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Societal Impact Statement Summary Humans and honey bees have a long history of interaction to yield valued products and services. However, honey bees are under pressure from changes in vegetation, agricultural practices and climate change. We investigate if pasture legumes can be harnessed to support honey bees. We use a diverse set of species that originated from the Mediterranean and western Europe but are now developed as commercial cultivars for the southern Australian livestock industries. We find that pasture legumes show great promise as a means to support honey bees and suggest that further targeted research is warranted. Globally, humans and honey bees (Apis mellifera) have a long history of interaction and this is now under pressure. In temperate southern Australia, exotic honey bees rely on an unstable native floral resource base, yet a diverse set of exotic pasture legume species are sown as staple rotational or permanent crops. We investigated if these pasture legumes could be strategically used to support honey bee populations for the apiary and horticulture industries in this region. A literature review on temperate pasture legumes and honey bees was undertaken and integrated with data from an Australian industry case study. Comparisons were made to the widely‐grown red clover (Trifolium pratense) and white clover (T. repens). We found that many southern Australian pasture legumes could provide significant floral resources as well as enhanced temporal and spatial stability for honey bees due to phenological variability and broad adaptation. Honey bees will likely recognise and be attracted to their flowers and other characteristics are comparable, or potentially superior, to red and white clover, such as floret number per inflorescence, floret length, nectar volume and sucrose‐dominance and pollen protein. The floral resource diversity from pasture legume mixes could aid honey bee health and support pollinator services for adjacent crops. We conclude that the exotic pasture legumes of temperate Australia show promise for development as a sown floral resource for honey bees. Globally, further investigation is merited to maximise benefits from the integration of a diverse range of pasture legumes into agricultural landscapes. [ABSTRACT FROM AUTHOR]

Temperate winters are getting warmer, the length of the growing season is increasing and mid‐winter fluctuations of warm and freezing temperatures are more frequent. Although typically winter dormant, some herbaceous perennials can maintain or grow green leaves during the winter and more species may do so as winters become warmer. For wintergreen leaves to provide an advantage, they must be able to photosynthesize during the warm spells but also withstand freezing temperatures and fluctuations between the two conditions. To understand the leaf traits of wintergreen herbs, we studied three widely distributed perennial species: Bellis perennis, Plantago lanceolata and Trifolium repens. For contrast between winter and growing season leaves, we measured three common leaf economic traits (LDMC, SLA and leaf N content). To assess freezing tolerance, we measured the ice nucleation temperature and LT50 after frost exposure in winter leaves. To confirm photosynthetic ability, we measured photosynthetic efficiency (Fv/Fm value) in winter and late spring. Additionally, we surveyed the green and dead leaf coverage for herbaceous species in a lawn community from winter to early summer (four surveys in total). We used our observations to determine phenological categories and compared them to historical observations of wintergreenness. All three species had denser leaves with lower nitrogen content in winter than in the growing season and relatively high tolerance of freezing temperatures (average of −8°C ice nucleation temperature and −15°C LT50). Both winter and growing season leaves were capable of photosynthesis, but the efficiency was greater for growing season leaves. In the community survey, a majority of the plant species had green leaves in winter, and many were without dead winter leaves. Many wintergreen herbs had previously been labelled ‘summer green’. With these initial investigations into the traits and phenology of wintergreen herbs, we confirm that wintergreen leaves have different traits from typical growing season leaves and are capable of both photosynthesis and freezing tolerance. Wintergreen herbs have a broad range of phenological strategies and differ from observations 20 years ago. In many temperate regions, winter is becoming a new growing season and thus should be included in phenological studies. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

Background: Soil salinization is a serious environmental hazard, limiting plant growth and production in different agro-ecological zones worldwide. Diethyl aminoethyl hexanoate (DA-6) as an essential plant growth regulator (PGR) exhibits a beneficial role in improving crop growth and stress tolerance. However, the DA-6-regulated effect and mechanism of salt tolerance in plants are still not fully understood. The objective of current study was to disclose salt tolerance induced by DA-6 in relation to changes in water and redox balance, photosynthetic function, ionic homeostasis, and organic metabolites reprogramming in white clover (Trifolium repens). Results: A prolonged duration of salt stress caused water loss, impaired photosynthetic function, and oxidative injury to plants. However, foliar application of DA-6 significantly improved osmotic adjustment (OA), photochemical efficiency, and cell membrane stability under salt stress. In addition, high salinity induced massive accumulation of sodium (Na), but decreased accumulation of potassium (K) in leaves and roots of all plants. DA-6-treated plants demonstrated significantly higher transcript levels of genes involved in uptake and transport of Na and K such as VP1, HKT8, SOS1, NHX2, NHX6, and SKOR in leaves as well as VP1, HKT1, HKT8, H+-ATPase, TPK5, SOS1, NHX2, and SKOR in roots. Metabolomics analysis further illustrated that DA-6 primarily induced the accumulation of glucuronic acid, hexanoic acid, linolenic acid, arachidonic acid, inosose, erythrulose, galactopyranose, talopyranose, urea, 1-monopalmitin, glycerol monostearate, campesterol, stigmasterol, and alanine. Conclusions: The DA-6 significantly up-regulated transcript levels of multiple genes associated with increased Na+ compartmentalization in vacuoles and Na+ sequestration in roots to reduce Na+ transport to photosynthetic organs, thereby maintaining Na+ homeostasis under salt stress. The accumulation of many organic metabolites induced by the DA-6 could be attributed to enhanced cell wall and membrane structural stability and functionality, OA, antioxidant defense, and downstream signal transduction in leaves under salt stress. The present study provides a deep insight about the synergistic role of DA-6 in salt tolerance of white clover. [ABSTRACT FROM AUTHOR]

Mitigation practices for nitrogen leaching losses from livestock agriculture are needed to protect freshwater quality and increase the efficiency of agricultural production. Within New Zealand, the most common pasture type is a twospecies mix of perennial ryegrass (Lolium perenne) and white clover (Trifolium repens). Ecological theory suggests that increasing species and functional diversity improves ecosystem function, including nitrogen (N) retention. Use of more diverse pasture types, including a mix of pasture grasses, legumes and other forbs, particularly plantain (Plantago lanceolata), with functional traits, including winter activity, deep-rooting, N fixation, and biological inhibition of nitrification in the soil, is a potential mitigation practice that requires further verification with long-term field measurements. Here we utilize a network of large lysimeters to make field-based measurements of N leaching from 5-8 species diverse pasture, including plantain, under a range of soil, climate and management conditions, for comparison with losses from traditional ryegrass-clover pasture. Over 3 years of measurements, leaching from fully established diverse pasture was 2-80 kg N ha-1 y-1. No differences were observed in dry matter production or N leaching of diverse pasture compared to ryegrass-clover lysimeters. Large losses, up to 120 kg N ha-1, were observed during periods when pasture was not fully established, including cultivation and sowing of new pasture, depending on season. Timing of management activities could be optimized to minimize these losses. These data provide critical assessment of diverse pasture as a mitigation approach for reducing N losses. Further work on diverse pastures should include higher diversity mixes as well as consideration of animal mediated effects of diverse pasture diets on N inputs. [ABSTRACT FROM AUTHOR]

This document is a response to comments on a review of the ability of plantain to mitigate nitrogen leaching from pasture systems. The authors thank the respondents for their feedback and note that there is general agreement on many points. The authors argue that dilution is the main mechanism by which plantain reduces nitrogen leaching. They address specific points raised by the respondents and emphasize the need for further research and discussion in the scientific community. The New Zealand Journal of Agricultural Research is a reputable publication that covers a wide range of topics related to agriculture, providing valuable insights for researchers and practitioners in the agricultural industry. [Extracted from the article]

BACKGROUND: Low rumen pH is proposed to be a major mechanism for low methane (CH4) emissions from sheep fed forage rape. However, it is difficult to separate this from other in vivo factors, such as rumen passage rate. The objective of this study was to determine the effect of pH alone on CH4 production in vitro using different pH buffers. Ryegrass, white clover and forage rape were incubated in vitro using three different incubation buffers with starting pH values of 5.5, 6.2 and 6.8. RESULTS: Decreasing pH reduced overall in vitro CH4 emission relative to fermented hexoses (CH4/FHex) by up to 54% and overall fermentation by 40%. pH also changed fermentation profiles where the acetate + butyrate to propionate + valerate ratio decreased when pH decreased. Within the three forages, forage rape led to the lowest CH4/FHex, but only in pH 5.5 and 6.2 buffer, and this was enhanced when the pH fell below 6. CONCLUSION: Reducing pH in vitro decreased CH4 production and overall fermentation across all forages. The lower pH reached by forage rape compared to ryegrass and white clover appears to drive the lower CH4 production relative to the extent of fermentation from forage rape compared to the other forages. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

White clover (Trifolium repens L.) is an important forage and aesthetic plant species, but it is susceptible to drought and heat stress. The phytohormone auxin regulates several aspects of plant development and alleviates the effects of drought stress in plants, including white clover, by involving auxin/indole acetic acid (Aux/IAA) family genes. However, Aux/IAA genes and the underlying mechanism of auxin-mediated drought response remain elusive in white clover. To extend our understanding of the multiple functions of Aux/IAAs, the current study described the characterization of a member of the Aux/IAA family TrIAA27 of white clover. TrIAA27 protein had conserved the Aux/IAA family domain and shared high sequence similarity with the IAA27 gene of a closely related species and Arabidopsis. Expression of TrIAA27 was upregulated in response to heavy metal, drought, salt, NO, Ca2+, H2O2, Spm, ABA, and IAA treatments, while downregulated under cold stress in the roots and leaves of white clover. TrIAA27 protein was localized in the nucleus. Constitutive overexpression of TrIAA27 in Arabidopsis thaliana led to enhanced hypocotyl length, root length, plant height, leaf length and width, and fresh and dry weights under optimal and stress conditions. There was Improved photosynthesis activity, chlorophyll content, survival rate, relative water content, endogenous catalase (CAT), and peroxidase (POD) concentration with a significantly lower electrolyte leakage percentage, malondialdehyde (MDA) content, and hydrogen peroxide (H2O2) concentration in overexpression lines compared to wild-type Arabidopsis under drought and salt stress conditions. Exposure to stress conditions resulted in relatively weaker roots and above-ground plant growth inhibition, enhanced endogenous levels of major antioxidant enzymes, which correlated well with lower lipid peroxidation, lower levels of reactive oxygen species, and reduced cell death in overexpression lines. The data of the current study demonstrated that TrIAA27 is involved in positively regulating plant growth and development and could be considered a potential target gene for further use, including the breeding of white clover for higher biomass with improved root architecture and tolerance to abiotic stress. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate the functional properties of muffins fortified with white clover flowers (Trifolium repens L.), which were added to the dough in the following amounts: (i) 0% (control); (ii) 2.5%; (iii) 5.5%; (iv) 7.5%; and (v) 10%. The organoleptic properties were assessed by a panel of consumers. Additionally, the following parameters were also tested: basic chemical composition, total polyphenols, the antioxidant activity together with antiproliferative effects on the A375 melanoma cell line, starch nutritional fractions and the in vitro glycemic index. As a result, replacing wheat flour with white clover flour significantly affected the color, aroma and taste of the muffins. The content of proteins, fats, total ash, dietary fiber, resistant starch (RS), slowly digestible starch (SDS),total polyphenols and antioxidant activity increased statistically significantly with the elevated amount of white clover flour added to the dough. At the same time, the content of free glucose (FG), rapidly available glucose (RAG) and rapidly digestible starch (RDS), the value of the in vitro glycemic index and the viability of melanoma cancer cells decreased significantly. The muffins enriched with white clover flowers might constitute an interesting proposition and extension of the existing assortment of confectionery products. [ABSTRACT FROM AUTHOR]

The Fabaceae genus Trifolium comprises around 250 species widely distributed worldwide, with the temperate Northern Hemisphere exhibiting the highest variety. The plants in this genus are widely used as livestock fodder crops and are particularly significant economically. This study's objective included isolating bacteria from the root nodules of the Trifolium repens plant and diagnosing it at the molecular and microbiological levels. T. repens root nodules were used as the source of an endophytic bacteria isolated on Yeast Extract Mannitol (YEM) media that had solidified and diagnosed at the molecular level by DNA Sequencing technique for analysis of the sequence of the nitrogenous bases of 16S rRNA gene with the global database. The isolated bacteria were characteristic of greyish-white color after 48 hours of growth and appeared as a circular shape, slightly convex and gram-negative. The bacteria were resistant to the antibiotics 20µg/ml Aztreonam. The DNA sequencing technique for analysis of the sequence of the nitrogenous bases of 16S rRNA gene with the global database of the National Center for Biotechnology Information (NCBI) showed that the isolated bacteria was at least 96.22% similar to the species Acinetobacter baumannii As a result, it was recorded for the first time as Acinetobacter sp. AZS1 strain in NCBI. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the phytotoxic, genotoxic, cytotoxic and antimicrobial effects of the Mentha arvensis L. essential oil (EO). The biological activity of M. arvensis EO depended on the analyzed variable and the tested oil concentration. Higher concentrations of EO (20 and 30 µg mL−1) showed a moderate inhibitory effect on the germination and growth of seedlings of tested weed species (Bellis perennis, Cyanus segetum, Daucus carota, Leucanthemum vulgare, Matricaria chamomilla, Nepeta cataria, Taraxacum officinale, Trifolium repens and Verbena × hybrida). The results obtained also indicate that the EO of M. arvensis has some genotoxic, cytotoxic and proliferative potential in both plant and human in vitro systems. Similar results were obtained for antimicrobial activity against eight bacteria, including multidrug-resistant (MDR) strains [Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Escherichia coli, extended-spectrum beta-lactamase-producing (ESBL) E. coli, Pseudomonas aeruginosa and Salmonella enterica subsp. enterica serovar Enteritidis], with the effect on multidrug-resistant bacterial strains. Research indicates that the EO of M. arvensis shows phytotoxic, genotoxic, cytotoxic and antimicrobial effects, as well as its potential application as a herbicide and against various human diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Understanding the effects of short-term artificial revegetation on preventing soil degradation in erosion gullies of black soil areas is essential to choosing the most suitable species of vegetation for controlling the development of erosion gullies. A field experiment with short-term artificial revegetation with herbaceous species (Medicago sativa L., Glycyrrhiza pallidiflora Maxim., Elytrigia repens (L.) Desv. ex Nevski, Rheum palmatum L., Asparagus officinalis L., Trifolium repens L., Bromus inermis Leyss., Elymus dahuricus Turcz.) and a runoff scouring test were conducted in a typical erosion gully in a black soil area. Soil erosion, physicochemical characteristics, and shoot/root characteristics were measured to evaluate the effects of short-term artificial revegetation. Short-term artificial revegetation significantly decreased (p < 0.05) sediment yield by 91.1% ± 7.2% compared with that of bare soil. Soil total nitrogen (TN), total potassium (TP), available phosphorus (AP), cation exchange capacity (CEC), water-stable aggregates > 0.25 mm (WR0.25), and aggregate mean weight diameter (MWD) and mean geometric diameter (GWD) were significantly correlated with vegetated treatments, indicating they were factors sensitive to short-term artificial revegetation. Except for total potassium (TK), the other soil characteristics decreased in vegetated treatments. In addition to increasing TK, vegetated treatments also increased soil available nitrogen (AN)/TN ratios in the short term. The overall effects of different herbaceous species on soil and water conservation, soil quality, and vegetation growth were evaluated, and Trifolium repens L. is the most suitable for preventing soil degradation in an erosion gully. The results of this study will provide a reference for the restoration and protection of the ecological environment in black soil areas with gully erosion. [ABSTRACT FROM AUTHOR]

Integrated forage–row cropping systems provide important agronomic and economic benefits to producers. However, little attention has been given to incorporating forages into row crop systems unique to the southeastern United States. This study assessed the viability of intercropping cotton (Gossypium hirsutum L.) on perennial, cool-season legumes during the summer months in the Southeast Coastal Plain over two production years. Treatments included a weedy fallow, annual ryegrass (ARG; Lolium multiflorum Lam.) monoculture, a red clover (RC; Trifolium pratense L.) and white clover (WC; Trifolium repens L.) mixture, and a three-species mixture of ARG, RC, and WC. Plots were established in fall 2020 with forage grown until May 2021 and 2022, when plots were strip-tilled and planted with cotton. Cotton was managed with minimal herbicide use to preserve perennial clovers. Results indicated WC was more persistent than RC (>40 plants m−2 vs. <40 plants m−2), and the presence of perennial clovers suppressed weeds at similar levels to the ARG monoculture (35 weeds m−2) during the second spring. Perennial clovers grew taller (4–5 cm) when mixed with ARG. The presence of clovers mixed with ARG during the second spring reduced acid detergent fiber (ADF) concentration and increased crude protein (CP) concentration (280 g ADF kg−1; 167 g CP kg−1) compared to the ARG monoculture (315 g ADF kg−1; 126 g CP kg−1). Benefits of intercropping perennial forages with cotton were maximized during the second year of growth, but future work is necessary to improve stand survival. [ABSTRACT FROM AUTHOR]

Weeds compete for environmental resources, leading to reduced soybean yield. In this context, integrated weed management strategies related to cultural control with the use of cover crops are necessary. Our aim was to evaluate weed occurrence in soybean systems with different cover crops. Field studies were conducted at Júlio de Castilhos, Santa Maria, Capão do Leão, Barra do Ribeiro, and Santo Ângelo, Rio Grande do Sul, Brazil. A randomized complete block design with four replications was used. Treatments consisted of black oats (Avena strigosa Schreb.), white oats (Avena sativa L.), rye (Secale cereale L.), hairy vetch (Vicia sativa L.), forage turnip (Raphanus sativus L.), and white clover (Trifolium repens L.) in pure stands or in mixtures. The analyzed variables were relative frequency, density, abundance, and importance value index, similarity index of weeds, dry shoot mass of cover crop, and soybean yield. Cover crops containing white or black oats reduced the relative importance value index of weeds, such as Lollium multiforum, Conyza spp., and Bidens pilosa. Forage turnip, hairy vetch, and white clover showed distinct responses. Black oats and forage turnip did not differ from cover crop mixtures in terms of dry shoot mass and grain yield, being superior to fallow, white clover, and hairy vetch. [ABSTRACT FROM AUTHOR]

Diversifying crop rotations by incorporating legumes is recommended to enhance the resilience of agricultural systems against environmental stresses and optimize nitrogen utilization. Nonetheless, ploughing forage legumes or grass-legumes poses a significant risk of nitrate leaching. The study aimed to assess the impact of strip tillage intercropping management on soil mineral nitrogen, water-extractable organic carbon, mobile humic substances content, and winter wheat (Triticum aestivum L.) grain yield compared to forage legume and winter wheat monocropping with conventional tillage. In the intercropping systems, the following bicrops were used: black medick (Medicago lupulina L.) with winter wheat, white clover (Trifolium repens L.) with winter wheat, and Egyptian clover (Trifolium alexandrinum L.) with winter wheat. Research was conducted in two experiments. The results indicated that after implementing strip tillage and winter wheat intercropping, the soil mineral nitrogen content was similar to or lower than that observed in conventional tillage and winter wheat sowing after forage legumes. Winter wheat grain yield in intercrops decreased compared to the legumes monocultures that were ploughed before winter wheat sowing. The highest amount of water- extractable organic carbon was in intercropping growing white clover and winter wheat bicrops or in all fields (except Egyptian clover and winter wheat bicrops) after applying strip tillage. During the research period, the quantities of mobile humic substances and mobile humic acids exhibited similar changes. Their content increased substantially in fields with white clover and Egyptian clover, regardless of whether the legumes were ploughed or grown with winter wheat. [ABSTRACT FROM AUTHOR]

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Abstract 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.

Introducing beneficial productivity and nutritional traits into food, feed and forage crops utilising the tools of biotechnology can lead to improvements via genetic modification that cannot be achieved by traditional plant breeding. The timelines and costs are significant, and the regulatory hurdles can lead to some promising traits failing to be commercialised. These challenges mean that large commodity crops are the primary beneficiaries of biotechnology. New Zealand being primarily a grazing pastoral agricultural market and small in international terms, faces greater challenges. The species used in pastoral agriculture have relatively small seed markets and therefore limited investment for genetic improvement. The nutritional quality of feed and forages both in composition and in energy density per ha has a major influence on animal productivity and the environmental impacts of agriculture. Through government and private industry support, AgResearch has developed novel approaches to improve plant photosynthesis, energy density, and nutritional quality that has applications in crops globally. This review discusses the different challenges and solutions to improving plant nutrient density and outlines the benefits of these novel biotechnology traits in animal forage and feed crops. [ABSTRACT FROM AUTHOR]

Alfalfa mosaic virus (AMV) is one of the most widely distributed viruses; it often exhibits combined infection with white clover mosaic virus (WCMV). Even so, little is known about the effects of co-infection with AMV and WCMV on plants. To determine whether there is a synergistic effect of AMV and WCMV co-infection, virus co-infection was studied by electron microscopy, the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and real-time fluorescence quantitative PCR (RT-qPCR) of AMV and WCMV co-infection in Nicotiana benthamiana. Meanwhile, measurements were carried out on the photosynthetic pigments, photosynthetic gas exchange parameters, and chlorophyll fluorescence parameters. The results showed that the most severe disease development was induced by AMV and WCMV co-infection, and the disease grade was scale 7. N. benthamiana leaves induced mottled yellow-green alternating patterns, leaf wrinkling, and chlorosis, and chloroplasts were observed to be on the verge of disintegration. The relative accumulation of AMV CP and WCMV CP was significantly increased by 15.44-fold and 10.04-fold upon co-infection compared to that with AMV and WCMV single infection at 21 dpi. In addition, chlorophyll a, chlorophyll b, total chlorophyll, the net photosynthetic rate, the water use efficiency, the apparent electron transport rate, the PSII maximum photochemical efficiency, the actual photochemical quantum yield, and photochemical quenching were significantly reduced in leaves co-infected with AMV and WCMV compared to AMV- or WCMV-infected leaves and CK. On the contrary, the carotenoid content, transpiration rate, stomatal conductance, intercellular CO2 concentration, minimal fluorescence value, and non-photochemical quenching were significantly increased. These findings suggest that there was a synergistic effect between AMV and WCMV, and AMV and WCMV co-infection severely impacted the normal function of photosynthesis in N. benthamiana. [ABSTRACT FROM AUTHOR]

This study examines the potential of 23 plant species, comprising 10 legumes, 9 grasses, and 4 forbs, as cover crops to enhance carbon (C) sequestration and soil nitrogen (N) in vineyards. After a 120-day evaluation period, cover crop biomass was incorporated into the soil, and grapevine seedlings were planted in its place. Among the established cover crops, the C input potential ranged from 0.267 to 1.69 Mg ha−1, and the N input potential ranged from 12.3 to 114 kg ha−1. Legume species exhibited up to threefold greater shoot dry weight (SDW) compared to grass species. Ladino white clover, Dutch white clover, and Clover blend were superior in SDW, total dry weight (TDW), total C content, and total N content. Legumes exhibited slightly higher root dry weight (RDW) than grasses, with the exception of Fall rye leading at 15 g pot−1, followed by Ladino white clover and Dutch white clover at an average of 12 g pot−1. Legumes, particularly clover blend and Alsike clover, displayed high shoot N concentration at an average of 2.95%. Root N concentration in Legumes (Fabaceae) were significantly higher at 1.82% compared to other plant families at 0.89%, while their root C/N ratio was lower at 18.3, contrasting with others at 27.7, resulting in a faster turnover. Biomass production exhibited a negative relationship (R2 = 0.51) with soil residual NO3−. Fall rye, Winfred brassica, and buckwheat had the highest N utilization efficiency (NUtE) values (ava. 121 g g−1). Alsike clover, Ladino white clover, and clover blend showed the highest N uptake efficiency (NUpE) values (ava. 75%). The Readily Available N (RAN) Reliance Index (RANRI) is introduced as a novel indicator for quantifying the extent to which a plant relies on RAN for its total N requirement. The RANRI value represents the percentage of the plant's total N sourced from RAN, ranging from 11% for legumes to 86% for grasses. This implies a substantial influx of nitrogen through a pathway independent of RAN in legumes. Grape shoot N concentration positively correlated with soil NO3− (R2 = 0.31) and cover crop C/N ratio (R2 = 0.17) but negatively correlated with cover crop TDW (R2 = 0.31). This study highlights legume plants as more effective in C and N assimilation during establishment but cautions about potential soil mineral N depletion before reaching their full biological N fixation capacity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Historically, white clover (Trifolium repens) seed was included in turfgrass seed mixtures to provide biodiversity and nitrogen (N) to lawns. White clover dicultures have been studied recently for inclusion in both warm- and cool-season turfgrasses, with the goals of reducing fertilizer applications and providing pollinator forage in lawns; however, other clovers have not been as widely researched in turfgrass. The objectives of this study were to evaluate 1) if white, strawberry (T. fragiferum), crimson (T. incarnatum), and rose (T. hirtum) clovers can persist in dicultures with Kentucky bluegrass (Poa pratensis); 2) if clover inclusion in dicultures impacts broadleaf weed cover; and 3) if low levels of N fertilization impact clover persistence or quality of clover-bluegrass dicultures. Kentucky bluegrass was grown as a monoculture or as a diculture with each of the four clover species. Each mono- or diculture was then treated with a low rate of N fertilizer (48.8 kgha-1 N) or no N fertilizer to determine quality and percentage of grass, clover, or weed and bare-soil cover. Dicultures contained similar or less weed and bare-soil cover, and maintained similar or greater quality compared with bluegrass monocultures, indicating clover and Kentucky bluegrass dicultures are suitable alternatives to Kentucky bluegrass monoculture lawns, and can potentially lead to reduced fertilizer and pesticide requirements. Fertilizer generally had no effect on cover, likely because of the low rates of N applied. [ABSTRACT FROM AUTHOR]

In the absence of chemical control with its negative side effects, fungal pathogens can cause large yield losses, requiring us to develop agroecosystems that are inherently disease resistant. Grassland biodiversity experiments often find plant species diversity to reduce pathogen pressure, but whether incorporating high biodiversity levels in agricultural fields have similar effects remains largely unknown.We tested if undersown plant species diversity could reduce barley disease, and whether the effect was mediated through above‐ or below‐ground mechanisms, by combining an agricultural field trial with a soil transplant experiment.As predicted, barley disease decreased in the presence of undersown plants. Undersown species richness had no effect, but their abundance led to early season disease reduction. Above‐ground mechanisms underpinned this disease reduction. Barley yield slightly decreased with increasing undersown species richness, and undersown species varied in their impact on yield.We identified two undersown species, Trifolium repens and T. hybridum, that contributed most to disease reduction and had the potential to increase barley yield. Furthermore, our results indicate that above‐ground mechanisms caused this. We show that agroecosystem functioning can be improved without trade‐offs on yield by targeted selection of undersown species. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

[Objective] The study aimed to explore the effects of four weed populations on apple photosynthetic characteristics and fruit quality formation, and screen out suitable grass for planting in Zhaotong orchards. [Method] We conducted a field experiment with Fuji apples and set orchard clear-cutting as the control, and explored the effects of Artemisia absinthium(Artemisia lavandulaefolia DC), Artemisia stellate (Stellaria vestita Kurz.), Ryegrass (Lolium perenne L.) and White-flowered cheatgrass (Trifolium repens Linn) on the leaf chlorophyll content, photosynthetic characteristics and fruit quality of the fruit trees. [Result] White-flowered cheatgrass treatment could increase leaf total chlorophyll content, net photosynthetic rate (P), intercellular CO₂ concentration (C₁), stomatal conductance(G) and carboxylation efficiency (CE), while the water use efficiency (WUE) of the fruit trees was significantly reduced. The Artemisia absinthium treatment could increase leaf G, and improve C, to a certain extent, but it would reduce the total chlorophyll content and leaf CE. Artemisia stellate treatment could increase C, and the total chlorophyll content as well as leaf transpiration rate (T,) were reduced. Ryegrass treatment significantly increased WUE and CE, but significantly reduced T. Quality aspect, the various raw grass treatments increased fruit solids content by 0.88% 13.87%, reduced reducing sugar content by 2.30% 15.71%, increased total antioxidant capacity by 10.03%-40.92%, and the ryegrass treatment reduced hydrogen peroxide content by 41.80% and superoxide anion scavenging capacity by 12.92%, as compared to clear tillage. Using fuzzy membership function analysis, the comprehensive evaluation value of clear tillage, white clover, mugwort, stellaria and ryegrass were 0.4858, 0.6044, 0.5548, 0.4894 and 0.6059, respectively. [Conclusion] Different weed populations can improve the photosynthetic capacity of fruit leaves and improve fruit quality to a certain extent. Scientific evaluation of four weed populations is beneficial to screen out more reasonable grass types in orchards. [ABSTRACT FROM AUTHOR]

In the experimental field of the Research Institute of Mountain Stockbreeding and AgricultureTroyan during the period 2020-2022, the following meadow legume crops were studied: bird’s foot trefoil (Lotus corniculatus L.), hybrid clover (Trifolium hibridum L.), alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.) and white clover (Trifolium repens L.). The purpose of the present research is to make a statistical analysis based on average data on yield, morphological features and quality, and determine dependencies between them. High correlations were found between plant height and crude fiber content (r = 0.88); crude fiber and the relative proportion of legumes in the grass stand (r = 0.77); acid detergent fibers and cellulose content (r = 0.92). These results make it possible to derive regression dependencies between them, which are represented by linear equations and coefficients of determination. [ABSTRACT FROM AUTHOR]

Azotobacter chroococcum and Bacillus subtilis were selected as fermentation strains, and biogas residue after anaerobic digestion of kitchen waste and residual sludge was used as fermentation substrate. A single factor optimization test was used to optimize the solid-state fermentation parameters of biogas residue with the number of viable bacteria and the number of spores as indexes. The results showed that the optimum inoculation conditions involved the following: 55% initial moisture content, 15% initial inoculation amount, 30 ℃, and 1:1 initial inoculation ratio for 13 days. Pot experiment showed that the prepared three kinds of bacterial fertilizers could not only effectively promote the growth of white clover, improve the composition of soil nutrients, but also change the structure of soil bacterial community, which is of great significance to the health of soil ecosystem in white clover. [ABSTRACT FROM AUTHOR]

Extreme heat poses a major threat to plants and pollinators, yet the indirect consequences of heat stress are not well understood, particularly for native solitary bees. To determine how brief exposure of extreme heat to flowering plants affects bee behaviour, fecundity, development and survival we conducted a no-choice field cage experiment in which Osmia lignaria were provided blueberry (Vaccinium corymbosum), phacelia (Phacelia tanacetifolia) and white clover (Trifolium repens) that had been previously exposed to either extreme heat (37.5°C) or normal temperatures (25°C) for 4 h during early bloom. Despite a similar number of open flowers and floral visitation frequency between the two treatments, female bees provided with heat-stressed plants laid approximately 70% fewer eggs than females provided with non-stressed plants. Their progeny received similar quantities of pollen provisions between the two treatments, yet larvae consuming pollen from heat-stressed plants had significantly lower survival as larvae and adults. We also observed trends for delayed emergence and reduced adult longevity when larvae consumed heat-stressed pollen. This study is the first to document how short, field-realistic bursts of extreme heat exposure to flowering host plants can indirectly affect bee pollinators and their offspring, with important implications for crop pollination and native bee populations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aims: To develop a methodology to study uptake and redistribution by plants of NH4+ from deep soil, applying it to investigate deep root N uptake by cultivated grassland species. Methods: A slow-release 15NH4+ label adsorbed to clinoptilolite was placed into soil (depth 42 cm) well below the densest root zone in well-established monospecific stands of five grass and two clover species. Species showing a variety of deep rooting patterns, N acquisition strategy, forage qualities, and persistence in hemiboreal conditions were chosen. The label was placed in early spring and tracked throughout one or two growing seasons in two repeated experiments. Results: After two growing seasons ~ 90% of the label was tracked in the soil and harvested herbage of grasses, less in clovers. Deep N uptake was limited in spring, increased during mid-season, and was strongest in autumn in all species, despite lower herbage yield in autumn. Species differed in ability to recover and maintain 15N in the soil–plant system. In one growing season, Lolium perenne L., Phleum pratense L., Schedonorus pratensis (Huds.) P.Beauv. and Schedonorus arundinaceus (Schreb.) Dumort herbage recovered ~ 65% of the label, Poa pratensis L. 54%, and Trifolium pratense L. and Trifolium repens L. 36–48%. Label transport to topsoil was observed, mainly attributable to plant nutrient redistribution rather than physical diffusion. Conclusions: The innovative slow-release 15N label enabled tracing species differences and seasonal changes in uptake of NH4+ from deep soil. Among the tall-growing grasses, growth vigor appeared as important for deep N uptake as expected root depth. [ABSTRACT FROM AUTHOR]

Purpose: Dissolved organic matter (DOM) plays a key role in agroecosystems' carbon cycling. However, the DOM evolutionary signatures and the timing of equilibrium being achieved in dryland inter-planting systems remain unclear. This study aimed to determine the characteristics of DOM dynamics using a three-dimensional fluorescence excitation-emission matrix combined with parallel factor analysis (EEMs-PARAFAC). Methods: DOM contents, different related components accumulation were assessed using clear tillage (CT) as the control, white clover (Trifolium repens L.) of three different ages (4, 8, 12 years), representing a chronosequence, in apple orchards on the Loess Plateau. Results: At a depth of 0–10 cm, DOM concentration decreased with depth and significantly increased with longer inter-planting, and the five fluorescence components contents of soil DOM showed a similar trend. Moreover, the relative abundances of humic-like (C2, C3) was higher than CT, but the protein-like was lower. The ultraviolet–visible spectral and optical indices indicated that DOM mainly originated from mixed sources, and gradually tended towards greater DOM molecular complexity with increasing time since sowing. Microbial biomass nitrogen and soil organic carbon (SOC) were the most important two factors affecting DOM over time, contributing to 87% and 77% of the total variation. Conclusions: This study demonstrated that inter-planting time and soil depth played important roles in determining DOM content and component accumulation. Overall, inter-planting with white clover in orchards can effectively improve the accumulation of macro-molecular humic-like DOM, which probably promotes SOC storage. Long-term experiments are required exploring DOM dynamics and the timing of equilibrium being reached. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) are commonly used as silage inoculants. Manganese sulphate (MnSO4) has been proposed to protect certain LAB during oxygen exposure and improve silage aerobic stability (ASTA). The aim of the present study was to assess the efficacy of inoculants in preservation of different forages. A short‐term screening trial investigated the effect of 24 additive treatments, compared to a negative control, on ensiling perennial grass in mini‐silos (2.75 L) for 7 d. The additive treatments comprised different ratios of Lactiplantibacillus plantarum [IMI 507026, IMI 507027, and IMI 507028] and Pediococcus pentacaceus [IMI 507024 and IMI 507025], with or without MnSO4. Target parameters, such as low levels of fresh matter loss, pH, acetic acid, ammonia‐nitrogen fraction (NH3–N/Total N), and high levels of lactic acid and ASTA, were considered for treatment selection. Eight treatments were selected based on a principal component analysis and hierarchical clustering of the target parameters. These selected treatments subsequently underwent a 100 d long‐term trial using lucerne, perennial grass, and perennial grass‐white clover forages. All silages treated with the eight additives exhibited an improved homolactic fermentation and a lower (p <.001) pH, acetic acid, and ethanol content than the control. Although DM losses and ASTA markedly improved in the treatments containing MnSO4 in the short‐term trial, these effects were not observed in the long‐term trial. These outcomes suggest that the selected additives can improve silage fermentation quality across a broad range of forages, but further studies are required to assess the impact of MnSO4 on ASTA. [ABSTRACT FROM AUTHOR]

Urine patches from grazing ruminants contain high concentrations of nitrogen (N) and are the main source of N leaching from grazed pastoral farming systems. While there have been various options identified to substantially reduce N leaching, in general these practices increase the cost of production or reduce production per hectare. In New Zealand, multi‐species pastures were evaluated as a cheaper option that would potentially retain productivity. Early research showed these pastures had lower urinary N excretion from grazing animals and increased plant N uptake, compared with standard New Zealand perennial ryegrass/white clover (PR‐WC) pastures. Further research highlighted the beneficial attributes of the pasture herb narrow‐leaved plantain (Plantago lanceolata; PL), specifically: reduced urine N concentration, reduced urine N excretion, and reduced rate of soil nitrification. Significant reductions (14%–89%) in N leaching were found from cow urine applied to lysimeters with pastures including PL, compared with PR‐WC pasture. Paddock‐scale measurements confirmed the effects of PL, with 20%–60% lower N leaching when PL comprised 30%–40% of the dry matter (DM) of PL‐PR‐WC grazed pastures. There were no negative effects on milk production and composition when feeding PL to dairy cows, but small positive effects on fatty acid profiles. However, weed and pest management, palatability and persistence remain challenging in pastures containing PL on commercial farms. Management options need to be clarified to ensure successful establishment and maintenance of PL. Additionally, the cultivar differences in secondary compounds and their impact on nitrification rate and N leaching need to be better understood. [ABSTRACT FROM AUTHOR]

Sprouts' consumption has become popular due to their wide availability, easy cultivation process, and proven biological activity. Moreover, stress factors, such as limited access to light or disturbed gravity during growth, may contribute to the increased activity and the synthesis of bioactive compounds. In this study, for the first time, the examination of the impact of darkness and simulated microgravity conditions on the white clover sprouts from the Fabaceae family was conducted. Among several species, used in the preliminary attempts, only white clover was satisfactory sprouting in the disturbed gravity conditions, and thus was chosen for further examination. A random positioning machine setup was used during the cultivation process to simulate microgravity conditions. Additionally, the sprouts were cultivated in total darkness. Simulated microgravity and/or darkness during the first few days of the sprouts' growth caused biomass reduction, the increased synthesis of bioactive compounds (isoflavones and phenolics), and changes in the level of abscisic acid and phenylalanine ammonia-lyase. Moreover, it increased the antioxidant properties of the sprouts, while the enhancement of their cytotoxic impact was observed only for androgen-dependent prostate cancer LNCaP cells. To conclude, the presented results are promising in searching for novel functional food candidates and further studies are necessary, directed at other plant families. [ABSTRACT FROM AUTHOR]

Parthenium hysterophorus L. has become a weed of global concern owing to its fast expansion and invasive character. In order to study the status of this noxious weed and its impact on floristic diversity in Dhauladhar foothills, the study was conducted during the year 2021–2022 in culturable wastelands of Dhauladhar ranges in Kangra district of Himachal Pradesh, India. The impact of Parthenium hysterophorus L. on associated species at different altitudes and aspects was observed. Our observations depicted that Parthenium hysterophorus L. has been growing more vigorously in the northern aspect than the southern aspect with the density of 37.78 m−2 and 21.62 m−2, respectively. The highest density of this noxious weed was recorded in the altitudinal range of 600–1200 m (34.32 m−2) while it was not observed beyond 1805 m above sea level. The invasion of Parthenium hysterophorus L. significantly affected the plant density of other species. The descending order of the species as per dominance was observed as Cynodon dactylon, Trifolium repens, Oxalis latifolia, Parthenium hysterophorus L., and Ageratum houstonianum. The average number of species and species density were observed more in non-invaded sites (9.35 and 27.67 m−2) than in invaded sites (7.10 and 20.60 m−2). Species abundance and plant cover were observed more in non-invaded sites (28.73 and 657.90 m2 ha−1) than in invaded sites (22.70 and 322.30 m2 ha−1). Species diversity, richness, and evenness were reported to decline in invaded sites (1.56, 0.95, and 0.81, respectively) with respect to the non-invaded sites (1.94, 1.16, and 0.88, respectively). The study highlights the significant concerns associated with the invasive weed within the plant communities. Understanding its invasive status holds considerable implications for local afforestation initiatives, forest management strategies, and conservation policies. Furthermore, this investigation lays a foundational groundwork for implementing effective measures to get rid of this alien weed. [ABSTRACT FROM AUTHOR]

Abstract Background White clover (Trifolium repens L.) is an excellent leguminous cool-season forage with a high protein content and strong nitrogen-fixing ability. Despite these advantages, its growth and development are markedly sensitive to environmental factors. Indole-3-acetic acid (IAA) is the major growth hormone in plants, regulating plant growth, development, and response to adversity. Nevertheless, the specific regulatory functions of Aux/IAA genes in response to abiotic stresses in white clover remain largely unexplored. Results In this study, we identified 47 Aux/IAA genes in the white clover genome, which were categorized into five groups based on phylogenetic analysis. The TrIAAs promoter region co-existed with different cis-regulatory elements involved in developmental and hormonal regulation, and stress responses, which may be closely related to their diverse regulatory roles. Collinearity analysis showed that the amplification of the TrIAA gene family was mainly carried out by segmental duplication. White clover Aux/IAA genes showed different expression patterns in different tissues and under different stress treatments. In addition, we performed a yeast two-hybrid analysis to investigate the interaction between white clover Aux/IAA and ARF proteins. Heterologous expression indicated that TrIAA18 could enhance stress tolerance in both yeast and transgenic Arabidopsis thaliana. Conclusion These findings provide new scientific insights into the molecular mechanisms of growth hormone signaling in white clover and its functional characteristics in response to environmental stress.

ABSTRACT: The objective of this study was to quantify the farm gate nitrogen (N) offset potential of perennial ryegrass (Lolium perenne L.; PRG) white clover (Trifolium repens L.; WC) swards by comparing the herbage and milk production from dairy farmlets that were simulations of full farming systems. A study was established where 120 cows were randomly assigned to 4 farmlets of 10.9 ha (stocking rate: 2.75 cow/ha), composed of 20 paddocks each. Cows were fed 526 kg of DM of concentrate on average each year. The 4 grazing treatments were PRG-only at 150 or 250 kg of N/ha and PRG-WC at 150 or 250 kg of N/ha. Cows remained in their treatment group for an entire grazing season and were re-randomized as they calved across treatments each year. As cows calved in the spring as standard practice in Ireland, they were rotationally grazed from early February both day and night (weather permitting) to mid-November, to a target postgrazing sward height of 4.0 cm. Mean sward WC content was 18.1% and 15.4% for the 150 and 250 kg of N/ha PRG-WC treatments, respectively over the 3-yr period. When WC was included, lowering the N rate did not reduce pregrazing yield, pregrazing height, or herbage removed, but those factors decreased significantly when WC was absent. Total annual herbage DM production was 13,771, 15,242, 14,721, and 15,667 kg of DM/ha for PRG-only swards receiving 150 or 250 kg of N/ha and PRG-WC swards receiving 150 or 250 kg of N/ha, respectively. In addition, when WC was present, compressed postgrazing sward heights were lower (4.10 vs. 4.21 cm) and herbage allowance (approximately 17 kg/cow feed allocation per cow per day) higher than the high-N control (+ 0.7 kg of DM/cow per day). There was a significant increase in milk production, both per cow and per hectare, when WC was included in PRG swards. Over the 3-yr study, cows grazing PRG-WC had greater milk (+304 kg) and milk solids (+31 kg of fat + protein) yields than cows grazing PRG-only swards. This significant increase in milk production suggests that the inclusion of WC in grazing systems can be effectively used to increase milk production per cow and per hectare and help offset nitrogen use. This result shows the potential to increase farm gate N use efficiency and reduce the N surplus compared with PRG-dominant sward grazing systems receiving 250 kg of N/ha, without negatively affecting milk solids yield or herbage production, thus increasing farm profit by €478/ha.

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. [ABSTRACT FROM AUTHOR]

Planting a cover crop living mulch between plastic mulch beds in fresh market vegetable production can reduce soil erosion and runoff, and offers an opportunity to grow an income generating cash crop alongside a soil building cover crop. However, potential negative impacts on yield, variable weed control, unclear impacts on soil health, and limited management recommendations challenge adoption of this practice, despite grower interest. In a two-year study in southwest Michigan, living mulches were evaluated in the production of plasticulture organic summer squash (Cucurbita pepo cv. Lioness) and bell pepper (Capsicum annuum cv. Paladin). Strategies evaluated included three common grower practices (cultivation, dead straw mulch, mowing ambient weeds) and three mowed living mulch treatments (Italian ryegrass [Lolium multiflorum] monoculture, rye [Secale cereale] monoculture, and a Dutch white clover [Trifolium repens]/rye mixture). We determined the impact of these strategies on weed control, organic matter inputs, vegetable crop performance, nitrogen retention, and soil microbial communities. We found that cultivation and dead mulch provided superior in-season weed control, reducing weed biomass by an average of 86% compared to 18% among living mulch treatments, with associated reductions in the weed seedbank compared to living mulches and mowed weeds. In most cases, living mulch establishment was a challenge and weed biomass on average accounted for 99, 74, and 94% of organic matter inputs in rye, ryegrass, and clover/rye treatments, respectively. Squash performance was unaffected by our treatments, but pepper yield reductions ranged from 41 to 54% in all treatments relative to cultivation in one of two years. Living mulches and the weedy treatment showed the ability to reduce end-of-season potentially leachable nitrogen by 61% compared to cultivation and dead mulching. Soil microbial abundance and functional diversity were similar across treatments, but extracellular enzyme activity was higher in dead mulch, living mulch and weedy treatments compared to cultivation. Results from this study help quantify tradeoffs between common grower practices for managing weeds and soil between plastic mulch beds, highlight key challenges with living mulch alternatives, and identify areas for future research. [ABSTRACT FROM AUTHOR]

During the period 2020-2022, the following types of meadow legume crops were tested on light gray pseudopodzolic soil on the territory of the experimental field of Research Institute of Mountain Stockbreeding and Agriculture-Troyan: Lotus corniculatus L., Trifolium hibridum L., Medicago sativa L., Trifolium pretense L., Trifolium repens L. The matter yield, the biodiversity of the species in the grass stand and the impact of the factors, such as conditions of the year and the type of grass stands were studied to determine the acclimatization of meadow legume crops under mountain conditions. It was established that the tested legume forage species realized a high yield of dry matter under mountain conditions, which varied from 836.68 to 971.13 kg/da, which determined their good adaptability for cultivation under the conditions of light gray pseudopodzolic soils. The low values of their variation coefficients by year and average yield of 909.35 kg/da of all species show that they are suitable for inclusion in the structure of farms in mountain conditions. Alfalfa proved to be the most rational for cultivation, followed by red clover. The botanical composition of the grass stands depends on the rate of growth and development of the herbaceous species. The high productivity of alfalfa corresponds to its high presence in the grass stand, which also indicates its high productivity. Natural factors determine the productivity of the grass stands of the various legume species, and subsequently the economic efficiency in the production of fodder. [ABSTRACT FROM AUTHOR]

Urbanization frequently alters the biotic and abiotic environmental context. Variation in both the biotic and abiotic environment can result in selection mosaics that lead to locally adapted phenotypes. Ecological changes to urban environments can result in evolutionary responses across a diverse array of plants and animals, but the effects of urbanization on local adaptation and coevolution between species remain largely unstudied.Using a common garden experiment of 30 populations and 1080 plants, we tested for local adaptation in the mutualism between white clover (Trifolium repens) and rhizobia (Rhizobium leguminosarum symbiovar trifolii) along an urbanization gradient. We asked: (Q1) are white clover and rhizobia locally adapted? (Q2) Does nitrogen (N) addition mediate the strength of local adaptation? (Q3) Is the strength of local adaptation in host plants related to the strength of local adaptation in rhizobia, as expected if white clover and rhizobia are coadapting? And (Q4) how does the degree of local adaptation vary with urbanization?We found evidence for local adaptation for both white clover and rhizobia, with stronger signatures of local adaptation for rhizobia than white clover (Q1). While N addition positively affected plant biomass and negatively affected nodule density, N addition did not consistently mediate patterns of local adaptation (Q2). The strength of local adaptation was positively related between white clover and rhizobia under N addition, suggesting that soil N mediates coadaptation between white clover and rhizobia (Q3). We did not find a consistent relationship between measures of urbanization and local adaptation (Q4). Additionally, fitness responses for white clover and rhizobia were unrelated to Rhizobium abundance and local adaptation was unrelated to the broader bacterial microbiome in the soil inoculants or root endosphere.Synthesis. Our results show a spatial mosaic in local adaptation, with stronger local adaptation for rhizobia than white clover. While urbanization does influence the ecology of plant–microbe interactions, our study suggests urbanization does not disrupt local adaptation and coevolution among mutualists. [ABSTRACT FROM AUTHOR]

Bacteria belonging to the genus Micromonospora are recognized as microorganisms with the potential to be used in biotechnology processes, given their beneficial influence on plant growth and the biocontrol of phytopathogens. In this study, nineteen Micromonospora isolates originating from the root nodules of white clover plants were taxonomically assigned based on the phylogenetic analysis of the 16S rRNA gene and four housekeeping genes. The antifungal properties of the bacteria against phytopathogenic Botrytis cinerea, Fusarium oxysporum, Fusarium equiseti, Sclerotinia sclerotiorum, and Verticillium albo-atrum were tested with the agar plug test and the dual culture test. The ability to produce various metallophores was determined with the agar plug diffusion test on modified chrome azurol S (CAS) agar medium. International Streptomyces Project-2 medium (ISP2) broth amended with 0.2% L-tryptophan was used to indicate the bacterial ability to produce auxins. The strains belonging to M. tulbaghiae, M. inaquosa, and M. violae showed in vitro potential as antimicrobial agents against the tested fungi. M. inaquosa strain 152, M. violae strain 126, M. violae strain 66, and M. violae strain 45 were recognized as the most efficient metallophore producers. M. alfalfae strain 55 and M. lupini strain 5052 were identified as the most promising auxin compound producers and, therefore, show potential as plant-growth-promoting bacteria. [ABSTRACT FROM AUTHOR]

Excessive aluminum (Al) in acidic soils is a primary factor that hinders plant growth. The objective of the present study was to investigate the effect and physiological mechanism of exogenous silicon (Si) in alleviating aluminum toxicity. Under hydroponic conditions, 4 mM Al significantly impeded the growth of white clover; however, pretreatments with 1 mM Si mitigated this inhibition, as evidenced by notable changes in growth indicators and physiological parameters. Exogenous silicon notably increased both shoot and root length of white clover and significantly decreased electrolyte leakage (EL) and malondialdehyde (MDA) content compared to aluminum treatments. This positive effect was particularly evident in the roots. Further analysis involving hematoxylin staining, scanning electron microscopy (SEM), and examination of organic acids (OAs) demonstrated that silicon relieved the accumulation of bioactive aluminum and ameliorated damage to root tissues in aluminum-stressed plants. Additionally, energy-dispersive X-ray (EDX) analysis revealed that additional silicon was primarily distributed in the root epidermal and cortical layers, effectively reducing the transport of aluminum and maintaining the balance of exchangeable cations absorption. These findings suggest that gradual silicon deposition in root tissues effectively prevents the absorption of biologically active aluminum, thereby reducing the risk of mineral nutrient deficiencies induced by aluminum stress, promoting organic acids exudation, and compartmentalizing aluminum in the outer layer of root tissues. This mechanism helps white clover alleviate the damage caused by aluminum toxicity. [ABSTRACT FROM AUTHOR]

In this work, we present and describe a spectral library (SL) with 15 vascular plant species from Montesinho Natural Park (MNP), a protected area in Northeast Portugal. We selected species from the vascular plants that are characteristic of the habitats in the MNP, based on their prevalence, and also included one invasive species: Alnus glutinosa (L.) Gaertn, Castanea sativa Mill., Cistus ladanifer L., Crataegus monogyna Jacq., Frangula alnus Mill., Fraxinus angustifolia Vahl, Quercus pyrenaica Willd., Quercus rotundifolia Lam., Trifolium repens L., Arbutus unedo L., Dactylis glomerata L., Genista falcata Brot., Cytisus multiflorus (L'Hér.) Sweet, Erica arborea L., and Acacia dealbata Link. We collected spectra (300–2500 nm) from five records per leaf and leaf side, which resulted in 538 spectra compiled in the SL. Additionally, we computed five vegetation indices from spectral data and analysed them to highlight specific characteristics and differences among the sampled species. We detail the data repository information and its organisation for a better understanding of the data and to facilitate its use. The SL structure can add valuable information about the selected plant species in MNP, contributing to conservation purposes. This plant species SL is publicly available in Zenodo platform. [ABSTRACT FROM AUTHOR]

Background: White clover (Trifolium repens L.) is an excellent leguminous cool-season forage with a high protein content and strong nitrogen-fixing ability. Despite these advantages, its growth and development are markedly sensitive to environmental factors. Indole-3-acetic acid (IAA) is the major growth hormone in plants, regulating plant growth, development, and response to adversity. Nevertheless, the specific regulatory functions of Aux/IAA genes in response to abiotic stresses in white clover remain largely unexplored. Results: In this study, we identified 47 Aux/IAA genes in the white clover genome, which were categorized into five groups based on phylogenetic analysis. The TrIAAs promoter region co-existed with different cis-regulatory elements involved in developmental and hormonal regulation, and stress responses, which may be closely related to their diverse regulatory roles. Collinearity analysis showed that the amplification of the TrIAA gene family was mainly carried out by segmental duplication. White clover Aux/IAA genes showed different expression patterns in different tissues and under different stress treatments. In addition, we performed a yeast two-hybrid analysis to investigate the interaction between white clover Aux/IAA and ARF proteins. Heterologous expression indicated that TrIAA18 could enhance stress tolerance in both yeast and transgenic Arabidopsis thaliana. Conclusion: These findings provide new scientific insights into the molecular mechanisms of growth hormone signaling in white clover and its functional characteristics in response to environmental stress. [ABSTRACT FROM AUTHOR]