Impact of Environmental Factors on Seed Germination and Seedling Emergence of White Clover (Trifolium repens L.)

White clover (Trifolium repens L.) is cultivated as a forage crop and planted in various landscapes for soil conservation. There are numerous reports of failed white clover stands each year. A good understanding of the seed germination biology of white clover in relation to environmental factors is essential to achieve successful stand establishment. A series of experiments were conducted to investigate the impacts of light, temperature, planting depth, drought, and salt stress on seed germination and the emergence of white clover. White clover is negatively photoblastic, and seed germination averaged 63 and 66% under light and complete dark conditions 4 weeks after planting (WAP), respectively. Temperature affected the seed germination speed and rate. At 1 WAP, seeds incubated at 15 to 25 °C demonstrated a significantly higher germination rate than the low temperatures at 5 and 10 °C; however, the germination rate did not differ among the temperature treatments at 4 WAP. The results suggest that white clover germination decreases with increasing sowing depths, and the seeds should be sown on the soil surface or shallowly buried at a depth ≤1 cm to achieve an optimal emergence. White clover seeds exhibited high sensitivity to drought and salinity stress. The osmotic potential and NaCl concentration required to inhibit 50% seed germination were −0.19 MPa and 62.4 mM, respectively. Overall, these findings provide quantifiable explanations for inconsistent establishment observed in field conditions. The results obtained in this research can be used to develop effective planting strategies and support the successful establishment of white clover stands.

Two Agrobacterium-mediated Transformation Protocols of White Clover (Trifolium Repens) Through the Callus System

White clover (Trifolium repens) is one of the most widely cultivated livestock forage legumes co-cultivated worldwide with pasture grass in a mixed-sward setting, however, its persistence and aesthetic quality are severely affected by abiotic stresses. In this study, regeneration of white clover plants was conducted through a callus system for 4-5 months with a regeneration frequency of 36-41%. Inoculating 4-day-old cotyledons into MS media fortified with 0.4 mg·L-1 6-BA and 2 mg·L-1 2,4-D significantly increased the callus formation rate. Roots and cotyledons were better induced, followed by hypocotyls, leaves, and petioles. The development of differentiated structures performed effectively on MS supplemented with 1 mg·L-1 6-BA and 0.1 mg·L-1 NAA. Further, we determined factors affecting the Agrobacterium tumefaciens-mediated transient transformation for root-derived callus and 4-day-old cotyledons. The parameters that facilitated transient transformation were: Agrobacterium suspension density of 0.5 (OD600), 20 mg·L-1 AS, and 4-days co-cultivation duration. Subsequently, we developed two transformation protocols: transformation after callus formation in root segments (Protocol A) and transformation before callus initiation in 4-day-old cotyledons (Protocol B). The transformation frequencies varied from 1.92% to 3.17% in Protocol A and from 2.76% to 3.47% in Protocol B. We offer the possibility to regenerate multiple transgenic white clover from a single genetic background. In addition to assistance in identification of functional genes associated with yield, resistance and aesthetic quality, our research will also contribute to successful genetic manipulation and genome editing in white clover.

Condensed Tannins in White Clover (Trifolium repens) Foliar Tissues Expressing the Transcription Factor TaMYB14-1 Bind to Forage Protein and Reduce Ammonia and Methane Emissions in vitro

Grazing ruminants contribute to global climate change through enteric methane and nitrous oxide emissions. However, animal consumption of the plant polyphenolics, proanthocyanidins, or condensed tannins (CTs) can decrease both methane emissions and urine nitrogen levels, leading to reduced nitrous oxide emissions, and concomitantly increase animal health and production. CTs are largely absent in the foliage of important temperate pasture legumes, such as white clover (Trifolium repens), but found in flowers and seed coats. Attempts at enhancing levels of CT expression in white clover leaves by mutagenesis and breeding have not been successful. However, the transformation of white clover with the TaMYB14-1 transcription factor from Trifolium arvense has resulted in the production of CTs in leaves up to 1.2% of dry matter (DM). In this study, two generations of breeding elevated foliar CTs to >2% of DM. The CTs consisted predominantly of prodelphinidins (PD, 75–93%) and procyanidins (PC, 17–25%) and had a mean degree of polymerization (mDP) of approximately 10 flavan-3-ol subunits. In vitro studies showed that foliar CTs were bound to bovine serum albumin and white clover proteins at pH 6.5 and were released at pH 2.-2.5. Using rumen in vitro assays, white clover leaves containing soluble CTs of 1.6–2.4% of DM significantly reduced methane production by 19% (p ≤0.01) and ammonia production by 60% (p ≤ 0.01) relative to non-transformed wild type (WT) controls after 6 h of incubation. These results provide valuable information for further studies using CT expressing white clover leaves for bloat prevention and reduced greenhouse gas emissions in vivo.

Agricultural Greenhouse Gas Fluxes Under Different Cover Crop Systems

Cultivated lands that support high productivity have the potential to produce a large amount of GHG emissions, including carbon dioxide (CO2), nitrous oxide (N2O), and methane (CH4). Intensive land management practices can stimulate CO2, N2O, and CH4 emissions from the soil. Cover crop establishment is considered as one of the sustainable land management strategies under warm and humid environmental conditions. To better understand how the incorporation of cover crops affect three major GHGs, we compared trace gas fluxes in a no-till maize field over the whole growing season in 2018 in a no cover crop (Tr) system and three cover crop systems: crimson clover (CC), cereal rye (CR), and living mulch (LM) using white clover. In 2019, we further explored potential differences in the three GHGs between in-row (IR) and between-row (BWR) of maize for LM and Tr systems during the early growing season. Measurements were taken using a cavity ring-down spectroscopy gas analyzer in Watkinsville, GA. In 2018, the highest CO2 flux (7.00 μmol m−2 s−1) was observed from BWR of maize for LM. The maximum N2O flux observed in LM on June 20th in 2018 was when soil N increase rate was the largest. Soils served as sinks for CH4 and Tr system served as the smallest CH4 sink compared to the other three cover crop systems. For N2O, the highest fluxes were observed from the TrIR plot (4.13 μmol m−2 hr−1) in 2019 with the greatest N inputs. In 2019, we observed a smaller CH4 sink in TrIR (−0.13 μmol m−2 hr−1) compared to TrBWR (−0.67 μmol m−2 hr−1) due potentially to greater NH4+ inhibition effects on CH4 consumption from greater N fertilizer inputs. The net carbon equivalent (CE) from May 23rd to Aug 16th in 2018, taking into account the three GHG fluxes, soil carbon content, and fertilizer, irrigation, and herbicide application, were 32–97, 35–101, 63–139, and 40–106 kg ha−1 yr−1 for CC, CR, LM, and Tr, respectively. LM had the lowest net CE after removing white clover respiration (−16–60 kg ha−1 yr−1). Our results show that implementing different types of cover crop systems and especially the LM system have some potential to mitigate climate change.

γ-Aminobutyric Acid (GABA) Priming Improves Seed Germination and Seedling Stress Tolerance Associated With Enhanced Antioxidant Metabolism, DREB Expression, and Dehydrin Accumulation in White Clover Under Water Stress

As an important plant growth regulator, the role of γ-aminobutyric acid (GABA) in regulating seeds germination was less well elucidated under water stress. The present study was conducted to investigate the impact of GABA pretreatment on seeds germination of white clover (Trifolium repens) under water deficient condition. Results demonstrated that seeds pretreated with 2μmol/l GABA significantly alleviated decreases in endogenous GABA content, germination percentage, germination potential, germination index, root length, and fresh weight along with marked reduction in mean germination time after 7days of germination under drought stress. In addition, seeds priming with GABA significantly increased the accumulation of soluble sugars, non-enzymatic antioxidants [reduced ascorbate, dehydroascorbic acid, oxidized glutathione (GSSG), and reduced glutathione (GSH)], and enzymes [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathioe reductase, and monodehydroasorbate reductase (MDHR)] activities involved in antioxidant metabolism, which could be associated with significant reduction in osmotic potential and the accumulation of superoxide anion, hydrogen peroxide, electrical leakage, and malondialdehyde in seeds under drought stress. The GABA-pretreated seeds exhibited significantly higher abundance of dehydrin (DHN, 56 KDa) and expression levels of DHNs encoding genes (SK2, Y2K, Y2SK, and Dehydrin b) and transcription factors (DREB2, DREB3, DREB4, and DREB5) than the untreated seeds during germination under water-limited condition. These results indicated that the GABA regulated improvement in seeds germination associated with enhancement in osmotic adjustment, antioxidant metabolism, and DREB-related DHNs expression. Current study will provide a better insight about the GABA-regulated defense mechanism during seeds germination under water-limited condition.

Reducing Phenanthrene Contamination in Trifolium repens L. With Root-Associated Phenanthrene-Degrading Bacterium Diaphorobacter sp. Phe15

Some root-associated bacteria could degrade polycyclic aromatic hydrocarbons (PAHs) in contaminated soil; however, their dynamic distribution and performance on root surface and in inner plant tissues are still unclear. In this study, greenhouse container experiments were conducted by inoculating the phenanthrene-degrading bacterium Diaphorobacter sp. Phe15, which was isolated from root surfaces of healthy plants contaminated with PAHs, with the white clover (Trifolium repens L.) via root irrigation or seed soaking. The dynamic colonization, distribution, and performance of Phe15 in white clover were investigated. Strain Phe15 could efficiently degrade phenanthrene in shaking flasks and produce IAA and siderophore. After cultivation for 30, 40, and 50 days, it could colonize the root surface of white clover by forming aggregates and enter its inner tissues via root irrigation or seed soaking. The number of strain Phe15 colonized on the white clover root surfaces was the highest, reaching 6.03 Log CFU⋅g–1 FW, followed by that in the roots and the least in the shoots. Colonization of Phe15 significantly reduced the contents of phenanthrene in white clover; the contents of phenanthrene in Phe15-inoculated plants roots and shoots were reduced by 29.92–43.16 and 41.36–51.29%, respectively, compared with the Phe15-free treatment. The Phe15 colonization also significantly enhanced the phenanthrene removal from rhizosphere soil. The colonization and performance of strain Phe15 in white clove inoculated via root inoculation were better than seed soaking. This study provides the technical support and the resource of strains for reducing the plant PAH pollution in PAH-contaminated areas.

Changes in sward structure, plant morphology and growth of perennial ryegrass–white clover swards over winter

White clover (Trifolium repens L.) is at a disadvantage to perennial ryegrass (Lolium perenne L.; PRG) due to its limited cold tolerance and low growth rates at colder temperatures, which can affect subsequent spring herbage dry matter (DM) availability. The effect of PRG ploidy on white clover morphology and growth over winter, and its subsequent recovery in spring and the following growing season, is poorly understood. The objective of this study was to compare the effect of white clover inclusion and PRG ploidy on sward structure, plant morphology and growth of PRG–white clover swards over winter. Four swards (diploid PRG only, tetraploid PRG only, diploid PRG–white clover and tetraploid PRG–white clover) were evaluated over a full winter period (November–February) at a farmlet scale. The PRG ploidy had no effect on herbage DM production, white clover content or tissue turnover (P > 0.05) over winter. However, white clover inclusion caused a significant decrease in herbage DM production (P < 0.001; −254 kg DM/ha) and tiller density (P < 0.001; −1,953 tillers/m2) over winter. Stolon mass was not affected by PRG ploidy (P > 0.05); however, stolon length and number of leaves per stolon were affected by PRG ploidy (P < 0.05). Including white clover in PRG swards can alter winter sward dynamics, potentially causing difficulties in subsequent spring management and performance due to the reduced over-winter growth rate when compared with PRG.

Wood vinegar for control of broadleaf weeds in dormant turfgrass

Wood vinegar, a product of pyrolysis, can induce phytotoxicity on plants when applied at an adequate rate and concentration. The objective of this research was to investigate wood vinegar obtained from the pyrolysis of apple tree branches for weed control in dormant zoysiagrass. In environment-controlled growth chambers, white clover visual injury and shoot mass reduction compared to the nontreated control were evaluated after wood vinegar application at 1000, 2000, or 4000 L ha-1 under 10 or 30 C temperature conditions. Averaged across rates, wood vinegar rapidly desiccated white clover and caused 83 and 71% visual injury at 10 and 30 C, respectively, at 1 d after treatment (DAT). Averaged across temperatures, wood vinegar at 1000, 2000, and 4000 L ha-1 reduced white clover shoot mass by 56, 81, and 98% from the nontreated control at 10 DAT, respectively. In field experiments, weed control increased as wood vinegar rates increased from 1000 to 5000 L ha-1 in dormant zoysiagrass. The effective application dose of wood vinegar required to provide 90% control (ED90) of annual fleabane, Persian speedwell, and white clover was determined to be 2450, 2300, and 4020 L ha-1, respectively, at 2 weeks after treatment. Turf quality did not differ among the wood vinegar treatments and the nontreated control when zoysiagrass completely recovered from dormancy. Overall, results illustrate that wood vinegar resulting from the pyrolysis of apple tree branches can be used as a nonselective herbicide in dormant turfgrass, offering a new non-synthetic herbicide option for weed control in managed turf.

Assessing Competitiveness of Fine Fescues (Festuca L. spp.) and Tall Fescue (Schedonorus arundinaceous (Schreb.) Dumort) Established with White Clover (Trifolium repens L., WC), Daisy (Bellis perennis L.) and Yarrow (Achillea millefolium L.)

Regulatory restrictions on herbicide use for managing turfgrass weeds has prompted the search for alternative control strategies. Fescue (Festuca) species were identified for their potential to interfere with growth of annual and perennial weeds. In a study conducted in 2018 and 2019, six fescue cultivars were tested from five different species for interference with the growth of three common turfgrass weeds: white clover (Trifolium repens L., WC), daisy (Bellis perennis L.) and yarrow (Achillea millefolium L.). Fine (Festuca L. spp.) and tall fescues (Schedonorus arundinaceus (Schreb.) Dumort.) were sown and grown in a field trial for 14 days before overseeding with different weeds. vigor and visual quality of grasses, weed cover, and vegetation cover was recorded regularly for 84 days. Differences in mean temperatures and precipitation between the two years of the study resulted in differences in growth of grasses and weeds, as well as in the extent of weed interference of fescue cultivars. Cultivars Musica (F. rubra L. ssp. commutata Gaudin) and Barpearl (F. rubra L. ssp. littoralis) were least affected by weed growth during both years, but there was overlap with other cultivars for the measured parameters. Melyane (tall fescue) was deemed unsuitable for natural weed suppression because growth and vigor declined after first mowing, ultimately leading to unacceptable visual quality. Turfgrass visual scores were moderately negatively correlated to weed cover in both years. Future research should focus on F. rubra L. ssp. rubra Gaudin and F. rubra L. ssp. littoralis subspecies and identify the mechanisms used to interfere with weed growth.