Significance of honeybee pollination in increasing seed yield of Trifolium alexandrinum (Fabales: Fabaceae) and its impact on economic sustainability of smallholder farmers.

A major limitation to producing high seed yields in berseem clover (Trifolium alexandrinum L.) is failure to set seed, predominantly due to lack of pollination. Despite the importance of berseem clover as a leading forage legume, the contribution of pollinators to seed set is scarce. In Pakistan, the honeybee population is declining mainly because of the extensive use of neonicotinoid pesticides and habitat fragmentation. This, combined with the region's harsh environment and the use of inferior, locally bred genotypes, has resulted in low seed yields. Insufficient seed availability leads to limited forage supply, resulting in poor livestock nutrition, which subsequently impacts livestock health and productivity, and reduces farmers' income. The present study estimated the seed production of 3 berseem clover genotypes resulting from honeybees [Apis mellifera L. (Hymenoptera: Apidae)] pollination in 2 growing seasons (2012-2014) in the central Punjab region of Pakistan. Experiments had 2 pollination treatments (open pollination and honeybee inclusion) and 3 seed genotypes, viz. farmer own-saved, market, and the improved variety cultivars. For both growing seasons, honeybee pollination resulted in significant increases in seed yields ranging from 35% to 67%, regardless of seed genotype. With the exception of the number of seed heads/m2, all seed yield parameters also increased significantly in response to honeybee inclusion. The combination of improved variety and honeybee inclusion resulted in the production of a maximum number of seeds per head (45.3), 1,000-seed weight (3.7 g), and estimated seed yield (375.5 kg/ha). In addition, the increase in estimated net income of seed ranged from PKR 82,485 Rs/ha (US$844/ha) to PKR 168,975 Rs/ha (US$1728/ha) with the use of honeybees as an insect pollinator across all the seed genotypes. Honeybee pollination has broader implications for mixed farming systems by playing a key role in preserving biodiversity and promoting sustainable agriculture. It also enhances the quality and quantity of berseem crops by increasing the production of high-quality seeds and forage leading to improved livestock productivity and family food security which strengthens the economic resilience of rural communities.

Effect of sward species diversity combined with a reduction in nitrogen fertilizer on the performances of spring calving grazing dairy cows.

The objective of this study was to evaluate the impact of sward diversification combined with a reduction of chemical nitrogen (N) fertilizer on the performance of spring calving grazing dairy cows within a farm systems experiment. Three farmlets were created; a monoculture of perennial ryegrass (Lolium perenne L.; PRG) fertilized with 250 kg N/ha (PRG-250N), a PRG - white clover (Trifolium repens; WC) sward fertilized with 125 kg N/ha (PRGWC-125N) and a multispecies sward (MSS) comprising of grasses, legumes and herbs also fertilized with 125 kg N/ha (MSS-125N). Each farmlet had its own herd of dairy cows on a total area of 18.7 ha divided into 20 paddocks. Each herd was comprised of pure Holstein-Friesian (HF) and HF Jersey crossbred (JFX) animals and randomly assigned through the 3 treatments. For 3 years (2021 to 2023), the performances of both swards (grass yield, botanical composition, nutritive value) and grazing animals (milk production and composition, body weight (BW) and body condition score (BCS)) were recorded. There were no significant differences in pasture production or sward nutritive value between sward systems and grazing season length was also similar (264 d). On average over the 3 years, PRGWC-125N contained 150 g/kg DM of legumes and the MSS-125N contained 160 g/kg DM legumes, 130 g/kg DM plantain and 40 g/kg DM chicory. Both individual cow milk and fat plus protein (Milk solids; MS) yield were lower for PRG-250N (5,018 and 452 kg, respectively), intermediate for PRGWC-125N (5,139 and 463 kg, respectively) and highest for MSS-125N (5,297 and 476 kg, respectively) while milk and MS production per hectare from grazing were similar during the study period (11,523 and 1,016 kg/ha, respectively). Breed also had a significant effect with the JFX having lower milk yield but higher fat and protein concentration compared with HF. This resulted in higher MS production per kg of BW for the JFX compared with HF (0.96 and 0.87 kg MS/kg BW, respectively). The results of this study highlight the possibility for more diverse pastures to reduce chemical N fertilizer input requirements and maintain pasture productivity while increasing animal performance within pasture-based spring calving systems.

Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer packaging HEK-293 cell lines used for rAAV production.

In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.

The Physiological Mechanism of Exogenous Melatonin on Improving Seed Germination and the Seedling Growth of Red Clover (Trifolium pretense L.) under Salt Stress.

Salt stress can affect various physiological processes in plants, ultimately hindering their growth and development. Melatonin (MT) can effectively resist multiple abiotic stresses, improving plant stress resistance. To analyze the mechanism of exogenous MT to enhance salt tolerance in red clover, we conducted a comprehensive study to examine the influence of exogenous MT on various parameters, including seed germination indices, seedling morphological traits, and physiological and photosynthetic indicators, using four distinct red clover varieties (H1, H2, H3, and H4). This investigation was performed under various salt stress conditions with differing pH values, specifically utilizing NaCl, Na2SO4, NaHCO3, and Na2CO3 as the salt stressors. The results showed that MT solution immersion significantly improved the germination indicators of red clover seeds under salt stress. The foliar spraying of 50 µM and 25 µM MT solution significantly increased SOD activity (21-127%), POD activity, soluble sugar content, proline content (22-117%), chlorophyll content (2-66%), and the net photosynthetic rate. It reduced the MDA content (14-55%) and intercellular CO2 concentration of red clover seedlings under salt stress. Gray correlation analysis and the Mantel test further verified that MT is a key factor in enhancing seed germination and seedling growth of red clover under salt stress; the most significant improvement was observed for NaHCO3 stress. MT is demonstrated to improve the salt tolerance of red clover through a variety of mechanisms, including an increase in antioxidant enzyme activity, osmoregulation ability, and cell membrane stability. Additionally, it improves photosynthetic efficiency and plant architecture, promoting energy production, growth, and optimal resource allocation. These mechanisms function synergistically, enabling red clover to sustain normal growth and development under salt stress.

Isoflavones obtained from red clover improve both dyslipidemia and menopausal symptoms in menopausal women: a prospective randomized placebo-controlled trial.

OBJECTIVE: This study aimed to investigate the effects of red clover isoflavones on menopausal symptoms and the lipid profile in menopausal females. METHODS: This study included postmenopausal women with dyslipidemia. The red clover group (n = 39) received 40 mg isoflavone red clover capsule twice daily for 6 months, while placebo (n = 36) was 40 mg starch capsule twice daily. Data were collected at baseline, 3 months and 6 months. The Menopause Rating Scale (MRS) was applied to calculate subdimension and total scores. RESULTS: The two groups were similar in terms of age, MRS and lipid profile at baseline. In the red clover group, MRS scores decreased significantly at both 3 and 6 months. Similarly, total cholesterol, low-density lipoprotein cholesterol (LDL-C) and triglyceride levels decreased at both 3 months and 6 months. High-density lipoprotein cholesterol increased significantly from baseline to 3 months and 6 months. Except for LDL-C and MRS urogenital score at 3 months, the improvements were significantly in favor of red clover isoflavone treatment. CONCLUSIONS: Red clover treatment for 3-6 months demonstrated significant improvements in lipid profiles and menopausal symptoms. While promising, further research is crucial to ascertain long-term safety and recommend the use of red clover isoflavones during menopause.

Exploring microbial diversity and function in companion planting systems of white clover and orchard grass.

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.

Metabolomics combined with physiology reveal how white clover (Trifolium repens L.) respond to 6PPD stress.

As a ubiquitous tire antioxidant, N-(1,3-Dimethyl-butyl)-N'-phenyl-p-phenylene- diamine (6PPD) exists widely in various environmental media and has been detected at high levels in the environment. However, the effects of 6PPD on plants are still poorly understood. In this study, a hydroponic experiment was carried out to investigate the response of white clover (Trifolium repens L.) stressed by 6PPD on physiology and metabolomics. The results indicated that the length of stem and root, as well as biomass were significantly reduced after 500 µg L-1 6PPD treatment. Photosynthetic performances including photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr) and chlorophyll content of leaves decreased in all treatments except 500 µg L-1 of 6PPD. The malondialdehyde (MDA) content in the shoot of white clover increased by 66.33 % when exposed to 500 µg L-1 of 6PPD compared to control group (CK). Hydrogen peroxide and superoxide anion presented a U-shape trend and began to increase at 500 µg L-1. Besides, peroxidase and catalase significantly decreased compared to CK after exposure to 500 µg L-1. Metabolic analysis of clover showed that 6PPD treatment induced changes in 10 metabolic pathways of white clover. Metabolites were significantly down-regulated after exposure to 500 µg L-1 in shoot, while significantly down-regulated in all treatment groups except 500 µg L-1 in root. These findings may provide a novel perspective for phytotoxicity assessment and phytoremediation of 6PPD.

Optimization of process parameters for Trifolium pratense L. seed granulation coating using GA-BP neural network.

Regarding the issue of low granulation qualification rates during the granulation coating of red clover seeds, this study theoretically analyzed the force conditions of seeds and powder particles under the action of liquid to obtain the main factors affecting seed coating quality. During the seed granulation coating process, an intermittent powder supply method combined with continuous liquid supply was utilized to control the ratio of powder to liquid. Using the granulation qualification rate as the evaluation index, single-factor experiments were conducted to investigate the effects of coating pan fill ratio, single powder supply amount, powder supply interval, and liquid supply amount on the quality of red clover seed granulation coating. Based on the results of the single-factor experiments, orthogonal experiments were conducted, revealing that the interaction of factors would influence the experimental results. To further optimize the quality of seed granulation coating, the mechanisms of powder and liquid in the adhesion process on granulation coating were explored. Orthogonal experiments were conducted on the process parameters of the granulation coating machine, and the GA-BP model was employed for optimization and solution. The optimal process parameter combination obtained was a coating pan fill ratio of 33.78 %, a single powder supply amount of 5.17 g, a powder supply interval of 7.7 s, and a liquid supply amount of 0.42 mL/s. Under this optimal parameter combination, granulation coating experiments with red clover seeds were performed, and the seed granulation coating quality was relatively high, with a granulation qualification rate of 97.7 %. The research results can provide a reference for optimization experiments on coating irregular seeds.

Influence of flaxseed mucilage on the formation, composition, and properties of exopolysaccharides produced by different strains of lactic acid bacteria.

Microorganisms produce a wide variety of polysaccharides. Due to biosafety considerations, lactic acid bacteria (LAB) are popular producers of exopolysaccharides (EPS) for various applications. In this study, we analyzed the composition and properties of EPS produced by L. delbrueckii ssp. bulgaricus and LAB from clover silage (L. fermentum AG8, L. plantarum AG9) after growth on Man, Rogosa, and Sharpe broth (MRS) and with the addition of flaxseed mucilage (FSM) using chromatography, microscopy, and biochemical methods. We found that adding 0.4 % FSM does not drastically alter the medium's rheology but substantially increases EPS yield (by 3.1 to 3.8 times) and modifies the composition and macrostructure of EPS, as well as changes the spatial organization of LAB cells. The presence of FSM led to the production of xylose- and glucose-enriched EPS, which also contained varying proportions of fucose, rhamnose, arabinose, mannose, glycosamines, and uronic acids, depending on the strain. Most EPS had a low molecular weight (up to 32 kDa), except for EPS produced by L. fermentum AG8 in FSM-containing medium, which had molecular weight of 163 kDa. All EPS exhibited a porous microstructure and demonstrated scavenging capacity for OH- and DPPH-radicals, as well as high levels of α-glucosidase and lipase inhibitory activities, even at low concentrations (<1 g·L-1 of EPS). These characteristics make them promising for use in functional food production and medicine.

Alfalfa Mosaic Virus and White Clover Mosaic Virus Combined Infection Leads to Chloroplast Destruction and Alterations in Photosynthetic Characteristics of Nicotiana benthamiana.

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.

Genetics and plasticity of white leaf mark variegation in white clover (Trifolium repens L.).

BACKGROUND AND AIMS: Leaf variegation is common in plants and confers diverse adaptive functions. However, its genetic underpinnings remain largely unresolved; this is particularly true for variegation that arises through modified leaf tissue structure that affects light reflection. White clover is naturally polymorphic for structure-based white leaf mark variegation. It therefore provides a useful system to examine the genetic basis of this phenotype, and to assess potential costs to photosynthetic efficiency resulting from modified leaf structures. This study sought to map the loci controlling the white leaf mark in white clover and evaluate the relationship between white leaf mark, leaf thickness, and photosynthetic efficiency. METHODS: We generated a high-density genetic linkage map from an F3 mapping population, employing reference genome-based SNP markers. White leaf mark was quantified through detailed phenotypic evaluations alongside leaf thickness to test how tissue thickness may affect the variegation phenotype. Quantitative trait locus (QTL) mapping was performed to characterize their genetic bases. Photosynthetic efficiency measurements were used to test for physiological trade-offs between variegation and photosynthetic output. KEY RESULTS: The V locus, a major gene responsible for the white leaf mark polymorphism, was mapped to the distal end of chromosome 5, and several modifier loci were also mapped that contribute additively to variegation intensity. The presence and intensity of white leaf mark was associated with greater leaf thickness; however, increased variegation did not detectably affect photosynthetic efficiency. CONCLUSIONS: We have successfully mapped the major locus governing the white leaf mark in white clover, along with several modifier loci, revealing a complex basis for this structure-based variegation. The apparent absence of compromised photosynthesis in variegated leaves challenges the notion that variegation creates fitness trade-offs between photosynthetic efficiency and other adaptive functions. This finding suggests that other factors may maintain the white leaf mark polymorphism in white clover.

De novo genome assembly of white clover (Trifolium repens L.) reveals the role of copy number variation in rapid environmental adaptation.

BACKGROUND: White clover (Trifolium repens) is a globally important perennial forage legume. This species also serves as an eco-evolutionary model system for studying within-species chemical defense variation; it features a well-studied polymorphism for cyanogenesis (HCN release following tissue damage), with higher frequencies of cyanogenic plants favored in warmer locations worldwide. Using a newly generated haplotype-resolved genome and two other long-read assemblies, we tested the hypothesis that copy number variants (CNVs) at cyanogenesis genes play a role in the ability of white clover to rapidly adapt to local environments. We also examined questions on subgenome evolution in this recently evolved allotetraploid species and on chromosomal rearrangements in the broader IRLC legume clade. RESULTS: Integration of PacBio HiFi, Omni-C, Illumina, and linkage map data yielded a completely de novo genome assembly for white clover (created without a priori sequence assignment to subgenomes). We find that white clover has undergone extensive transposon diversification since its origin but otherwise shows highly conserved genome organization and composition with its diploid progenitors. Unlike some other clover species, its chromosomal structure is conserved with other IRLC legumes. We further find extensive evidence of CNVs at the major cyanogenesis loci; these contribute to quantitative variation in the cyanogenic phenotype and to local adaptation across wild North American populations. CONCLUSIONS: This work provides a case study documenting the role of CNVs in local adaptation in a plant species, and it highlights the value of pan-genome data for identifying contributions of structural variants to adaptation in nature.

Functional characterization of TrGSTF15, a glutathione S-transferase gene family member, on the transport and accumulation of anthocyanins and proanthocyanidins in Trifolium repens.

Anthocyanins and proanthocyanidins (PAs) are important secondary metabolites in plants, high contents of which are an important goal for quality breeding of white clover (Trifolium repens). However, the involvement of glutathione S-transferase (GST) in the transport of anthocyanins and PAs remains unexplored in white clover. This study identified 153 different TrGSTs in white clover. At the transcriptional level, compared to other TrGSTFs, TrGSTF10 and TrGSTF15 are highly expressed in the 'Purple' white clover, and they may work with the anthocyanin biosynthesis structural genes CHS and CHI to contribute to pigment buildup in white clover. Subcellular localization confirmed that TrGSTF10 and TrGSTF15 are located in the cytoplasm. Additionally, molecular docking experiments showed that TrGSTF10 and TrGSTF15 have similar binding affinity with two flavonoid monomers. Overexpression of TrGSTF15 complemented the deficiency of anthocyanin coloring and PA accumulation in the Arabidopsis tt19 mutant. The initial findings of this research indicate that TrGSTF15 encodes an important transporter of anthocyanin and PA in white clover, thus providing a new perspective for the further exploration of related transport and regulatory mechanisms.

Modified Clover Technique Using Automated Suture Placement and Securing Technology in a Passive Beating Heart Model.

Tricuspid regurgitation (TR) is a prevalent finding in echocardiography and in case of severe disease is associated with impaired patient outcome. Clover repair offers a surgical solution that can be applied for the treatment of primary and secondary TR. An ex vivo passive beating porcine heart model was created to test a modified clover technique using automated suturing devices and to compare this approach to standard ring annuloplasty. Secondary TR was induced in 10 porcine hearts and the backflow of fluid was assessed. The primary endpoint of this study was regurgitant volume measured in mL at the site right atrial cannula. The baseline regurgitation was 43.3 ± 10.8 mL. The mean regurgitant volume was significantly reduced after all repair procedures to 22.2 ± 5.9 mL with isolated ring annuloplasty, 12 ± 3.9 mL with the modified clover, and 7.6 ± 3.4 mL with the combined procedure (p < 0.0001). The modified clover technique shows how to effectively reduce TR in an ex vivo model. This method may be suitable to facilitate tricuspid repair, especially for totally endoscopic valve surgery.

Composition of Proteins Associated with Red Clover (Trifolium pratense) and the Microbiota Identified in Honey.

The nutritional composition of honey is determined by environmental conditions, and botanical and geographical origin. In addition to carbohydrates, honey also contain pollen grains, proteins, free amino acids, and minerals. Although the content of proteins in honey is low, they are an important component that confirms the authenticity and quality of honey; therefore, they became a popular study object. The aim of the study was to evaluate protein content and composition of monofloral red clover and rapeseed honey collected from five different districts of Lithuania. Forty-eight proteins were identified in five different origin honey samples by liquid chromatography. The number of red clover proteins identified in individual honey samples in monofloral red clover honey C3 was 39 in polyfloral honey S22-36, while in monofloral rapeseed honey S5, S15, and S23 there was 33, 32, and 40 respectively. Aphids' proteins and lactic acid bacteria were identified in all honey samples tested. The linear relationship and the strongest correlation coefficient (r = 0.97) were determined between the content of Apilactobacillus kunkeei and Apilactobacillus apinorum, as well as between the number of faba bean (Vicia faba) pollen and lactic acid bacteria (r = 0.943). The data show a strong correlation coefficient between the amount of lactic acid and aphid protein number (r = 0.693). More studies are needed to evaluate the relationship between the pollination efficiency of red clover by bees and the multiplicity of red clover proteins in honey protein, as well as microbiota diversity and the influence of nature or plant diversity on the occurrence of microbiota in honey.

The White Clover TrMYB33-TrSAMS1 Module Contributes to Drought Tolerance by Modulation of Spermidine Biosynthesis via an ABA-Dependent Pathway.

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.

Optimizing forage harvest and the nutritive value of Italian ryegrass-based mixed forage cropping under northwestern Himalayan conditions.

The scarcity of high-quality forage has a significant influence on the productivity and profitability of livestock. Addressing this concern, an investigation was undertaken to assess the effects of distinct Italian ryegrass genotypes, namely, Punjab ryegrass-1, Kashmir collection, and Makhan grass, in conjunction with varying seeding ratios of Italian ryegrass to Egyptian clover. The seeding ratios considered were 100:0 (Italian ryegrass to Egyptian clover), 75:25, 50:50, and 25:75. All possible combinations of Italian ryegrass and Egyptian clover with seeding ratios were set up in a randomized complete block design and replicated thrice. Co-cultivating Italian ryegrass and Egyptian clover at a 75:25 seeding ratio yields the best yield benefit, as determined by the land equivalent ratio. It is noteworthy that in this configuration, real yield loss is higher for Egyptian clover and for Italian ryegrass when the seeding ratio is 25:75. The higher competitiveness of Italian ryegrass in comparison to Egyptian clover is highlighted by the competitive ratio. Notably, the nutritive parameter, crude protein yield, was significantly higher in the Makhan grass-based 50:50 and 75:25 seeding ratio. Results of the study ascertained the compatibility of grass-legume co-cultivation with significantly higher quantity and quality forage harvested under mixed cropping systems whereas Makhan grass as the superior and dominant genotype in comparison to Kashmir collection. The outcomes of this study revealed that the 100:0 seeding ratio, coupled with the Makhan grass genotype, exhibited superior performance in terms of cumulative forage harvest, dry matter accumulation, net returns, and benefit-cost ratio.

Investigating the involvement of the NLRP3/ASC/caspase-1 and NF-κb/MAPK pathways in the pathogenesis of gouty arthritis: Insights from irradiated and non-irradiated Trifolium alexandrium L. extracts and some metabolites.

ETHNOPHARMACOLOGICAL RELEVANCE: Trifolium alexandrinum L. (TA), has traditionally been used in folk medicine for its anti-inflammatory properties against hyperuricemia and gout. However, the specific mechanisms of action of TA have not been thoroughly studied. AIM OF THE WORK: This study aimed to evaluate the protective effects of irradiated (TR25) and non-irradiated (TR0) Trifolium alexandrinum L. aqueous extract (TAAE), along with two isolated compounds, caffeine (CAF) and saponin (SAP), in a rat model of acute gouty arthritis (GA). MATERIALS AND METHODS: The GA model was established by injecting a monosodium urate (MSU) suspension into the knee joint. Synovial tissue pathology was assessed, and levels of TNF-α, IL-6, IL-1ß, NF-κB, mTOR, AKT1, PI3K, NLRP3, and ASC were measured by ELISA. mRNA expression of ERK1, JNK, and p-38 MAPK was detected using qRT-PCR, and Caspase-1 protein expression was assessed by immunohistochemical analysis. Knee swelling, uric acid levels, liver and kidney function, and oxidative stress markers were also evaluated. RESULTS: TAAE analysis identified 170 compounds, with 73 successfully identified using LC-HR-MS/MS, including caffeine citrate and theasapogenol B glycoside as the main constituents. The studied materials demonstrated significant protective effects against GA. TR25 administration significantly mitigated knee joint circumference compared to other treatments. It demonstrated potential in alleviating hyperuricemia, renal and hepatic impairments induced by MSU crystals. TR25 also alleviated oxidative stress and reduced levels of IL1ß, IL-6, TNF-α, and NF-κB. Weak Caspase-1 immune-positive staining was observed in the TR25 group. TR25 decreased NLRP3 and ASC expression, reducing inflammatory cytokine levels in GA. It effectively inhibited the PI3K, AKT, and mTOR signaling pathways, promoting autophagy. Additionally, TR25 suppressed ERK1, JNK, and p-38 MAPK gene expression in synovial tissue. These effects were attributed to various components in TAAE, such as flavonoids, phenolic acids, tannins, alkaloids, and triterpenes. CONCLUSION: Importantly, irradiation (25 KGy) enhanced the antioxidant effects and phtchemical contents of TAAE. Additionally, TR0, TR25, CAF, and SAP exhibited promising protective effects against GA, suggesting their therapeutic potential for managing this condition. These effects were likely mediated through modulation of the NLRP3/ASC/Caspase-1 and ERK/JNK/p-38 MAPK signaling pathways, as well as regulation of the PI3K/AKT/mTOR pathway. Further research is warranted to fully elucidate the underlying mechanisms and optimize their clinical applications.

Evaluating foliar insecticides and economic thresholds for Tychius picirostris (Coleoptera: Curculionidae) management in Oregon white clover seed production.

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.

Beyond cyanogenesis: Temperature gradients drive environmental adaptation in North American white clover (Trifolium repens L.).

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.