This study was designed to determine the effect of exogenous spermidine (Spd) (30 μM) on white clover seed germination under water stress induced by polyethylene glycol 6000. Use of seed priming with ...Spd improved seed germination percentage, germination vigor, germination index, root viability and length, and shortened mean germination time under different water stress conditions. Seedling fresh weight and dry weight also increased significantly in Spd-treated seeds compared with control (seeds primed with distilled water). Improved starch metabolism was considered a possible reason for this seed invigoration, since seeds primed with Spd had significantly increased α-amylase/β-amylase activities, reducing sugar, fructose and glucose content and transcript level of β-amylase gene but not transcript level of α-amylase gene. In addition, the physiological effects of exogenous Spd on improving seeds' tolerance to water deficit during germination were reflected by lower lipid peroxidation levels, better cell membrane stability and significant higher seed vigour index in seedlings. Enhanced antioxidant enzyme activities (superoxide dismutase, peroxidase, catalase and ascorbate peroxidase), ascorbate-glutathione cycle (ASC-GSH cycle) and transcript level of genes encoding antioxidant enzymes induced by exogenous Spd may be one of the critical reasons behind acquired drought tolerance through scavenging of reactive oxygen species (ROS) in water-stressed white clover seeds. The results indicate that Spd plays an important function as a stress-protective compound or physiological activator.
Abstract
The clover seed weevil, Tychius picirostris Fabricius, a serious pest of white clover, Trifolium repens L., grown for seed in western Oregon, causing feeding damage to flowers and developing ...seeds. Since 2017, white clover seed producers have anecdotally reported T. picirostris control failures using foliar pyrethroid insecticide applications. This mode of action (MoA) is an important chemical control option for T. picirostris management. To evaluate insecticide resistance selection to pyrethroids (bifenthrin) and other MoAs labeled for T. picirostris management (malathion and chlorantraniliprole), adult populations were collected from 8 commercial white clover grown for seed fields in the Willamette Valley, OR, in 2022 and 2023. Among collected Oregon populations, very high resistance ratios (RR50 = 178.00–725.67) were observed to technical grade bifenthrin and low to high resistance ratios (RR50 = 7.80–32.80) to malathion in surface contact assays compared to a susceptible Canadian field population. Moreover, >2.73 times the labeled rate of formulated product containing bifenthrin as the sole MoA was required to kill >50% of T. picirostris in topical assays. Synergistic assays with a mixed-function oxidase inhibitor, an esterase inhibitor, and a glutathione-S-transferase inhibitor revealed phase I and II detoxification enzymes are present in Oregon T. picirostris populations and confer metabolic resistance to bifenthrin. This is the first report of T. picirostris insecticide resistance selection to pyrethroid and organophosphate insecticides. Results will inform continued monitoring and insecticide resistance management strategies to slow the evolution of T. picirostris insecticide resistance selection in Oregon’s white clover seed production.
Rhizobial and arbuscular mycorrhizal (AM) symbioses each may consume 4–16% of recently photosynthetically-fixed carbon to maintain their growth, activity and reserves. Rhizobia and AM fungi improve ...plant photosynthesis through N and P acquisition, but increased nutrient uptake by these symbionts does not fully explain observed increases in the rate of photosynthesis of symbiotic plants. In this paper, we test the hypothesis that carbon sink strength of rhizobial and AM symbioses stimulates the rates of photosynthesis. Nutrient-independent effects of rhizobial and AM symbioses result in direct compensation of C costs at the source. We calculated the response ratios of photosynthesis and nutrient mass fraction in the leaves of legumes inoculated with rhizobial and/or AM fungi relative to non-inoculated plants in a number of published studies. On average, photosynthetic rates were significantly increased by 28 and 14% due to rhizobial and AM symbioses, respectively, and 51% due to dual symbiosis. The leaf P mass fraction was increased significantly by 13% due to rhizobial symbioses. Although the increases were not significant, AM symbioses increased leaf P mass fraction by 6% and dual symbioses by 41%. The leaf N mass fraction was not significantly affected by any of the rhizobial, AM and dual symbioses. The rate of photosynthesis increased substantially more than the C costs of the rhizobial and AM symbioses. The inoculation of legumes with rhizobia and/or AM fungi, which resulted in sink stimulation of photosynthesis, improved the photosynthetic nutrient use efficiency and the proportion of seed yield in relation to the total plant biomass (harvest index). Sink stimulation represent an adaptation mechanism that allows legumes to take advantage of nutrient supply from their microsymbionts without compromising the total amount of photosynthates available for plant growth.
El objetivo del presente estudio fue evaluar un análisis de crecimiento del trébol blanco (Trifolium repens L) y determinar el momento óptimo de cosecha por estación. El experimento se realizó en el ...Colegio de Postgraduados, Campus Montecillo, Texcoco, México. Se utilizaron 24 parcelas de 3.7 X 1.7 m, distribuidas en un diseño completamente al azar, con ocho tratamientos y tres repeticiones por estación. Los tratamientos consistieron en cortes semanales sucesivos, durante un ciclo de rebrote de ocho semanas, en cada estación del año. Al inicio del estudio se realizó un corte de uniformización y se determinó el forraje residual. Las variables evaluadas fueron: acumulación de materia seca, composición botánica y morfológica e índice de área foliar del trébol blanco. La mayor acumulación de forraje (P<0.05) se presentó en la octava semana en primavera (2,688 kg MS ha-1). La producción de hoja fue mayor (p < 0.05) en primavera, otoño e invierno. El mayor índice de área foliar se alcanzó en la octava semana en primavera (3.0; P< 0.05). Se recomienda aprovechar la pradera de trébol blanco en la sexta semana para primavera-verano y séptima semana en otoño e invierno.
Legumes play a crucial role in nitrogen supply to grass-legume mixtures for ruminant fodder. To quantify N transfer from legumes to neighbouring plants in multi-species grasslands we established a ...grass-legume-herb mixture on a loamy-sandy site in Denmark. White clover (Trifolium repens L.), red clover (Trifolium pratense L.) and lucerne (Medicago sativa L.) were leaf-labelled with 15N enriched urea during one growing season. N transfer to grasses (Lolium perenne L. and xfestulolium), white clover, red clover, lucerne, birdsfoot trefoil (Lotus corniculatus L.), chicory (Cichorium intybus L.), plantain (Plantago lanceolata L.), salad burnet (Sanguisorba minor L.) and caraway (Carum carvi L.) was assessed. Neighbouring plants contained greater amounts of N derived from white clover (4.8 gm−2). compared with red clover (2.2 gm−2) and lucerne (1.1 gm−2). Grasses having fibrous roots received greater amounts of N from legumes than dicotyledonous plants which generally have taproots. Slurry application mainly increased N transfer from legumes to grasses. During the growing season the three legumes transferred approximately 40 kg N ha−1 to neighbouring plants. Below-ground N transfer from legumes to neighbouring plants differed among nitrogen donors and nitrogen receivers and may depend on root characteristics and regrowth strategies of plant species in the multi-species grassland.
In addition to studying bioactive organic compounds in plants, it is increasingly important to determine the biological role of elements in plants growing in environmentally unfavorable areas. One of ...such regions in Russia is Kuzbass with its intensively developing chemical, metallurgical, and coal mining sectors. In this study, we assessed the plant materials of red clover (Trifolium pratense L.), alsike clover (Trifolium hybridum L.), and white clover (Trifolium repens L.) collected from their natural populations in Kuzbass.
The qualitative and quantitative composition of heavy metals in the clover samples was determined voltammetrically. The contents of molybdenum and phosphorus were measured by the photocolorimetric method. Total nitrogen and protein were determined by the Kjeldahl method. Nickel, cobalt, and chromium were quantified by spectrophotometry.
We analyzed the plant materials of the clover samples for heavy metals and found that the content of lead was the least in red clover and the highest in alsike clover. Copper varied in a larger range and was minimal in red clover compared to that in alsike and white clover. Zinc was found at higher concentrations of in white and red clover compared to that in alsike clover. The levels of cadmium exceeded the maximum permissible concentrations in all the clover samples. We also revealed that the clover samples contained different amounts of various amino acids, including arginine, valine, lysine, glycine, aspartic acid, and alanine.
The plant materials of the clover species growing in Kuzbass can be used to improve the fertility of soil and nitrogen regime. However, the clover species should not be used in bulk feed for farm animals because of high concentrations of cadmium.
An efficient method based on ultrafiltration high‐performance liquid chromatography coupled with photodiode array detector and electrospray ionization mass spectrometry for the rapid screening and ...identified of the ligands for activated from the extract of Trifolium repens L. Five major compounds, namely ononin, daidzein, genistein, formononetin, and biochanin A, were identified as potentially effective inhibitors. Subsequently, the specific binding ligands were separated by stepwise flow rate high‐speed countercurrent chromatography and semipreparative high‐performance liquid chromatography. This is the first report that T. repens extracts contain potent lipoxidase inhibitors. In summary, we systematically studied the active components in T. repens, evaluated their activity, separated and purified them, and identified their structure. This method is simple, fast, and efficient. It is suitable for the separation and purification of active compounds in T. repens, and provides a theoretical basis and technical platform for the development of natural medicines.
Nitric oxide (NO) is a stress-signaling molecule in plants that mediates a wide range of physiological processes and responses to metal toxicity. In this work, various NO modulators (NO donor: SNP; ...NO scavenger: cPTIO; NO synthase inhibitor: l-NAME; and SNP analogs: sodium nitrite/nitrate and sodium ferrocyanide) were investigated to determine the role of NO in Trifolium repens L. plants exposed to Cd. Cd (100μM) markedly reduced biomass, NO production and chlorophyll (Chl a, Chl b and total Chl) concentration but stimulated reactive oxygen species (ROS) and Cd accumulation in plants. SNP (50μM) substantially attenuated growth inhibition, reduced hydrogen peroxide (H2O2) and malonyldialdehyde (MDA) levels, stimulated ROS-scavenging enzymes/agents, and mitigated the H+-ATPase inhibition in proton pumps. Interestingly, SNP considerably up-regulated the levels of jasmonic acid (JA) and proline in plant tissues but down-regulated the levels of ethylene (ET) in both shoots and roots and the level of salicylic acid (SA) in roots only, which might be related to the elevated NO synthesis. Additionally, SNP (25–200μM) regulated mineral absorption and, particularly at 50μM, significantly enhanced the uptake of shoot magnesium (Mg) and copper (Cu) and of root calcium (Ca), Mg and iron (Fe). Nevertheless, the effects of SNP on plant growth were reversed by cPTIO and l-NAME, suggesting that the protective effect of SNP might be associated with NO synthesis in vivo. Moreover, SNP analogs did not display roles similar to that of SNP. These results indicated that NO depleted Cd toxicity by eliminating oxidative damage, enhancing minerals absorption, regulating proton pumps, and maintaining hormone equilibrium.
The hypothetical scheme illustration for nitric oxide functions in the regulation of the white clover plants response to metals (Cd) cytotoxicity. “?” mark indicates that the route has not yet been resolved. Dashed box in the upper left corner indicates the defense mechanism of reactive oxygen species (ROS) and antioxidants involve in the response to metals stress in plants. Plants possess several enzymes involved in NO production which are: cytosolic nitrate reductase (NR), plasma-membrane (PM)-nitrite: NO reductase (Ni: NOR), nitric oxide synthase (NOS) and xanthine dehydrogenase (XDH).
Abbreviations for enzymes or compounds/agents: AA–ascorbic acid; APX–ascorbate peroxidase; ATP–Adenosine triphosphate; Ca–calcium; CAT–catalases; Cd–cadmium; cGMP–cyclic guanosinc monophosphate; cPTIO-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidaz-oline-1-oxyl-3-oxide; Cu–copper; DHAR–dehydroascorbate reductase; ET–ethylene; Fe–iron; GPX–glutathione peroxidase; GR–glutathione reductase; GSH–glutathione; GSSG–oxidized glutathione; H2O2–hydrogen peroxide; JA–jasmonic acid; K–potassium; L-NAME–NG-nitro-L-Arg-methyl ester; MDAR–monodehydroascorbate reductase; Mg–magnesium; NADPH–triphosphopyridine nucleotide; NO–Nitric oxide; NPT–non-protein thiols; OH•–hydroxyl radical; O2•––superoxide radicals; SA–salicylic acid; SNP–sodium nitroprusside; SOD–superoxide dismutase.
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•100μM Cd markedly reduced the growth attributes of Trifolium repens L. plants.•The accumulation of endogenous NO and ROS reveled reciprocal changes.•SNP application depleted the deleterious effects of Cd on plant growth.•SNP upregulated the levels of jasmonic acid (JA) and proline in Cd treated plants.•The protective effects of SNP in plants were reversed by cPTIO and l-NAME.
Increased transcriptional levels of genes encoding antioxidant enzymes play important protective roles in coping with excessive accumulation of reactive oxygen species (ROS) in plants exposed to ...various abiotic stresses. To fully elucidate different evolutions and functions of ROS-scavenging enzymatic genes, we isolated iron superoxide dismutase (FeSOD), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) from white clover for the first time and subsequently tested dynamic expression profiles of these genes together with previously identified other antioxidant enzyme genes including copper zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD), glutathione reductase (GR), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) in response to cold, drought, salinity, cadmium stress and exogenous abscisic acid (ABA) or spermidine (Spd) treatment. The cloned fragments of FeSOD, DHAR and MDHAR genes were 630, 471 and 669 bp nucleotide sequences encoding 210, 157 and 223 amino acids, respectively. Phylogenetic analysis indicated that both amino acid and nucleotide sequences of these three genes are highly conservative. In addition, the analysis of genes expression showed the transcription of GR, POD, MDHAR, DHAR and Cu/ZnSOD were rapidly activated with relatively high abundance during cold stress. Differently, CAT, APX, FeSOD, Cu/ZnSOD and MnSOD exhibited more abundant transcripts compared to others under drought stress. Under salt stress, CAT was induced preferentially (3-12 h) compared to GR which was induced later (12-72 h). Cadmium stress mainly up-regulated Cu/ZnSOD, DHAR and MDHAR. Interestingly, most of genes expression induced by ABA or Spd happened prior to various abiotic stresses. The particular expression patterns and different response time of these genes indicated that white clover differentially activates genes encoding antioxidant enzymes to mitigate the damage of ROS during various environmental stresses.
White clover (
Trifolium repens
L.) is an important perennial legume forage with high productivity and quality. To strengthen the basic research on the genetic characteristics, fingerprint ...identification and adaptability of white clover germplasm resources, Simple sequence repeat (SSR) molecular markers were applied to 10 white clover cultivars to assess the genetic diversity and related lines of white clover at the molecular level in order to lay a theoretical foundation for the selection of high-quality seeds and cultivars of white clover. A total of 120 different bands were amplified by 29 pairs of SSR primers with good polymorphism, of which 103 (89.5%) were polymorphic. Meanwhile, the PIC of each primer was 0.181–0.588, with an average of 0.329. Analysis of molecular variance revealed that 57% of the genetic variation occurred within cultivars and 43% occurred among cultivars. The results of cluster analysis and the principal coordinate analysis revealed that the parental relationships of the 10 cultivars, with the ‘Purple’ cultivar very distantly related to the other 9 cultivars and the closest parental relationship between ‘Ladino’ and ‘Sulky’. The fingerprints constructed by three representative primers (gtrs679, gtrs319, and gtrs678) have a strong identification ability. In summary, the SSR markers had good polymorphism and could be used for DNA fingerprint analysis of white clover cultivars.
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