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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]

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]

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]

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.

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]

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]

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]

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. [ABSTRACT FROM AUTHOR]

Research was carried out to assess the yield and quality of fruits from the new Polish apple tree 'Chopin'—a 'green peel', scab-resistant cultivar under grass living mulch management. Blue fescue and red fescue were tested in experiment no. 1. In experiment no. 2, meadow grass and perennial ryegrass were used. Every species of grass was sown in two doses of 50 and 150 kg per ha. Herbicide fallow was introduced as a control in both experiments. Strongly variable temperatures and precipitations in the years of evaluation made it possible to estimate year—a function of variable environmental conditions—as an additional experimental factor. An unexpected effect of the presence of living mulch was its significant impact on the appearance of blush on the 'green peel' fruit. However, living mulches had little effect on weight and fruit size. Cool days during apple ripening enhanced the process of fruit skin red coloration. The effect of both agrotechnical and environmental factors on fruit quality was more visible in the case of less vigorous trees, which were more susceptible to experimental, stressful conditions. However, increasing the sowing dose of each grass seed did not influence red blushing, weight, or fruit size. An additional difficulty for the trees was the competition caused by the early germination of these living grass mulches, reinforced by the presence of Trifolium repens L. [ABSTRACT FROM AUTHOR]

Weed control and replenishing soil moisture and fertility are important areas in fruit production. The number of studies focused to determine whether living mulches in tree rows can fulfil these tasks increases recently. In the paper the results of an 11-year experiment on the effect of two such mulches (Trifolium repens L. and Agrostis capillaris L.) on the growth, yield, and fruit quality of three pear cultivars ('Alfa', 'Dolores', 'Amfora') in relation to herbicide fallow are presented. In the experiment, a single sowing of covering plants without additional treatments was used. A statistically significant reduction in yield (20-22%) was found for both mulches used, which was related to weakening the vegetative growth of trees. The average fruit mass did not change significantly, but the percent share of large fruits increased significantly (about 17%) in the A. capillaris mulch. Fruits from trees growing in T. repens contained significantly more soluble solids and Ca. However, that mulch became heavily infested with weeds after a few years. The obtained results allow us to recommend the use of the living mulch A. capillaris in the tree rows of a commercial pear orchard. The 'Dolores' and 'Amfora' cvs are particularly recommended. [ABSTRACT FROM AUTHOR]

Ramularia sphaeroidea was primarily identified based on the characteristics of its conidia and several sequences. The fungus causes severe leaf spot disease on hairy vetch (Vicia villosa var. glabrescens) in Yunnan Province in China. The growth, sporulation, fungicide efficacy, and host range of the pathogen were evaluated to aid in disease management. Different types of culture media and carbon and nitrogen sources were used to evaluate the growth of R. sphaeroidea. Oatmeal, maltose, and potassium nitrate agar had a higher amount of sporulation. Difenoconazole (10%) was the most effective fungicide against the leaf disease caused by R. sphaeroidea. In addition, foliar inoculation sprays were used to assess the host range of R. sphaeroidea in six different plant species, including alfalfa (Medicago sativa L.), sainfoin (Onobrychis viciifolia Scop.), erect milkvetch (Astragalus adsurgens Pall.), common vetch (Vicia sativa L.), red clover (Trifolium pratense L.), and white clover (Trifolium repens L.). R. sphaeroidea successfully infected these plants, indicating that it has a wider host range than hairy vetches. [ABSTRACT FROM AUTHOR]

The scientific aim of this article is to investigate the potential benefits of implementing a multi-cropping system, specifically focusing on the incorporation of caraway, to improve soil agrochemical and biological properties, prevent soil degradation and erosion, and ultimately enhance soil quality and health to better adapt to climate change. This study aims to provide valuable insights into the comparative analysis of various soil parameters and biological indicators to showcase the promising perspectives and importance of perennial crop production for improving soil quality and agricultural sustainability. These crops are designed to provide multiple benefits simultaneously, including improved yields, enhanced ecosystem services, and reduced environmental effects. However, an integrated assessment of their overall effects on the agroecosystem is crucial to understand their potential benefits and trade-offs. The field experiment was conducted over three consecutive vegetative seasons (2017 to 2021) at the Experimental Station of Vytautas Magnus University Agriculture Academy (VMU AA) in Kaunas district, Lithuania. The experimental site is located at 54°53′7.5″ N latitude and 23°50′18.11″ E longitude. The treatments within a replicate were multi-cropping systems of sole crops (spring barley (1), spring wheat (2), pea (3), caraway (4)), binary crops (spring barley–caraway (5), spring wheat–caraway (6), pea–caraway (7)), and trinary crops (spring barley–caraway–white clover (8), spring wheat–caraway–white clover (9), pea–caraway–white clover (10)) crops. However, an integrated assessment of their impact on the agroecosystem is needed to understand their potential benefits and processes. To determine the complex interactions between indicators, the interrelationships between indicators, and the strength of impacts, this study applied an integrated assessment approach using the comprehensive assessment index (CEI). The CEI values showed that integrating caraway (Carum carvi L.) into multi-cropping systems can have several positive effects. The effect of the binary spring barley and caraway and the trinary spring barley, caraway, and white clover crops on the agroecosystem is positively higher than that of the other comparative sole, binary, and trinary crops. Caraway, after spring wheat together with white clover, has a higher positive effect on the agroecosystem than caraway without white clover. Specifically, this study addresses key aspects, such as soil health, nutrient cycling, weed management, and overall agricultural sustainability, within the context of multi-cropping practices. By evaluating the effects of these cropping systems on soil agrochemical properties and ecosystem dynamics, the research provides valuable insights into sustainable agricultural practices that promote environmental conservation and long-term soil health. [ABSTRACT FROM AUTHOR]

Trifolium repens L. (white clover) and Lolium perenne L. (ryegrass) are green manures widely used in conservation tillage systems worldwide. Eleusine indica L. (goosegrass) is a globally recognized noxious weed. Herein, we investigated the effects of aqueous extracts, decomposed liquids, and different straw-to-soil ratios on the germination and growth of goosegrass. The results showed that high concentrations (= 30%) of aqueous extracts or decomposed liquids of both green manures significantly inhibited germination-related parameters of goosegrass. The strongest inhibitory effect was observed for the 7-day decomposition treatment, and white clover's inhibitory effect was greater than ryegrass's. A pot experiment showed that non-photochemical quenching, catalase, and peroxidase activity levels of goosegrass leaves were significantly increased. At the same time, the net photosynthetic rate significantly decreased. Seedling growth was inhibited when the straw-to-soil ratio was greater than 3:100. The ryegrass treatments inhibited goosegrass seedlings more than the white clover treatments. This study demonstrated the inhibitory potential of white clover and ryegrass straw return on seed germination and seedling growth of goosegrass. The study has also helped to identify weed-resistant substances in these green manures so that their weed-control properties can be used more effectively and herbicide usage can be reduced. [ABSTRACT FROM AUTHOR]

White clover (Trifolium repens L.) is an allopolyploid plant and an excellent perennial legume forage. However, white clover is subjected to various stresses during its growth, with cold stress being one of the major limiting factors affecting its growth and development. Beta-amylase (BAM) is an important starch-hydrolyzing enzyme that plays a significant role in starch degradation and responses to environmental stress. In this study, 21 members of the BAM gene family were identified in the white clover genome. A phylogenetic analysis using BAMs from Arabidopsis divided TrBAMs into four groups based on sequence similarity. Through analysis of conserved motifs, gene duplication, synteny analysis, and cis-acting elements, a deeper understanding of the structure and evolution of TrBAMs in white clover was gained. Additionally, a gene regulatory network (GRN) containing TrBAMs was constructed; gene ontology (GO) annotation analysis revealed close interactions between TrBAMs and AMY (α-amylase) and DPE (4-alpha-glucanotransferase). To determine the function of TrBAMs under various tissues and stresses, RNA-seq datasets were analyzed, showing that most TrBAMs were significantly upregulated in response to biotic and abiotic stresses and the highest expression in leaves. These results were validated through qRT-PCR experiments, indicating their involvement in multiple gene regulatory pathways responding to cold stress. This study provides new insights into the structure, evolution, and function of the white clover BAM gene family, laying the foundation for further exploration of the functional mechanisms through which TrBAMs respond to cold stress. [ABSTRACT FROM AUTHOR]

Aim. Develop a Life Cycle Analysis (LCA) in livestock farms with rotational management of the pasture according to botanical composition. Materials and methods: Dairy farms from Cotopaxi, at 2800-3590 meters above sea level, were compared. Farm 1: load of 1 and 1.2 AU/ha, with 50% Plantain (Plantago lanceolata), 50% White Clover (Trifolium repens). Farm 2: Load of 1 and 1.2 AU/ha, 85% Ryegrass (Lolium perenne)-15% White clover (Trifolium repens). Farm 3: Load of 1 and 2.1 AU/ha, 33% Ryegrass-33% Plantain-34% White Clover. Fertilization, balancing and crossbred, New Zealand and North American type Holstein animals and grass rest were used. Results: There were differences in land use, coinciding in farms 1 and 3, in family labor. On Farm 1, rest was 15 to 28 days and NDF from 47.38 to 28.03 and 1.96 to 2.45 Mcal of energy and in 3, Metabolizable Energy values from 1.99 to 40 days and 2 .11 to 35 days, less milk/cow than 3 and higher than 1. In milk production/ha, farms 1 and 3 higher with 24 kg vs 19 kg. This was positive for ACV on those farms. There was potential to convert more of the system's energy into products. Conclusion: In the LCA, a coincidence was found with indicators of specialized systems in milk production, nitrogen balances, energy and their relationships with the environment and emissions, although efficiency can be recovered with improvements in management and without extra inputs to the farm. [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.

AbstractThe GRAS transcription factor (TF) is a plant-specific regulator that plays a critical role in plant growth, development and response to various abiotic stresses. While the functions of many GRAS TFs have been extensively studied in numerous model plants with fully sequenced genomes, the GRAS TFs in white clover (Trifolium repens L.) remain elusive despite the sequencing of its genome. To bridge this knowledge gap, homologs of GRAS TFs from Arabidopsis were employed and subjected to a genome-wide blast against white clover proteins. This analysis identified and characterized 102 putative GRAS genes, designated as TrGRAS, based on the presence of GRAS domains in positive blast hits. Concurrently, an exploration of their gene structures, classification, evolutionary relationships and conservative motifs provided insights into the distinctive features of TrGRAS in white clover. Furthermore, gene duplication analysis revealed an expansion of TrGRAS genes in the PAT1 and LISCL subfamilies, indicating functional diversity within the white clover genome. Lastly, RNA-seq and quantitative reverse transcription-PCR (qRT-PCR) experiments confirmed their higher expression during the early or intermittent responses to cold stress, suggesting their potential and crucial roles in determining the cold tolerances of white clover. The analysis of TrGRAS genes, coupled with expression studies, contributes to the functional characterization of TrGRAS genes in white clover. This knowledge is pivotal for informing genetic improvement strategies in future breeding efforts aimed at enhancing cold tolerance in white clover.

Plant functional traits serve as a bridge between plants, the environment, and ecosystem function, playing an important role in predicting the changes in ecosystem function that occur during ecological restoration. However, the response of grassland ecosystem function to plant functional traits in the context of ecological restoration in areas of karst desertification remains unclear. Therefore, in this study, we selected five plant functional traits [namely, plant height (H), specific leaf area (SLA), leaf dry matter content (LDMC), root length (RL), and root dry matter content (RDMC)], measured these along with community-weighted mean (CWM) and functional trait diversity, and combined these measures with 10 indexes related to ecosystem function in order to investigate the differences in plant functional traits and ecosystem function, as well as the relationship between plant functional traits and ecosystem functions, under four ecological restoration models [Dactylis glomerata (DG), Lolium perenne (LP), Lolium perenne + Trifolium repens (LT), and natural grassland (NG)]. We found that: 1) the Margalef index and Shannon–Wiener index were significantly lower for plant species in DG and LP than for those in NG (P<0.05), while the Simpson index was significantly higher in the former than in NG (P<0.05); 2) CWMH, CWMLDMC, and CWMRDMC were significantly higher in DG, LP, and LT than in NG, while CWMSLA was significantly lower in the former than in NG (P<0.05). The functional richness index (FRic) was significantly higher in DG and LP than in NG and LT, but the functional dispersion index (FDis) and Rao’s quadratic entropy index (RaoQ) were significantly lower in DG and LP than in NG and LT (P<0.05), and there was no significant difference between DG and LP, or between NG and LT (P>0.05); 3) ecosystem function, including ecosystem productivity, carbon storage, water conservation and soil conservation, was highest in LT and lowest in NG; and 4) CWMLDMC (F=56.7, P=0.024), CWMRL (F=28.7, P=0.024), and CWMH (F=4.5, P=0.048) were the main factors affecting ecosystem function. The results showed that the mixed pasture of perennial ryegrass and white clover was most conductive to restoration of ecosystem function. This discovery has important implications for the establishment of vegetation, optimal utilization of resources, and the sustainable development of degraded karst ecosystems. [ABSTRACT FROM AUTHOR]

Pyrolysis is a thermochemical conversion process producing biochar, gas, and bio-oil at high temperatures in an oxygen-free environment. Specific pyrolysis conditions enable a significant production of the aqueous phase of bio-oil, commonly known as wood vinegar. Wood vinegar contains organic compounds such as acetic acid and phenols derived from bio-oil. These compounds have herbicidal properties against weeds and biostimulant properties for plant growth. This study reveals the potential for efficient management of cranberry residues consisting of stems and leaves by producing wood vinegar through pyrolysis at 475 °C with a humidity level of 20%. Membrane separation of wood vinegar, using nanofiltration (NF) and reverse osmosis (RO) membranes, yielded phenols in the retentate and acetic acid in the permeate with respective yields of 44.7% with NF membrane and 45% with RO membrane. Biostimulation tests using 2% of the retentate showed significant germination rates for basil, sage, and parsley plants. Additionally, using 40 mL of the wood vinegar permeate (30 mL injected at the base and 10 mL sprayed on the leaves) resulted in leaf damage, measured by conductivity (leakage of electrolytes released by the leaves), of 62.3% and 20.5% respectively for quack grass and white clover, two weeds found in cranberry production. [ABSTRACT FROM AUTHOR]

The main purpose of this study was to assess the influence of grass planting and jujube branch mulching on soil moisture levels and jujube tree transpiration rates, with the ultimate goal of improving jujube tree production in rain-fed orchards. The study encompassed four treatments: jujube branch mulching (JBM), jujube branch mulching with white clover planting (JBM + WCP), white clover planting (WCP), and clean cultivation (CC). During a two-year experiment, it was observed that the JBM treatment exhibited the highest capacity for moisture conservation. Specifically, it resulted in an average increase of 2.69% (in 2013) and 2.23% (in 2014) in soil moisture content compared with the CC treatment. The application of statistical analysis revealed significant differences (p < 0.05) between JBM and JBM + WCP, as well as highly significant differences (p < 0.01) between JBM and WCP in the year 2013. In 2014, JBM exhibited significant differences (p < 0.01) from both JBM + WCP and WCP. Between April and August, JBM exhibited the highest soil moisture content, followed by CC, with WCP showing the lowest levels. From September to October, JBM retained its status as the treatment with the highest soil moisture content, JBM + WCP ranked second, and CC experienced a decline and recorded the lowest soil moisture content. Under sunny conditions, all treatments showed a broad peak curve in the daily variation of sap flow velocity. In cloudy weather, a multi-peak wave-like curve was observed with similar trends across treatments. Between April and August, the monthly average sap flow velocity of JBM ranked the highest, followed by CC, while WCP showed the lowest velocity. During the period of September to October, JBM maintained its lead in sap flow velocity, while JBM + WCP rose to the second position, and CC's sap flow velocity dropped to the lowest level. JBM and WCP treatments showed significant differences (p < 0.01), and in 2014, JBM also had significant differences (p < 0.05) compared with JBM + WCP. The sap flow velocity was positively correlated with air temperature, vapor pressure deficit, wind velocity, photosynthetically active radiation, and soil temperature. Photosynthetically active radiation was identified as the main driving factor influencing jujube tree transpiration. In conclusion, the findings of this study demonstrate the effectiveness of using pruned jujube branches for coverage in rain-fed jujube orchards. This approach not only conserves mulching materials and diminishes the expenses associated with transporting pruned jujube tree branches away from the jujube orchard but also achieves multiple objectives, including increasing soil moisture, promoting jujube tree transpiration, and enhancing soil water utilization. These results have significant implications for the efficient utilization of rainwater resources in rain-fed jujube orchards and provide valuable insights for practical applications in orchard management. [ABSTRACT FROM AUTHOR]

The key problem in the cultivation of densely planted dwarf orchards is the removal of weeds—trees' competitors for habitat resources. There is an urgent need to look for ecological methods of weed control as an alternative to herbicides that are harmful to the environment. The use of living mulch (LM) in tree rows additionally improves soil quality but usually weakens tree growth and may reduce yield. The aim of this 11-year experiment was to assess the impact of the use of two different LMs in rows (Trifolium repens—Tr and Agrostis capillaris—Ac) on the growth, yield, and fruit quality of three pear cultivars on Quince S1 rootstock compared to herbicide fallow. The presence of LM did not significantly affect tree growth. There was no significant effect of either mulch on the cumulative yield. However, for the first 4–6 years, the yield was clearly lower than in the control, which changed in the later years of the experiment. When LMs were used, pear trees showed a significantly lower tendency to alternate fruiting. The average fruit weight was significantly lower in Tr, but the other parameters of external fruit quality did not differ significantly. Furthermore, a smaller share of ultra-small fruit was found with LM compared to the control. The LM did not significantly affect such parameters as the content of soluble solids, vitamin C, Ca, Mg, and P. The use of Ac in dwarf pear orchards with sowing in tree rows is recommended in the 2nd or 3rd year after planting at the earliest. [ABSTRACT FROM AUTHOR]

Biochar has been used to remediate contaminated-soil with heavy metals, however, less is known on how biochar interacts with planting density and nutrient fluctuation to affect the remediation. A pot experiment was conducted in the greenhouse to investigate the effects of biochar application (without vs. with 1% biochar, g/g substrate), nutrient fluctuation (constant vs. pulsed) and planting density (1-, 3- and 6-individuals per pot) on the growth, and cadmium (Cd) and nutrient uptake of Trifolium repens population. Our results found that the growth of T. repens population increased significantly with increasing planting density, and the increment decreased with increasing planting density. Both the Cd and nutrient uptake were higher at higher planting density (e.g., 3- and 6-individuals) than at lower planting density (e.g., 1-individual). Biochar application increased the biomass and shoot Cd uptake, but decreased the ratio of root to shoot and root Cd uptake of T. repens population, the effects of which were significantly influenced by planting density. Although nutrient fluctuation had no effect on the growth of T. repens population, but its interaction with planting density had significant effects on Cd uptake in tissues. Overall, the effects of biochar application and nutrient fluctuation on the growth and Cd uptake were both influenced by planting density in the present study. Our findings highlight that biochar application and constant nutrient supply at an appropriate planting density, such as planting density of 3-individuals per pot in the present study, could promote the growth, and Cd and nutrient uptake of T. repens population. [ABSTRACT FROM AUTHOR]

Simple Summary: Grapevine red blotch virus (GRBV) and grapevine Pinot gris virus (GPGV) are two of the most concerning grapevine viruses in Canada. Currently, the secondary spreading of these viruses (spreading via an insect vector) in Canada is poorly documented. To monitor the spreading of these viruses, we deployed virus-free grapevines onto two virus-infected vineyards with different agronomic practices. One vineyard was organic while the other was conventional. Using two independent diagnostic tools, namely the polymerase chain reaction (PCR) and the high-throughput sequencing technique (HTS), we have found that GPGV is spreading in both vineyards. The spread of GPGV was noticeably faster in the organic vineyard than in the conventional one, possibly due to a higher insect population. There was no evidence of the spreading of GRBV. A small collection of plant species in and around the vineyard was also tested by the two methods above. None of the non-grape species harbored any grapevine viruses but a large portion of the wild grape found on the edge of the vineyard carried GPGV. This study is the first direct proof of insect-mediated spreading of GPGV in North America. This study investigated the transmission of grapevine viruses, specifically grapevine red blotch virus (GRBV) and grapevine Pinot gris virus (GPGV), in vineyards in Niagara Region, Ontario, Canada. Forty sentinel vines that were confirmed free of GRBV and GPGV by both high-throughput sequencing (HTS) and endpoint polymerase chain reaction (PCR) were introduced to two vineyards (one organic and one conventional) that were heavily infected with both GRBV and GPGV. Four months post-introduction, the sentinel vines were relocated to a phytotron. The HTS results from 15 months post-introduction revealed a widespread infection of GPGV among the sentinel vines but did not detect any GRBV. The GPGV infection rate of sentinel vines in the organic vineyard (13/18) was higher than in the conventional vineyard (1/19). The possibility of an alternative viral reservoir was assessed by testing the most abundant plants in between rows (Medicago sativa, Trifolium repens, Cirsium arvense and Taraxacum officinale), perennial plants in border areas (Fraxinus americana, Ulmus americana, Rhamnus cathartica) and wild grape (unknown Vitis sp.). The HTS result showed that cover crops and perennial plants did not harbor any grapevine viruses, while 4/5 wild grapes tested positive for GPGV but not GRBV. A pairwise sequence identity analysis revealed high similarities between the GPGV isolates found in the established vines on the vineyard and the newly contracted GPGV isolates in the sentinel vines, implicating a recent transmission event. This work provides novel insights into the spread of grapevine viruses in Niagara Region and is also the first direct proof of the spread of GPGV in natural vineyard conditions in North America. [ABSTRACT FROM AUTHOR]

In addition to improving soil quality, the fertilisation of grassland with cattle slurry is often associated with seed dispersal. Most studies focus on the effects of cattle slurry on the germination and early development of weed species, but less is known about how slurry affects the germination process of grasses and forage legumes. The aim of Experiment I of our study was therefore to investigate the influence of soaking time in cattle slurry of different concentrations on Lolium multiflorum, Dactylis glomerata, Trifolium pratense and Trifolium repens. Seeds were soaked in undiluted (100%) and diluted cattle slurries (50% and 25%) for 14, 28, 42, 56, 70 and 84 days. Experiment II was conducted to study the initial growth of studied plants from seeds soaked in cattle slurry of different concentrations for 14 days. After the germination test, which was carried out under controlled conditions, the germination index (GI) was calculated. The results (Experiment I) show that a short soaking in cattle slurry (14 days) has no negative effect on the germination process for all species. However, a longer soaking resulted in significantly reduced and delayed germination, especially in undiluted slurry for grasses and diluted slurries for clovers. The slurry concentration (Experiment II) only influenced the root growth of L. multiflorum. Seedlings grown from seeds soaked in undiluted slurry had a 17% higher relative root length than the control and developed significantly longer root systems than the other two slurry concentrations. [ABSTRACT FROM AUTHOR]

Abstract Mining environmental liabilities generate environmental pollution. The objective of the present study was to determine the yield of white clover (Trifolium repens) and orchard grass (Dactylis glomerata) cultivated in mining environmental passives adding black earth and compost as a substrate in the Buenaventura Julcani Huancavelica Company. The treatments were the combinations: 4: 3.1: 3.1: 3:1.1 kg of RP: RP, TN: RP, C: RP, TN, C respectively. They were distributed in four treatments with twelve experimental units for each species of leguminous and gramineous grass, we worked according to the completely randomized design (DCA) with a 2 x 4 factorial arrangement, the experimental unit being a treatment with twelve repetitions. The variables evaluated were: germination percentage (TG) and stem survival percentage (TST). For the statistical analysis, the SPSS software was used.

Symbiotic microorganisms such as arbuscular mycorrhizal fungi (AMF) and rhizobia (Rh) both promote host plant N acquisition, but whether and how the combination affects host plant nitrogen (N) assimilation remains unclear. To clarify the role of AMF and rhizobia on N assimilation in white clover (Trifolium repens), the effect of single or combined inoculation with an arbuscular mycorrhizal fungus Paraglomus occultum and a rhizobium Rhizobium trifolii on plant growth, photosynthesis, leaf N and amino acid contents, and the activities of N assimilation-related enzymes were analyzed in white clover under potted conditions. The results obtained were as follows: (1) The plant height, stolon length, leaf number, shoot biomass, total biomass, chlorophyll b and total chlorophyll contents, steady-state light quantum efficiency, and leaf N contents of white clover were significantly increased by single inoculation with Paraglomus occultum, single Rhizobium trifolii, and combined inoculations of Paraglomus occultum and Rhizobium trifolii, along with the order of combined inoculations of Paraglomus occultum and Rhizobium trifolii > single Paraglomus occultum > single Rhizobium trifolii > non-inoculation control. (2) Combined inoculations of Paraglomus occultum and Rhizobium trifolii significantly increased the contents of various amino acids including alanine, arginine, asparagine, aspartate, glutamine, glutamic acid and histidine in leaves of white clover, and distinctly improved the activities of nitroreductase, nitrite reductase, glutamine synthetase, glutamate synthetase, glutamate dehydrogenase, asparagine synthetase, and aspartate aminotransferase. In addition, the introduction of Rhizobium trifolii significantly promoted the colonization of Paraglomus occultum to roots of white clover, suggesting the partnership between the two. Therefore, it is concluded that combined inoculations of AMF and rhizobia promote N assimilation, generate more amino acids, and further promote the growth of white clover by activating the activities of N assimilation-related enzymes. It is further demonstrated that AMF and rhizobia have synergistic effects on promoting N assimilation of white clover.

Spermidine is well known to accumulate in plants exposed to drought, but the regulatory network associated with its biosynthesis and accumulation and the underlying molecular mechanisms remain unclear. Here, we demonstrated that the Trifolium repens TrMYB33 relayed the ABA signal to modulate drought-induced spermidine production by directly regulating the expression of TrSAMS1, which encodes an S-adenosylmethionine synthase. This gene was identified by transcriptome and expression analysis in T. repens. TrSAMS1 overexpression and its pTRV-VIGS-mediated silencing demonstrated that TrSAMS1 is a positive regulator of spermidine synthesis and drought tolerance. TrMYB33 was identified as an interacting candidate through yeast one-hybrid library screening with the TrSAMS1 promoter region as the bait. TrMYB33 was confirmed to bind directly to the predicted TAACCACTAACCA (the TAACCA MYB binding site is repeated twice in tandem) within the TrSAMS1 promoter and to act as a transcriptional activator. Additionally, TrMYB33 contributed to drought tolerance by regulating TrSAMS1 expression and modulating spermidine synthesis. Additionally, we found that spermidine accumulation under drought stress depended on ABA and that TrMYB33 coordinated ABA-mediated upregulation of TrSAMS1 and spermidine accumulation. This study elucidated the role of a T. repens MYB33 homolog in modulating spermidine biosynthesis. The further exploitation and functional characterization of the TrMYB33–TrSAMS1 regulatory module can enhance our understanding of the molecular mechanisms responsible for spermidine accumulation during drought stress.

ABSTRACT: This experiment investigated the variation in enteric methane production and associated gas exchange parameters, nutrient digestibility, rumen fermentation, and rumen microbiome when a range of silages based on different forage types (grass or clover), and different species within the 2 types, were fed as the sole feed to heifers. Three grass species (perennial ryegrass, festulolium, and tall fescue) and 2 clover species (red clover and white clover) were included. Perennial ryegrass was harvested at 2 maturity stages in the primary growth, white clover was harvested once in the primary growth, and 4 cuts of festulolium and tall fescue and 3 cuts of red clover were harvested during the growing season, giving 14 different silage batches in total. Sixteen Holstein heifers 16 to 21 mo old and 2 to 5 mo in pregnancy were fed the silages ad libitum as the sole feed in an incomplete crossover design. Each silage was fed to 4 heifers, except for the 2 perennial ryegrass silages, which were fed to 8 heifers; in total 64 observations. The CH4 production was measured for 3 d in respiration chambers. Heifers fed clover silages had higher dry matter intake (DMI) compared with heifers fed grass silages, and heifers fed tall fescue silages had the numerically the lowest DMI. Compared with grass silages, feeding clover silages led to higher crude protein digestibility but lower neutral detergent fiber (NDF) digestibility. Rumen pH was higher in heifers fed clover silages compared with those fed grass silages. Based on composition analysis, the rumen microbiota of the heifers clustered clearly according to forage type and species. More specifically, 7 of the 34 dominating rumen bacterial genus-level groups showed higher relative abundances for the clover silages, whereas 7 genus-level groups showed higher abundances for the grass silages. Methane yield was higher for heifers fed grass silages than for those fed clover silages when methane production was related to dry matter and digestible organic matter intake, whereas the opposite was seen when related to NDF digestion. The gross energy lost as methane (CH4 conversion factor, %) reduced from 7.5% to 6.7%, equivalent to an 11% reduction. The present study gives the outlines for choosing the optimal forage type and forage species with respect to nutrient digestibility and enteric methane emission in ruminants.

Calcium-dependent protein kinases (CDPKs) are an important class of calcium-sensitive response proteins that play an important regulatory role in response to abiotic stresses. To date, little is known about the CDPK genes in white clover. White clover is a high-quality forage grass with high protein content, but it is susceptible to cold stress. Therefore, we performed a genome-wide analysis of the CDPK gene family in white clover and identified 50 members of the CDPK genes. Phylogenetic analysis using CDPKs from the model plant Arabidopsis divided the TrCDPK genes into four groups based on their sequence similarities. Motif analysis showed that TrCDPKs within the same group had similar motif compositions. Gene duplication analysis revealed the evolution and expansion of TrCDPK genes in white clover. Meanwhile, a genetic regulatory network (GRN) containing TrCDPK genes was reconstructed, and gene ontology (GO) annotation analysis of these functional genes showed that they contribute to signal transduction, cellular response to stimuli, and biological regulation, all of which are important processes in response to abiotic stresses. To determine the function of TrCDPK genes, we analyzed the RNA-seq dataset and found that most TrCDPK genes were highly up-regulated under cold stress, particularly in the early stages of cold stress. These results were validated by qRT-PCR experiments, implying that TrCDPK genes are involved in various gene regulatory pathways in response to cold stress. Our study may help to further investigate the function of TrCDPK genes and their role in response to cold stress, which is important for understanding the molecular mechanisms of cold tolerance in white clover and improving its cold tolerance.

The use of living mulch (LM) in corn (Zea mays L.) production must be evaluated under different N fertilization levels. The objective of this study was to determine the optimum amount of N fertilization in white clover (Trifolium repens L. (WC)) LM in corn. The study was conducted in Spring Hill, TN, from 2020 to 2021. The treatments were [C-0] no LM + no N, [C-135] no LM + 135 kg/ha N, [CLM-0] LM + no N, [CLM-45] LM + 45 kg/ha N, [CLM-90] LM + 90 kg/ha N, and [CLM-135] LM + 135 kg/ha N. We evaluated the LM mass and corn production (silage and grain). In general, the LM mass was higher with CLM-45, CLM-90, and CLM-135 during both years. Corn silage production was higher in 2020 compared with 2021 (6.8 and 2.8 Ton/ha, respectively), and showed differences among treatments. The production of corn grain showed the same response pattern with a higher value in 2020 than in 2021 (1.8 and 0.1 Ton/ha, respectively). Thus, the use of LM did not increase corn silage or grain production, even receiving a high N rate as in conventional systems. [ABSTRACT FROM AUTHOR]

Rhizobial associations with leguminous plants are some of the most important symbioses on Earth, and they have economic relevance in agriculture. Because their interactions are positive and have advantages for both partners, nitrogen-fixing rhizobia also demand significant carbohydrate allocation in exchange for key nutrients, and this demand is reflected in the anatomy of roots. In the current scenario of climate change, rhizobia–legume interactions can be affected, and plants may need to compensate for carbon loss when light availability is not correct. Under such conditions, roots can modify their anatomy to accommodate symbionts' needs, and the outcome of an interaction can switch from mutualism to parasitism, resulting in changes in root allocation. We experimented with two legume species originating from well-irradiated environments (Coronilla juncea L. and Ornithopus compressus L.) and two species from shaded environments (Trifolium repens L. and Vicia sativa L.). We applied high radiation, intermediate radiation, and low radiation to two treatments of microbial inoculation (inoculation and control). After an incubation period of 105 days, we quantified the root area, size, and complexity, as well as the nodule production and mass, plant relative growth, and below-ground allocation. For plants originating in shaded environments, nodulation, root complexity, and below-ground allocation were enhanced in inoculated plants when they were transferred to conditions of high irradiance. Strikingly, plants from environments exposed to high light radiation were less plastic when exposed to changing light availability, and the symbionts were less beneficial than expected in stress-free environments. Our study proved that the stress imposed on plants due to high irradiance is overcome when plants are inoculated, and the positive effect is more evident in plants that are usually grown in shaded environments (e.g., Trifolium repens and Vicia sativa). [ABSTRACT FROM AUTHOR]

White clover (Trifolium repens L.; Fabaceae) is an important forage and cover crop in agricultural pastures around the world and is increasingly used in evolutionary ecology and genetics to understand the genetic basis of adaptation. Historically, improvements in white clover breeding practices and assessments of genetic variation in nature have been hampered by a lack of high-quality genomic resources for this species, owing in part to its high heterozygosity and allotetraploid hybrid origin. Here, we use PacBio HiFi and chromosome conformation capture (Omni-C) technologies to generate a chromosome-level, haplotype-resolved genome assembly for white clover totaling 998 Mbp (scaffold N50 = 59.3 Mbp) and 1 Gbp (scaffold N50 = 58.6 Mbp) for haplotypes 1 and 2, respectively, with each haplotype arranged into 16 chromosomes (8 per subgenome). We additionally provide a functionally annotated haploid mapping assembly (968 Mbp, scaffold N50 = 59.9 Mbp), which drastically improves on the existing reference assembly in both contiguity and assembly accuracy. We annotated 78,174 protein-coding genes, resulting in protein BUSCO completeness scores of 99.6% and 99.3% against the embryophyta_odb10 and fabales_odb10 lineage datasets, respectively. [ABSTRACT FROM AUTHOR]

White clover is a widely grown temperate legume forage with high nutritional value. Research on the functional genomics of white clover requires a stable and efficient transformation system. In this study, we successfully induced calluses from the cotyledons and leaves of 10 different white clover varieties. The results showed that the callus formation rate in the cotyledons did not vary significantly among the varieties, but the highest callus formation rate was observed in 'Koala' leaves. Subsequently, different concentrations of antioxidants and hormones were tested on the browning rate and differentiation ability of the calluses, respectively. The results showed that the browning rate was the lowest on MS supplemented with 20 mg L−1 AgNO3 and 25 mg L−1 VC, respectively, and the differentiation rate was highest on MS supplemented with 1 mg L−1 6-BA, 1 mg L−1 KT and 0.5 mg L−1 NAA. In addition, the transformation system for Agrobacterium tumefaciens-mediated transformation of 4-day-old leaves was optimized to some extent and obtained a positive callus rate of 8.9% using green fluorescent protein (GFP) as a marker gene. According to our data, by following this optimized protocol, the transformation efficiency could reach 2.38%. The results of this study will provide the foundation for regenerating multiple transgenic white clover from a single genetic background. [ABSTRACT FROM AUTHOR]

SUMMARY: Maintenance of stable mitochondrial respiratory chains could enhance adaptability to high temperature, but the potential mechanism was not elucidated clearly in plants. In this study, we identified and isolated a TrFQR1 gene encoding the flavodoxin‐like quinone reductase 1 (TrFQR1) located in mitochondria of leguminous white clover (Trifolium repens). Phylogenetic analysis indicated that amino acid sequences of FQR1 in various plant species showed a high degree of similarities. Ectopic expression of TrFQR1 protected yeast (Saccharomyces cerevisiae) from heat damage and toxic levels of benzoquinone, phenanthraquinone and hydroquinone. Transgenic Arabidopsis thaliana and white clover overexpressing TrFQR1 exhibited significantly lower oxidative damage and better photosynthetic capacity and growth than wild‐type in response to high‐temperature stress, whereas AtFQR1‐RNAi A. thaliana showed more severe oxidative damage and growth retardation under heat stress. TrFQR1‐transgenic white clover also maintained better respiratory electron transport chain than wild‐type plants, as manifested by significantly higher mitochondrial complex II and III activities, alternative oxidase activity, NAD(P)H content, and coenzyme Q10 content in response to heat stress. In addition, overexpression of TrFQR1 enhanced the accumulation of lipids including phosphatidylglycerol, monogalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and cardiolipin as important compositions of bilayers involved in dynamic membrane assembly in mitochondria or chloroplasts positively associated with heat tolerance. TrFQR1‐transgenic white clover also exhibited higher lipids saturation level and phosphatidylcholine:phosphatidylethanolamine ratio, which could be beneficial to membrane stability and integrity during a prolonged period of heat stress. The current study proves that TrFQR1 is essential for heat tolerance associated with mitochondrial respiratory chain, cellular reactive oxygen species homeostasis, and lipids remodeling in plants. TrFQR1 could be selected as a key candidate marker gene to screen heat‐tolerant genotypes or develop heat‐tolerant crops via molecular‐based breeding. [ABSTRACT FROM AUTHOR]

Red clover (Trifolium pratense L.), a key perennial pastoral species used globally, can strengthen pastural mixes to withstand increasingly disruptive weather patterns from climate change. Breeding selections can be refined for this purpose by obtaining an in-depth understanding of key functional traits. A replicated randomized complete block glasshouse pot trial was used to observe trait responses critical to plant performance under control (15% VMC), water deficit (5% VMC) and waterlogged conditions (50% VMC) in seven red clover populations and compared against white clover. Twelve morphological and physiological traits were identified as key contributors to the different plant coping mechanisms displayed. Under water deficit, the levels of all aboveground morphological traits decreased, highlighted by a 41% decrease in total dry matter and 50% decreases in both leaf number and leaf thickness compared to the control treatment. An increase in root to shoot ratio indicated a shift to prioritizing root maintenance by sacrificing shoot growth, a trait attributed to plantwater deficit tolerance. Under waterlogging, a reduction in photosynthetic activity among red clover populations reduced several morphological traits including a 30% decrease in root dry mass and total dry matter, and a 34% decrease in leaf number. The importance of root morphology for waterlogging was highlightedwith low performance of red clover: there was an 83% decrease in root dry mass compared to white clover which was able to maintain root dry mass and therefore plant performance. This study highlights the importance of germplasm evaluation across water stress extremes to identify traits for future breeding programs. [ABSTRACT FROM AUTHOR]

Legume crop production has many benefits for agricultural systems. Through the rhizodeposition process, they release a significant amount of C and N into the soil, increasing soil organic C and reducing the use of N fertilizer. Rhizodeposition is known as a dynamic process influenced by many factors.The aim of this study was to study the contribution of root exudation and root senescence to the rhizodeposition of atmospheric C and N during vegetative and reproductive growth in annual and perennial legumes and to understand how this is linked to the fixation capacities of C and N and root functional traits.An original approach that combined 13CO2 labelling and the 15N dilution method was developed to measure the rhizodeposition of atmospheric C and N throughout plant growth by two annual grain legumes (pea and faba bean) and two perennial forage legumes (white and crimson clovers).C rhizodeposition was found to increase proportionally with N rhizodeposition during reproductive development and the differences observed between species were related to the C and N fixation abilities. The use of root traits such as specific root length, root tissue density and root dry matter content suggests a strong contribution of root exudation to C rhizodeposition at vegetative growth and a strong contribution of root senescence to both C and N rhizodeposition during reproductive growth.Synthesis. Both C and N rhizodeposition appeared to be controlled by traits indicative of resource acquisition and root development. [ABSTRACT FROM AUTHOR]

This study developed an unattended electric weeder (UEW) to control floor weeds in an orchard greenhouse. The UEW was a motor-driven dolly equipped with a spark exposer. The spark exposer was constructed by applying an alternating voltage (10 kV) to a conductor net (expanded metal net). The charged conductor net (C-CN) discharged into the surrounding space. Wild oat and white clover were used as test weed species. Weed seedlings growing on the floor were grounded by the biological conductor and were subjected to a spark from the C-CN when they reached the discharge space. The spark-exposed seedlings were singed and shrunk instantaneously. In the present experiment, the UEW was remotely controlled to move on the soil-cover metal nets, which were laid on the floor to make a flat surface, in a stop-and-go manner, and to eject a spark to the weed seedlings that emerged from the floor. All of the mono- and dicotyledonous weed seedlings, which had been artificially sown on the floor, were completely eradicated using this method. Thus, this study provides an experimental basis for developing an unattended technique for controlling floor weeds in an orchard greenhouse. [ABSTRACT FROM AUTHOR]

White clover (Trifolium repens L.) is one of the legume herbages with high feed quality, but it is sensitive to water deficiency. The objectives of this study were to compare morpho-physiological responses to drought stress and post-drought recovery in four-leafed white clover cultivars. Under well-watered conditions, drought stress (3 d, 6 d, 9 d and 12 d), and rehydration, the relative water content (RWC), membrane lipid permeability, osmoregulatory substances, photosynthetic characteristics and stomatal features of Chinese native Longping No.1 (LP, small-leafed) and three introduced cultivars, namely Pudi (PD, small-leafed), Rivendel (RD, medium-leafed) and Koala (KL, large-leafed), were evaluated. Results showed that small-leafed white clover maintained higher RWC and better membrane stability and osmotic regulation with increased drought intensities, compared to the medium-leafed and large-leafed cultivars. Following rewatering, small-leafed white clover recovered more rapidly with all of the parameters than the other cultivars. The increase of drought stress intensity led to the decline of net photosynthetic rate (Pn), transpiration rate (Tr) and stomatal conductance (Cs). However, the decreased range of Pn, Tr and Cs in the small-leafed white clover was significantly smaller than that in the medium-leafed and large-leafed white clovers. Meanwhile, the small-leafed white clover showed a smaller size and higher density of stoma either under normal or drought conditions than the intermediate- and large-leafed cultivars. Taken together, the results indicated that the adaptability of small-leafed white clover to drought stresses and post-drought recovery was better than that of medium-leafed and large-leafed white clovers. The study will provide better insights into the mechanism of drought response and recovery potential in different white clover cultivars. [ABSTRACT FROM AUTHOR]