified QTLs for locally adaptive fitness traits in North American white clover. Our findings suggest that local adaptation in white clover is likely determined primarily by genes controlling the timing of growth and flowering in response to local seasonal cues. More generally, this work suggests that caution is warranted when considering the importance of conspicuous phenotypic clines as primary determinants of local adaptation., (© 2024 John Wiley & Sons Ltd.)

Background: Ectomycorrhizal (ECM and ECM-like) structures associated with plant root systems are a challenge for scientists. The dispersion pattern of roots within the soil profile and the nutritional conditions are both favourable factors to motivate the plants to make ECM associations., Results: This study discusses the colonization of mycorrhizal associations in Kobresia and Polygonum species including Polygonum viviparum, Kobresia filicina, K. myosuroides, Alnus nitida, Betula pendula, Pinus sylvestris, and Trifolium repens grown naturally in cold stressed soils of Gilgit-Baltistan (high-altitude alpine Deosai plains), Hazara, Swat, Dir, and Bajaur. Sieved soil batches were exposed to +5 °C (control), -10, -20, -30, -40, -50, -125 °C for 5 h, and selected plants were sown to these soils for 10 weeks under favourable conditions for ECM colonization. Ectomycorrhizal associations were examined in the above mentioned plants. Some ECM fungi have dark mycelia that look like the mantle and Hartig net. Examples of these are Kobresia filicina, K. myosuroides, and Polygonum viviparum. Findings of this study revealed that K. myosuroides excelled in ECM root tip length, dry mass, and NH 4 concentration at -125 °C. Contrarily, A. nitida demonstrated the lower values, indicated its minimum tolerance. Notably, T. repens boasted the highest nitrogen concentration (18.7 ± 1.31 mg/g), while P. sylvestris led in phosphorus (3.2 ± 0.22 mg/g). The B. pendula showed the highest potassium concentration (9.4 ± 0.66 mg/g), emphasising species-specific nutrient uptake capabilities in extreme cold conditions. The PCA analysis revealed that the parameters, e.g., NH 4 in soil mix (NH 4 ), NO 3 in soil mix (NO 3 ), phosphorus in soil in species of Polygonum viviparum, Kobresia filicina, K. myosuroides, Alnus nitida, Betula pendula, Pinus sylvestris, and Trifolium repens are most accurately represented in cases of + 5 °C, -10 °C, and -20 °C temperatures. On the other hand, the parameters for ECM root tips (ECM) and Dry Mass (DM) are best described in -40 °C, -50 °C, and - 125 °C temperatures. All parameters have a strong influence on the variability of the system indicated the efficiency of ECM. The heatmap supported the nutrients positively correlated with ECM colonization with the host plants., Conclusion: At lower temperatures, hyphae and spores in roots were reduced, while soluble phosphorus concentrations of leaves were increased in cold stress soils. Maximum foliar nutrient concentrations were found in K. myosuroides at the lowest temperature treatments due to efficient functioning and colonization of ECM., (© 2024. The Author(s).)

The invasion of alien plant and the pollution caused by soil microplastics have emerged as significant ecological threats. Recent studies have demonstrated aggravating effect of non-biodegradable microplastics on plant invasion. However, the impact of biodegradable microplastics (BMPs) on plant invasion remains unclear. Therefore, it is imperative to explore the impact of BMPs on plant invasion. In this study, a 30-day potting experiment with Trifolium repens L. (an invasive plant) and Oxalis corniculata L. (a native plant) was conducted to evaluate the influence of BMPs on T. repens's invasion. The findings revealed that BMPs results in a reduction in available N and P contents, thereby facilitating the colonization of arbuscular mycorrhizal fungi on T. repens 's roots. Consequently, T. repens adjusted its N and P foraging strategy by increasing P absorption ratio, and enhancing the accumulation of N and P in leaves. This ultimately led to the decrease of relative neighbor effect index of T. repens, indicating an aggravated invasion by T. repens. This study significantly enhances and expands the understanding of mechanisms by which microplastics aggravate plant invasion., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024 Scandinavian Plant Physiology Society.)

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 IC 50 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 O 2 .- and H 2 O 2, 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 IC 50 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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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.

Some forage legumes synthesize phytoestrogens. We conducted a glasshouse study to investigate how water stress (drought and waterlogging) influences phytoestrogen accumulation in red clover and kura clover. Compared to the red clover control, the 20 day drought resulted in an over 100% increase in the phytoestrogens formononetin and biochanin A, which together accounted for 91-96% of the total phytoestrogens measured. Waterlogging resulted in elevated concentrations of daidzein, genistein, and prunetin but not formononetin or biochanin A. Concentrations of phytoestrogens in kura clover were low or undetectable, regardless of water stress treatment. Leaf water potential was the most explanatory single-predictor of the variation in concentrations of formononetin, biochanin A, and total phytoestrogens in red clover. These results suggest that drought-stressed red clover may have higher potential to lead to estrogenic effects in ruminant livestock and that kura clover is a promising alternative low- or no-phytoestrogen perennial forage legume.

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., (© 2022. The Author(s).)

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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The plant microbiome supports plant growth, fitness, and resistance against climate change. Trifolium pratense (red clover), an important forage legume crop, positively contributes to ecosystem sustainability. However, T. pratense is known to have limited adaptive ability toward climate change. Here, the T. pratense microbiomes (including both bacteria and fungi) of the rhizosphere and the root, shoot, and flower endospheres were comparatively examined using metabarcoding in a field located in Central Germany that mimics the climate conditions projected for the next 50-70 years in comparison with the current climate conditions. Additionally, the ecological functions and metabolic genes of the microbial communities colonizing each plant compartment were predicted using FUNGuild, FAPROTAX, and Tax4Fun annotation tools. Our results showed that the individual plant compartments were colonized by specific microbes. The bacterial and fungal community compositions of the belowground plant compartments did not vary under future climate conditions. However, future climate conditions slightly altered the relative abundances of specific fungal classes of the aboveground compartments. We predicted several microbial functional genes of the T. pratense microbiome involved in plant growth processes, such as biofertilization (nitrogen fixation, phosphorus solubilization, and siderophore biosynthesis) and biostimulation (phytohormone and auxin production). Our findings indicated that T. pratense microbiomes show a degree of resilience to future climate changes. Additionally, microbes inhabiting T. pratense may not only contribute to plant growth promotion but also to ecosystem sustainability., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

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., (© 2020 Society of Chemical Industry.)

Modern coexistence theory holds that stabilizing mechanisms, whereby species limit the growth of conspecifics more than that of other species, are necessary for species to coexist. Here, we used experimental and observational approaches to assess stabilizing forces in eight locally co-occurring, annual, legume species in the genus Trifolium. We experimentally measured self-limitation in the field by transplanting Trifolium species into each other's field niches while varying competition and related these patterns to the field coexistence dynamics of natural Trifolium populations. We found that Trifolium species differed in their responses to local environmental gradients and performed best in their home environments, consistent with habitat specialization and presenting a possible barrier to coexistence at fine scales. We found significant self-limitation for 5 of 42 pairwise species combinations measured experimentally with competitors absent, indicating stabilization through plant-soil feedbacks and other indirect interactions, whereas self-limitation was largely absent when neighbors were present, indicating destabilizing effects of direct plant-plant interactions. The degree of self-limitation measured in our field experiment explained year-to-year dynamics of coexistence by Trifolium species in natural communities. By assessing stabilizing forces and environmental responses in the full n-dimensional field niche, this study sheds light on the roles of habitat specialization, plant-soil feedbacks, and plant interactions in determining species coexistence at local scales., (© 2021 by the Ecological Society of America.)

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 (IC 50 ) value of 35.13 mg/L. An active compound was isolated from the 70% ethanol fraction and identified as hederagenin 3- o - β -D-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (Compound A). Compound A had an IC 50 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.

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.

Some rhizosphere bacteria could colonize on the root surface of plants, or even form biofilm to promote plant growth, enhance plant resistance to harsh external environments and block the soil contamination. In this study, to explore the effects of pyrene-degrading bacterium on root surface on plant uptake of pyrene, a pyrene-degrading bacterium Mycolicibacterium sp. Pyr9 was isolated from the root surface of Eleusine indica L. Gaertn. in PAH-contaminated fields; after antibiotic labeling, it was colonized onto the root surface of white clover (Trifolium repens L.), and its distribution and performance were monitored under different levels of pyrene contamination. Strain Pyr9 could degrade 98% of pyrene (with an initial concentration of 50 mg L -1 ) in culture solution within 8 d; it also owns a variety of plant growth promoting characteristics and appreciable tolerance to harsh environments. The transcription of pyrene catabolic genes in Pyr9 enhanced obviously when induced by pyrene. Pyr9 colonized and grew well on the root surface of white clover via root inoculation; some cells could even enter into the root tissues and move to the shoots. Compared with the Pyr9-free treatment, the pyrene contents in the roots and shoots of Pyr9-inoculated white clover decreased by 25%-30% and 33%-42%, respectively. Correspondingly, the pyrene accumulation and translocation factors in white clover decreased as well. These results indicate that Pyr9 would be a good potential to circumvent plant pyrene pollution. This research may provide a theoretical basis and technical support for the safety of agricultural products and human health in PAH-contaminated sites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The most economical method of drill cuttings disposal may be their application in land reclamation which allows for the wastes recovery. However, the wastes application into the soil should ensure that the quality of the environment would not be deteriorated. These investigations were aimed at identifying the effect of drill cuttings, which were the mixture of different types of drilling wastes, on the physicochemical properties of acidic soil and growth of red clover (Trifolium pratense L.). The experimental design comprised 5 treatments, which differed in a dose of the drill cuttings: 0% (control), 2.5%, 5%, 10% and 15% of dry weight. A six-week pot experiment was conducted to determine the influence of the wastes on the plant growth. The results showed that the drill cuttings addition significantly changed the chemical and physicochemical properties of the soil, such as: electrical conductivity (EC), pH, base saturation, content of carbonate, alkaline cations (Ca2+, Na+, K+, Mg2+), organic matter, total organic carbon (TOC), and available phosphorus form. However, the most important factors that influenced the growth of red clover were pH, base saturation, content of Mg2+ and plant available phosphorus. The red clover biomass was increased from 1.5 to 2.5 times depending on the dose of wastes. We concluded that the examined wastes can be used for reclamation of the acid and unfertile degraded soils, but the amount of wastes should not exceed 5% of the soil, because the highest total clover biomass was observed just at this dose., Competing Interests: The author(s) received no specific funding for this work.

The response of plant species to external factors depends partly on the interaction with the environment and with the other species that coexist in the same ecosystem. Several studies have investigated the main traits that determine the competitive capacity of plant species, and although the relevance of the traits is not clear, traits both from belowground and aboveground have been observed. In this paper, we grew Trifolium pratense and Agrostis capillaris in intra- and interspecific competition, analyzing the photosynthetic metabolism and nitrogen uptake, among other variables. The results indicated that T. pratense possesses better competition ability due to the higher competitive performance for soil resources compared to A. capillaris, explained by a higher root biomass and a higher nitrogen uptake rate in the former than in the latter. These traits permitted T. pratense to show higher photosynthetic rate than A. capillaris when both species were grown in mixture. Furthermore, the interspecific competition provoked A. capillaris to activate its antioxidant metabolism, through SOD activity, to detoxify the reactive oxygen species generated due to its lower capacity for using the photochemical energy absorbed. In this experiment, we conclude that the competitiveness seems to be more related with soil resources competition than with light competition, and that the photosynthetic rate decline in A. capillaris is more a secondary effect as a consequence of nitrogen limitation., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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., (© 2020 Society of Chemical Industry.)

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 ( v DIA) 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.

Intercropping legumes with cereals has been a common cropping system in short-season rainfed environments due to its increased productivity and sustainability. Intercropping barley (Hordeum vulgare L.) with Egyptian clover (Trifolium alexandrinum L.) could increase the grain yield of barley and improve resource use efficiency of the intercropping system. However, non-optimum planting geometry has been a hurdle in the adaptation of barley-based cropping systems. This study was aimed at optimizing the planting geometry, and assess the productivity and profitability of barley-Egyptian clover intercropping system. Ten different planting geometries, differing in number of rows of barley, width and number of irrigation furrows and planting method were tested. Intercropping barley with Egyptian clover improved 56-68% grain yield of barley compared with mono-cropped barley. Barley remained dominant crop in terms of aggressiveness, relative crowding coefficient and competitive ratio. The amount of water used was linearly increased with increasing size of barley strip from 3 to 8 rows. The highest water use efficiency (4.83 kg/cf3) was recorded for 8-row barley strip system with 120 cm irrigation furrows compared to rest of the planting geometries. In conclusion, 8-rows of barley planted on beds with Egyptian clover in 120 cm irrigation furrows had the highest net income and cost benefit ratio. Therefore, it is recommended that this planting geometry can be used for better economic returns of barley-Egyptian clover intercropping system. However, barley strips with >8 rows were not included in this study, which is limitation of the current study. Therefore, future studies with >8 barley rows in strip should be conducted to infer the economic feasibility and profitability of wider barley strips., Competing Interests: The authors have declared that no competing interests exist.

The incorporation of cover crops into the maize (Zea mays L.)-soybean [Glycine max (L.) Merr.] rotation in the U.S. upper Midwest may improve sustainability. Long, cold winters in the region make identifying successful cover crop species and management practices a challenge. Two experiments were conducted in Minnesota, USA from fall 2016 through spring 2019 to examine the effect of cover crops interseeded at four- to six-leaf collar (early-interseeded) and dent to physiological maturity (late-interseeded) on biomass and grain yield of maize. Annual ryegrass (Lolium multiflorum L.) and cereal rye (Secale cereale L.) were evaluated as monocultures and in mixtures with crimson clover (Trifolium incarnatum L.) and forage radish (Raphanus sativus L.). Differences in canopy cover and biomass of late-interseeded cover crops were observed at the southernmost location in 2018. Additional accumulated growing-degree days in fall 2018 did not translate into increased cover crop canopy coverage of late-interseeded cover crops. Differences in cover crop canopy cover and biomass of early-interseeded cover crops were observed by fall frost at all locations in 2017 and at the northernmost location in 2018. Cover crop canopy cover and biomass at termination before planting maize, soil moisture at maize planting as well as maize aboveground biomass and yield were not affected by spring cereal rye regrowth of cover crops late-interseeded the previous year. Similarly, early-interseeded cover crops did not affect maize aboveground biomass or yield. We attribute these results to limited cover crop growth. This highlights the potential of a variety of cover crop strategies interseeded into maize in the U.S. upper Midwest; however, efforts to fine-tuning cover crop management and weather conditions are needed to benefit from such practice., Competing Interests: The authors have declared that no competing interests exist.

Plant protection products (PPPs) undergo rigorous regulatory assessment to ensure that they do not pose unacceptable risks to the environment. Elucidation of their fate and behavior in soil is an integral part of this environmental risk assessment. The active substance degradation in soil of PPPs is first assessed in laboratory studies (typically following Organisation for Economic Co-operation and Development [OECD] test guideline 307). Conditions in guideline laboratory studies are far removed from those occurring under agricultural use, and the contribution of crop roots has currently not been assessed. We integrated viable plant root systems, representative of 3 different crop types, into the OECD test guideline 307 design to assess their impact on the dissipation of the herbicide prometryn. Significantly faster decline of parent residue and higher formation of nonextractable residues were observed in all 3 planted systems. This led to a reduction in the time required for 50% of the compound to dissipate (DT50) of approximately one-half in the presence of rye grass and hot pepper and of approximately one-third in the presence of red clover. These findings imply that plants and their associated root networks can have a significant influence on PPP dissipation. Based on these data, greater environmental realism could be added to the standardized laboratory study design by the inclusion of plant root systems into higher tier studies, which, in turn, could serve to improve the environmental risk assessment process. Environ Toxicol Chem 2020;39:450-457. © 2019 SETAC., (© 2019 SETAC.)

Subterranean clover (Trifolium subterraneum L.) is a diploid self-pollinated annual pasture legume native to the Mediterranean region and widely sown in southern Australia and other countries with Mediterranean-type climates. This study utilised a core collection of 97 lines, representing around 80% of the genetic diversity of the species, to examine morphological diversity within subterranean clover. A total of 23 quantitative agro-morphological and 13 semi-quantitative morphological marker traits were assayed on the core collection and 28 diverse Australian cultivars as spaced plants in a replicated common garden experiment. Relationships between these traits and 24 climatic and edaphic parameters at their sites of origin were also examined within the core collection. Significant diversity was present for all traits. The Australian cultivars had similar levels of diversity to the core collection for several traits. Among the agro-morphological traits, time to flowering, leaf size and petiole diameter in mid-winter, plant area in late winter, maximum stem length, content of the oestogenic isoflavone biochanin A and total isoflavone content, were correlated with seven or more environmental variables. These can be considered highly adaptive, being the result of strong environmental selection pressure over time. For the first time in a clover species, morphological markers, including leaf mark, anthocyanin pigmentation and pubescence traits, have been associated with rainfall and soil parameters. This suggests they either have an adaptive role or the genes controlling them may be linked to other genes controlling adaptive traits. This study demonstrated the value of core collections to examine diversity within much larger global collections. It also identified adaptive traits from wild plants that can be utilised to develop more productive and persistent subterranean clover cultivars. The high heritability of these traits indicates that selection gains can be readily made., Competing Interests: The authors have declared that no competing interests exist.

Nitric oxide (NO), γ-aminobutyric acid (GABA), and mannose (MAS) could be important regulators of plant growth and adaptation to water stress. The application of sodium nitroprusside (SNP, a NO donor), GABA, and MAS improved plant growth under water-sufficient conditions and effectively mitigated water stress damage to white clover. The metabonomic analysis showed that both SNP and GABA application resulted in a significant increase in myo-inositol content; the accumulation of mannose was commonly regulated by SNP and MAS; GABA and MAS induced the accumulation of aspartic acid, quinic acid, trehalose, and glycerol under water deficit. In addition, citric acid was uniquely up-regulated by SNP associated with tricarboxylic acid (TCA) cycle under water stress. GABA specially induced the accumulation of GABA, glycine, methionine, and aconitic acid related to GABA shunt, amino acids metabolism, and TCA cycle in response to water stress. MAS uniquely enhanced the accumulation of asparagine, galactose, and D-pinitol in association with amino acids and sugars metabolism under water stress. SNP-, GABA-, and MAS-induced changes of metabolic profiles and associated metabolic pathways could contribute to enhanced stress tolerance via involvement in the TCA cycle for energy supply, osmotic adjustment, antioxidant defense, and signal transduction for stress defense in white clover., (© 2019 Institute of Botany, Chinese Academy of Sciences.)

Main Conclusion: β-sitosterol influences amino acids, carbohydrates, organic acids, and other metabolite metabolism and homeostasis largely contributing to better tolerance to water stress in white clover. β-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.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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 F 1 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 (F 2 -F 5 ) 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.

Endophyte-assisted phytoremediation is considered to be an effective approach for bioremediation of heavy metal-contaminated soil; however, few information is available on Trifolium repens L. and its endophytes to remediate heavy metal-polluted soils. In this study, heavy metal-resistant endophytes were isolated from T. repens growing in mining smelter and identified by BIOLOG system. The isolate was also evaluated for promoting plant growth in heavy metal-contaminated soils in pot experiments. A total of eight Cd 2+ -resistant endophytes were isolated and these isolates preferred to grow on L-aspartic acid and α-D-glucose. All the isolates had at least two plant growth-promoting properties including siderophore production, phosphate solubilization activity, indole acetic acid (IAA) production, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. Strain RE02, identified as Pseudomonas putida by Biolog system, showed the highest Cd tolerance and could reduce Cd concentration from 20 to 1.84 mg L -1 in about 49 h in liquid medium, amounting to about 90.8%. Among the five endophytes which have positive effect on the growth of T. repens, RE02, whose IAA production ability was 7.06 mg L -1 and phosphate solubilization was 134.76 mg L -1 , could improve T. repens root and shoot biomass by 25.9% and 37.7% in cadmium-contained soil, respectively. Our research may provide a new microbial-enhanced phytoremediation of heavy metal-polluted soils and improve the remediation efficiency.

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&#95;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., (© 2019 John Wiley & Sons Ltd.)

Catch crop (CC) mixtures of non-legumes (nL) and legumes (L) have been promoted as a strategy to achieve two different goals: to decrease the risk of nitrate leaching and to enhance the nitrogen supply to the subsequent crop. To investigate if two-component mixtures of nL + L have advantages over pure nL stands experiments were carried out over a two year period (2013-2015) at two contrasting field sites in Denmark. Nitrogen (N) uptake by the CCs was measured by aboveground biomass sampling, and N leaching by ceramic suction cups. When grown in pure stands, white clover (Trifolium repens) on coarse sand and common vetch (Vicia sativa) on sandy loam were less effective at reducing N leaching than perennial ryegrass (Lolium perenne) and fodder radish (Raphanus sativus). When the proportion of the nL + L in mixtures was similar or favored the nL, leaching was not significantly different from the nL in the pure stand. However, during one of the years on the sandy loam L (vetch) almost outperformed nL (fodder radish), resulting in N leaching from nL + L similar to L. The yield of the following spring barley was only significantly different from the yield in the plots with previously bare soil in one of the years on the coarse sandy soil. It is concluded that in nL + L mixtures L can take over and thereby lower the effect of the CCs on N leaching while not necessarily enhancing the N supply for the subsequent crop., (Copyright © 2019. Published by Elsevier Ltd.)

Cover crops have the potential to be agricultural nitrogen (N) regulators that reduce leaching through soils and then deliver N to subsequent cash crops. Yet, regulating N in this way has proven difficult because the few cover crop species that are well-studied excel at either reducing N leaching or increasing N supply to cash crops, but they fail to excel at both simultaneously. We hypothesized that mixed species cover crop stands might balance the N fixing and N scavenging capabilities of individual species. We tested six cover crop monocultures and four mixtures for their effects on N cycling in an organically managed maize-soybean-wheat feed grain rotation in Pennsylvania, USA. For three years, we used a suite of integrated approaches to quantify N dynamics, including extractable soil inorganic N, buried anion exchange resins, bucket lysimeters, and plant N uptake. All cover crop species, including legume monocultures, reduced N leaching compared to fallow plots. Cereal rye monocultures reduced N leaching to buried resins by 90% relative to fallow; notably, mixtures with just a low seeding rate of rye did almost as well. Austrian winter pea monocultures increased N uptake in maize silage by 40 kg N ha-1 relative to fallow, and conversely rye monocultures decreased N uptake into maize silage by 40 kg N ha-1 relative to fallow. Importantly, cover crop mixtures had larger impacts on leaching reduction than on maize N uptake, when compared to fallow plots. For example, a three-species mixture of pea, red clover, and rye had similar maize N uptake to fallow plots, but leaching rates were 80% lower in this mixture than fallow plots. Our results show clearly that cover crop species selection and mixture design can substantially mitigate tradeoffs between N retention and N supply to cash crops, providing a powerful tool for managing N in temperate cropping systems., Competing Interests: The authors have declared that no competing interests exist.

Charcoal is a ubiquitous legacy of wildfire in terrestrial systems that often contributes to rapid revegetation following disturbance; the use of charcoal soil amendments, or "biochars", to promote plant growth has received recent research attention and increasing applied use. Despite its widespread use, well-resolved quantitative estimates of dose-response relationships for biochar effects on plant growth are nonexistent, and studies of biochar dosage effects on plant ecophysiology are minimal. We investigated the effects of biochar dosage on plant growth and ecophysiology in a glasshouse experiment involving two common early-successional plants, Abutilon theophrasti and Trifolium repens. Plants were grown in disturbed temperate soils with increasing dosages of wood biochars: 0, 2, 4, 6, 8, 10, 20, 30, 40, 50 t/ha. We measured leaf-level gas-exchange traits (A max , g s , WUE), chlorophyll concentration, and leaf area growth throughout the experiment. At the end of the experiment, we measured biomass, foliar nutrition, and soil properties (pH, EC, C and N). Responses of biomass and physiological traits were highly dose-dependent, followed primarily unimodal forms, and differed in some traits between species. Increases in the uptake of K, P, and Mg, were responsible for accelerated growth. Biochars also generally increased the concentration of micronutrients, especially B. As a result, nutrient stoichiometry shifted substantially: in A. theophrasti, biochars increased C:N, P:N, and K:N ratios, suggesting nitrogen dilution or induced deficiency at higher dosages. This work supports the general hypothesis that ecophysiological responses to biochar are dose-dependent and driven mainly by changes in nutrient availability. Additional work is necessary to understand the broader ecological impacts of heterogeneity in soil pyrogenic C levels to succession and ecosystem function., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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.

Phenolics are important mediators in plant-environment interactions. The presence and concentration of phenolic compounds and their antioxidant activity were evaluated in leaves and flowers of a set of Trifolium species originating from contrasting environments encompassing lowland and mountain sites. The current germplasm proved a great reservoir of phenolic compounds, with different chemical structure and, possibly, diversified biological activity. Germplasm groups with specific phenolic composition were observed. In some cases, different patterns bore a taxonomic meaning. Lowland germplasm showed higher concentration of total phenolics in leaves than mountain accessions (50.30 vs. 34.19 mg/g dry matter (DM)), while the latter had higher concentration in flowers (114.16 vs. 57.44 mg/g DM). Outstanding concentration of isoflavones was observed in leaves of lowland germplasm (24.19 mg/g DM), and of both proanthocyanidins and flavonoids in flowers of mountain germplasm (53.81 and 56.62 mg/g DM, respectively). The pattern of phenolic composition in lowland and mountain germplasm was suggestive of different adaptive strategies. Three assays of antioxidant activity were tested, which were characterised by rather different reactivity towards phenolic composition. The scavenging activity was higher for leaf extracts of lowland germplasm, and for flower extracts of mountain germplasm. Besides identifying germplasm of interest, this study also suggested possible links between environmental factors and concentration and composition of phenolic compounds.

The development and reproductive potential of Spodoptera eridania (Stoll, 1782) (Lepidoptera, Noctuidae) was compared on natural and artificial diets. Its biology was determined under laboratory conditions, providing an artificial diet, leaves of white clover and the peach cultivar 'Chimarrita' and fruits and leaves of the apple cultivar 'Gala'. Larvae of S. eridania could not complete their biological cycle when fed on apple fruits or peach leaves. The artificial diet provided the shortest development time, with a cycle of 28.5±0.14 days from egg to adult and 37.3% total viability. Apple leaves led to a longer development time from egg to adult (62.8±1.22 days) and lower total viability (23%) and white clover provided the highest total viability (54.3%). The results showed that S. eridania can use white clover and apple leaves to develop normally in apple orchards. The reduction of food sources such as white clover used by the larvae results in infestation and damage to fruits from "test bites".

People rely on the synthetic chemical pesticides in most of the agricultural practices, which have a serious problem on human health and ecosystem. Essential oil (EO), derived from wild plants, offers a new opportunity to explore eco-friendly green biocides. This study aimed to characterize the chemical constituents of EO from Egyptian ecospecies of Cleome droserifolia (Forssk.) Delile and evaluate its antioxidant and allelopathic potential. The EO was extracted from aerial parts by hydrodistillation and analyzed by GC/MS. To assess the allelopathic potential, EO concentrations from 50 to 200 μL L -1 were tested on Trifolium repens and three weeds. Antioxidant activity was determined using DPPH. Hydrodistillation yielded 0.64 % of dark yellow oil, which comprises 35 compounds, in which sesquiterpene was a major class and represented by 61.97 % of the total essential oil. Moreover, the dominant sesquiterpenes are cis-nerolidol, α-cadinol, δ-cadinene, and γ-muurolene. The speed of germination index, shoot and root length of clover and weeds were reduced in a concentration-dependent manner. The IC 50 values of C. droserifolia EO on germination of T. repens, Cuscuta trifolii, Melilotus indicus, and Chenopodium murale were 181.6, 183.5, 159.0, and 157.5 μL L -1 , respectively. From the obtained data, we concluded that C. droserifolia EO could provide a hope to produce environment-friendly bioherbicide as well as a natural resource of antioxidants., (© 2018 Wiley-VHCA AG, Zurich, Switzerland.)

This study was designed to examine the effects of NaCl pretreatment on the seed germination of white clover ( Trifolium repens cv. Ladino) under water stress induced by 19% polyethylene glycol (PEG) 6000. Lower concentrations of NaCl (0.5, 1, and 2.5 mM) pretreatment significantly alleviated stress-induced decreases in germination percentage, germination vigor, germination index, and radicle length of seedlings after seven days of germination under water stress. The soaking with 1 mM of NaCl exhibited most the pronounced effects on improving seed germination and alleviating stress damage. NaCl-induced seeds germination and growth could be associated with the increases in endogenous gibberellic acid (GA) and indole-3-acetic acid (IAA) levels through activating amylases leading to improved amylolysis under water stress. Seedlings pretreated with NaCl had a significantly lower osmotic potential than untreated seedlings during seed germination, which could be related to significantly higher soluble sugars and free proline content in NaCl-treated seedlings under water stress. For antioxidant metabolism, NaCl pretreatment mainly improved superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione reductase activities, transcript levels of FeSOD , APX , and DHAR , and the content of ascorbic acid, reduced glutathione, and oxidized glutathione during seed germination under water stress. The results indicated that seeds soaking with NaCl could remarkably enhance antioxidant metabolism, thereby decreasing the accumulation of reactive oxygen species and membrane lipid peroxidation during germination under water stress. In addition, NaCl-upregulated dehydrin-encoded genes SK2 expression could be another important mechanism of drought tolerance during seeds germination of white clover in response to water stress., Competing Interests: The authors declare no conflict of interest.

Agricultural grasslands are often cultivated as mixtures of grasses and legumes, and an extensive body of literature is available regarding interspecific interactions, and how these relate to yield and agronomic performance. However, knowledge of the impact of intraspecific diversity on grassland functioning is scarce. We investigated these effects during a 4-year field trial established with perennial ryegrass (Lolium perenne) and red clover (Trifolium pratense). We simulated different levels of intraspecific functional diversity by sowing single cultivars or by combining cultivars with contrasting growth habits, in monospecific or bispecific settings (i.e. perennial ryegrass whether or not in combination with red clover). Replicate field plots were established for seven seed compositions. We determined yield parameters and monitored differences in genetic diversity in the ryegrass component among seed compositions, and temporal changes in the genetic composition and genetic diversity at the within plot level. The composition of cultivars of both species affected the yield and species abundance. In general, the presence of clover had a positive effect on the yield. The cultivar composition of the ryegrass component had a significant effect on the yield, both in monoculture, and in combination with clover. For the genetic analyses, we validated empirically that genotyping-by-sequencing of pooled samples (pool-GBS) is a suitable method for accurate measurement of population allele frequencies, and obtained a dataset of 22,324 SNPs with complete data. We present a method to investigate the temporal dynamics of cultivars in seed mixtures grown under field conditions, and show how cultivar abundances vary during subsequent years. We screened the SNP panel for outlier loci, putatively under selection during the cultivation period, but none were detected., Competing Interests: The authors declare that they have no competing interests.

Background and Aims: Grassland-based livestock systems in cool maritime regions are commonly dominated by grass monocultures receiving relatively high levels of fertilizer. The current study investigated whether grass-legume mixtures can improve the productivity, resource efficiency and robustness of yield persistence of cultivated grassland under extreme growing conditions over a period of 5 years., Methods: Monocultures and mixtures of two grasses (Phleum pratense and Festuca pratensis) and two legumes (Trifolium pratense and Trifolium repens), one of which was fast establishing and the other temporally persistent, were sown in a field trial. Relative abundance of the four species in the mixtures was systematically varied at sowing. The plots were maintained under three N levels (20, 70 and 220 kg N ha-1 year-1) and harvested twice a year for five consecutive years. Yields of individual species and interactions between all species present were modelled to estimate the species diversity effects., Key Results: Significant positive diversity effects in all individual years and averaged across the 5 years were observed. Across years, the four-species equi-proportional mixture was 71 % (N20: 20 kg N ha-1 year-1) and 51 % (N70: 70 kg N ha-1 year-1) more productive than the average of monocultures, and the highest yielding mixture was 36 % (N20) and 39 % (N70) more productive than the highest yielding monoculture. Importantly, diversity effects were also evident at low relative abundances of either species group, grasses or legumes in the mixture. Mixtures suppressed weeds significantly better than monocultures consistently during the course of the experiment at all N levels., Conclusions: The results show that even in the less productive agricultural systems in the cool maritime regions grass-legume mixtures can contribute substantially and persistently to a more sustainable agriculture. Positive grass-legume interactions suggest that symbiotic N2 fixation is maintained even under these marginal conditions, provided that adapted species and cultivars are used.