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Loss of chlorophyll (Chl) is a hallmark of leaf senescence, which may be regulated by Chl catabolic genes, including NON-YELLOW COLORING 1 (NYC1)-like (NOL). The objective of this study was to determine molecular factors and metabolic pathways underlying NOL regulation of leaf senescence in perennial grass species. LpNOL was cloned from perennial ryegrass (Lolium perenne L.) and found to be highly expressed in senescent leaves. Transient overexpression of LpNOL accelerated leaf senescence and Chl b degradation in Nicotiana benthamiana. LpNOL RNA interference (NOLi) in perennial ryegrass not only significantly blocked Chl degradation in senescent leaves, but also delayed initiation and progression of leaf senescence. This study found that NOL, in addition to functioning as a Chl b reductase, could enact the functional stay-green phenotype in perennial grass species, as manifested by increased photosynthetic activities in NOLi plants. Comparative transcriptomic analysis revealed that NOL-mediated functional stay-green in perennial ryegrass was mainly achieved through the modulation of Chl catabolism, light harvesting for photosynthesis, photorespiration, cytochrome respiration, carbohydrate catabolism, oxidative detoxification, and abscisic acid biosynthesis and signaling pathways.

Background: The so-called “thunderstorm asthma” (TA) is an uncommon but dramatic outbreak of asthma attacks occurring during a thunderstorm in the pollen and moulds season. Mechanisms which make the pollen able to enter the deeper airways and provoke severe asthma symptoms are still unclear. Objective: To test the hypothesis that sub-pollen particles (SPPs) originated from the rupture by an osmotic shock of pollen associated with TA contain allergens. Methods: After hydration, SPPs released from pollen grains of grass, pellitory, olive, cypress, ragweed and birch were isolated and determined by microscopy. Allergens were determined by in vitro ELISA inhibition tests indirectly using the sera from 10 polyreactive patients. An inhibition 75% complete. Results: The inhibition experiments showed that the SPPs from birch and cypress were unable to inhibit serum IgE reactivity to Bet v 1 and Cup a 1, respectively. Ragweed SPPs inhibited ragweed pollen extract and Amb a 1 by 75.8±0.11% and 81.2±0.15%, respectively. Olive and pellitory SPPs retained almost the whole IgE-binding capability in all cases tested. Grass SPPs inhibited 32±0.06% of Lolium perenne Lol p 1 and 65% of Phleum pratense extracts, but results were highly variable for individual allergens (97.5%-0.03% for Phl p 2, 45.3±0.12% for Phl p 5, 24.7±0.22% for Phl p 6, and 38.3±0.2% for Phl p 1). Conclusions: Inhibition experiments confirm the hypothesis that SSPs obtained after the osmotic shock of pollen involved in TA, namely grass, pellitory and olive tree pollen, contain allergens and therefore they can induce severe asthma attacks during thunderstorms.

Background This study aimed to evaluate the effect of L-tryptophan (L-TRP) used in the cultivation of Lolium perenne on the yield, chemical and microbiological quality of its herbage and silage. L-TRP was applied in doses of 5 kg ha-1 , 0.5 kg ha-1 , 0.05 kg ha-1 . The experiment was conducted with a control group (C) and a comparative control group (C+) with higher nitrogen fertilization. Results The dose of 5 kg ha-1 had a significant effect on its herbage yield, which increased by 15% compared to group C and approximated the value achieved in group C+. The treatment with L-TRP caused a significant increase in water-soluble carbohydrate (WSC) content only in Tr5 (165g kg-1 DM), which was reflected in a more beneficial course of fermentation, lower pH (4.59), and a higher sum of fermentation acids, including lactic acid (94.7 g kg-1 DM). Ryegrass treatment with the high L-TRP dose effectively reduced loads of Clostridium spp. and fungi, and increased the count of Bacillus spp. The L-TRP significantly reducing N-NH3 content in Tr05 (98.6 g kg-1 TN) compared to C+ (123g kg-1 TN) and butyric acid content in Tr05 (from 1.35 g kg-1 DM in the C to 0.38 g kg-1 DM). Conclusion The most effective turned out to be dose of 5 kg ha-1 , which allowed achieving higher yield and a better fermentation course. This work presents the feasibility of using L-TRP to optimize nutrient consumption by Lolium perenne and ultimately affect the quality of its silage as a feedstuff. This article is protected by copyright. All rights reserved.

As a conserved microRNA (miRNA) family in plants, miR408 is known to be involved in different abiotic stress responses, including drought. Interestingly, some studies indicated a species- and/or cultivar-specific drought-responsive characteristic of miR408 in plant drought stress. Moreover, the functions of miR408 in perennial grass species are unknown. In this study, we investigated the role of miR408 in perennial ryegrass (Lolium perenne L.) by withholding water for 10 days for both wild type and transgenic plants with heterologous expression of rice (Oryza sativa L.) miR408 gene, Os-miR408. The results showed that transgenic perennial ryegrass plants displayed morphological changes under normal growth conditions, such as curl leaves and sunken stomata, which could be related to decreased leaf water loss. Moreover, transgenic perennial ryegrass exhibited improved drought tolerance, as demonstrated by maintaining higher leaf relative water content (RWC), lower electrolyte leakage (EL), and less lipid peroxidation compared to WT plants under drought stress. Furthermore, the transgenic plants showed higher antioxidative capacity under drought. These results showed that the improved drought tolerance in Os-miR408 transgenic plants could be due to leaf morphological changes favoring the maintenance of water status and to increased antioxidative capacity protecting against the reactive oxygen species damages under stress. These findings implied that miR408 could serve as a potential target for genetic manipulations to engineer perennial grass plants for improved water stress tolerance.

Nitrogen (N) fertilization is the major contributor to nitrous oxide (N2O) emissions from agricultural soil, especially in post‐harvest seasons. This study was carried out to investigate whether ryegrass serving as cover crop affects soil N2O emissions and denitrifier community size. A microcosm experiment was conducted with soil planted with perennial ryegrass (Lolium perenne L.) and bare soil, each with four levels of N fertilizer (0, 5, 10 and 20 g N m−2; applied as calcium ammonium nitrate). The closed‐chamber approach was used to measure soil N2O fluxes. Real‐time PCR was used to estimate the biomass of bacteria and fungi and the abundance of genes involved in denitrification in soil. The results showed that the presence of ryegrass decreased the nitrate content in soil. Cumulative N2O emissions of soil with grass were lower than in bare soil at 5 and 10 g N m−2. Fertilization levels did not affect the abundance of soil bacteria and fungi. Soil with grass showed greater abundances of bacteria and fungi, as well as microorganisms carrying narG, napA, nirK, nirS and nosZ clade I genes. It is concluded that ryegrass serving as a cover crop holds the potential to mitigate soil N2O emissions in soils with moderate or high NO3− concentrations. This highlights the importance of cover crops for the reduction of N2O emissions from soil, particularly following N fertilization. Future research should explore the full potential of ryegrass to reduce soil N2O emissions under field conditions as well as in different soils. Highlights This study was to investigate whether ryegrass serving as cover crop affects soil N2O emissions and denitrifier community size; Plant reduced soil N substrates on one side, but their root exudates stimulated denitrification on the other side; N2O emissions were lower in soil with grass than bare soil at medium fertilizer levels, and growing grass stimulated the proliferation of almost all the denitrifying bacteria except nosZ clade II; Ryegrass serving as a cover crop holds the potential to mitigate soil N2O emissions., China Scholarship Council http://dx.doi.org/10.13039/501100004543, The National Science Project for University of Anhui Province

Compared to monocultures, multi‐species swards have demonstrated numerous positive diversity effects on aboveground plant performance, such as yield, N concentration, and even legacy effects on a following crop. Whether such diversity effects are seen in the soil microbiome is currently unclear. In a field experiment, we analyzed the effect that three plant species (a grass, forb, and legume), and mixtures of these, had on soil fungal and bacterial community structures, as well as their associated legacy effects under a following crop, the grass Lolium multiflorum. We utilized six sward types, three monocultures (Lolium perenne, Cichorium intybus and Trifolium pratense), two bi‐species mixtures, and a mixture of the three species. Soil samples were taken from these swards in March (at the end of a three year conditioning phase) and in June, August, and September after L. multiflorum was established, that is, the legacy samplings. When present, the differing monocultures had a significant effect on various aspects of the fungal community: structure, OTU richness, the relative abundance of the phylum Glomeromycota, and indicator OTUs. The effect on bacterial community structure was not as strong. In the multi‐species swards, a blending of individual plant species monoculture effects (identity effect) was seen in (a) fungal and bacterial community structure and (b) fungal OTU richness and the relative abundance of the Glomeromycota. This would indicate that plant species identity, rather than diversity effects (i.e., the interactions among the plant species), was the stronger determinant. During the legacy samplings, structural patterns in the fungal and bacterial communities associated with the previous swards were retained, but the effect faded with time. These results highlight that plant species identity can be a strong driver of soil microbial community structures. They also suggest that their legacy effect on the soil microbiome may play a crucial role in following crop performance., We investigated the effect of differing plant functional types, that is, grass, forb, and legume as well as various combinations of these, and their associated legacy effects on microbial community structure using amplicon‐based metagenomics.

Several studies have reported the presence of smithsonite (ZnCO3 ) in soils polluted by zinc mining. The present study aimed to determine upper critical threshold values of Zn phytotoxicity in a substrate spiked with ZnCO3 and to compare them with those obtained in field-collected soils. We studied Zn toxicity to perennial ryegrass (Lolium perenne L.) grown in pots with unpolluted peat treated with increasing concentrations of ZnCO3 that produced nominal total Zn concentrations of 0, 0.7, 1.3, 2.0, 2.6, and 3.3%. To keep constant near-neutral pH value in all the treatments, we used decreasing concentrations of dolomitic lime. In the treatment with total soil Zn of 3.3% (pH 6.8), the foliar Zn concentration of L. perenne was 1914 ± 211 mg kg-1 , falling into the range of 2400 ± 300 mg kg-1 reported for Lolium species grown under similar laboratory conditions in a polluted soil (total soil Zn 5.4%, pH 7.3) collected near a Zn smelter. The value of 92 ± 98 mg kg-1 was obtained for the median effective concentration (EC50) values of 0.01 M KNO3 -extractable Zn using the responses of shoot dry biomass, shoot length, and total pigments. This value falls within the range of 95 ± 46 mg kg-1 reported in other studies for the EC50 values of salt-extractable Zn using field-collected soils. The application of ZnCO3 for spiking was able to mimic foliar Zn concentrations of Lolium species observed in field-collected soils. The effective concentrations of soil Zn obtained in the present study are comparable to those obtained in field-collected soils. Future research should determine effective concentrations of metals using soils spiked with metal-containing compounds that mimic a real source of contamination. Environ Toxicol Chem 2020;39:1790-1796. © 2020 SETAC.

Gramineae and Leguminosae forages are widely grown in the semiarid region of northwest China. To understand the changes in root morphology in forage seedlings under drought stress, a nursery experiment was conducted with and without water stress using four Gramineae species (Bromus inermis, Agropyron mongolicum, Lolium perenne, and Bothriochloa ischaemum) and four Leguminosae species (Lespedeza bicolor, Medicago sativa, Astragalus adsurgens and Melilotus suaveolens). The forages were grown in rhizoboxes under drought stress (15% polyethylene glycol stress) and well‐watered (85% of field capacity) conditions. The results showed that the root length, root area, and specific root length of the Gramineae species were higher than those of the Leguminosae species at the 30–60 cm soil depth. The fine roots of eight species in the 0~0.5 mm diameter class had the largest total root length and total root area, and the total root volume and root tips in the 0.5~1.0 mm diameter class were the highest. Based on the subordinate function method results, B. inermis had the highest comprehensive score, while M. suaveolens had the lowest. Our results implied that Gramineae seedlings distributed more roots than Leguminosae seedlings in the 30–60 cm soil layer under drought stress. Under drought stress, fine roots in the 0~0.5 mm and 0.5~1.0 mm diameter classes were the main organs sustaining the growth of the eight species when suffered drought stress. Among the eight species, B. inermis had the strongest drought tolerance and could act as an optimal ecological restoration forage in the semiarid region of northwest China.

Recent years have seen a decline in herbage production and tiller populations in New Zealand's perennial ryegrass (Lolium perenne) dairy pastures. One hypothesis is that modern genotypes are less suited to the warmer, drier weather experienced under changing climate patterns. In this study, a combination of long‐term trial data (2011–2017) and a process‐based pasture model (BASGRA) was used to explore the causes and possible mitigation of the observed production and population loss at three sites (dryland sites in Northland and Waikato and an irrigated site in Canterbury). Bayesian calibration was used to identify the model parameter sets that were consistent with the trial data and to identify differences in plant morphology and responses between sites. The model successfully simulated the observed differences in tiller numbers between the dryland sites, where populations and production declined rapidly after the second year and the irrigated site where populations and production were maintained. Analysis of the model calibrations along with preliminary scenario simulations suggests that increased tiller mortality associated with drought was the main cause of persistence failure at the dryland sites and that decreasing grazing pressure or breeding for tolerance to higher temperatures may not be successful in preventing this.

BACKGROUND: The research focused on the evaluation of the impact of cover cropping on trace metals (Fe, Mn, Cu, Zn, Pb, Co and Cd) and nutrients in vineyard soils and Vitis vinifera L. For this purpose, two types of cover crops (Lolium perenne L. and Medicago sativa L.) and their mixture were planted between vine rows of Muscat white in the vineyard in South Crimea. Trace elements, nutrients and other parameters were analyzed in the soil layers, leaves and grapevines of control and cover cropped plots. RESULTS: The effect of cover cropping was dependent on applied plant species. Ryegrass (L. perenne L.) seems to compete with V. vinifera L. for nutrients – these were lower in the soil and vines of the treated plot. In parallel, lead (Pb) bioconcentration in grapevines was reduced. In contrast, under lucerne (M. sativa L.), nitrogen in the soil and vines, and trace metal bioconcentration (Fe, Pb and Co) were higher. CONCLUSIONS: Our results indicate that cover cropping can influence the chemical composition of soil and vines. This should be considered when selecting cover crops. © 2019 Society of Chemical Industry

Agricultural grass cultivars bred for increased forage yield commonly have extensive root systems. As roots are an important input of organic matter into the soil, it follows that such cultivars could lead to important increases in soil organic carbon (SOC), but this, and the mechanisms involved, are poorly understood with little empirical field evidence. We assessed the effect of two cultivars, Lolium perenne L. cv. ‘AberMagic’ and L. perenne L. × Festuca pratensis Huds. cv. ‘Prior’, in comparison with mixed-species permanent pasture (‘Pasture’) in a field experiment on temperate alluvial soil in the southwest UK. Root and SOC stocks, and specific plant-derived lipids as mechanistic biomarkers for shoot and root inputs of C, were measured on intact 1-m volumetric soil cores collected on four dates in 2013-2015. Total grass yields were greater for both grass cultivars (40-43 Mg ha–1) than Pasture (39 Mg ha–1), and by 2015 root stocks in the upper 0.1 m were significantly greater by more than 4.7 Mg ha–1. At 0.1-0.3 m depth, SOC stocks (per 0.1-m depth) were significantly greater under the two grass cultivars (24-50 Mg ha–1) compared to Pasture (18-32 Mg ha–1). In the full 1-m profile, mean SOC stocks were 137, 133 and 110 Mg ha–1 for AberMagic, Prior and Pasture, respectively, matching root biomass distribution. Odd-chain n-alkane concentrations were significantly greater for shoots than roots (274 vs. 64 mg kg–1, respectively), and roots had significantly greater concentrations than shoots of even-chain hydroxy fatty acids (1829 vs. 305 mg kg–1, respectively) and diacids (317 vs. 93 mg kg–1, respectively). This confirmed lipids as candidate biomarkers for distinguishing shoot and root inputs into the soil, with the latter being the dominant source of SOC. Based on the observations of increased forage yield and SOC stock, high-yielding forage grass cultivars can contribute to future strategies for climate-smart agriculture in grassland-dominated temperate regions.

Rock flour (RF) is a fine‐grained material produced naturally by glacial movement and resulting bedrock abrasion. In Greenland fluvial transported RF from the inland ice sheet sediments in riverbeds and marine outflows. This fine‐sized RF (50% < 9.8 µm) has a high reactivity and may therefore potentially be used to rejuvenate nutrient poor soils and provide nutrients to plants. The aim of this study was to evaluate the ability of a RF from Greenland to supply P, K, Mg, and S to plants. A double‐pot system was used, in which ryegrass (Lolium perenne L.) could take up nutrients from both a hydroponic solution and a soil‐compartment with or without RF amendment; a soil mixture or pure sand was used in the soil‐compartment to estimate RF‐soil interaction effects. Omission of single nutrients from the hydroponic solution allowed assessment of which nutrients the RF in the soil‐compartment was able to supply. Ryegrass biomass was harvested four times during 62 days. We found that RF could supply K continuously to plants grown in soil or sand, but insufficient to fully circumvent K deficiency. During 62 days 5.8% and 4.3% of the applied K from RF was accumulated in the aboveground plant tissue in soil and sand, respectively. Mg was supplied from RF to plants in sand, but no significant effects were observed in soil, possibly due to background soil Mg availability. The amounts of P and S supplied to plants were insignificant. These results indicate the potential of Greenland RF to act as a slow release K and Mg fertilizer.

The formation of a compacted layer in cultivated organic soils reduces water infiltration and can lead to the formation of a perched water table. To test the hypothesis that integrating deep-rooted crops in a short-term rotation could improve soil physical properties, a 3-yr trial was conducted on two Histosols. A lettuce (Lactuca sativa L.) control and three crops combinations—corn (Zea mays L.) and common vetch (Vicia sativa L.), sorghum [Sorghum bicolor (L.) Moench] and faba bean (Vicia faba L.) , and a mix of tillage radish (Raphanus sativus L.), ryegrass (Lolium perenne L.) and crimson clover (Trifolium incarnatum L.)—were planted for 2 yr, on subsoiled and not subsoiled plots. Then, lettuce was reintroduced on all plots the third year of the experiment. Measurements of soil penetration resistance (SPR), saturated hydraulic conductivity (Kₛ and Kfₛ), water table level, and matric potential were performed over time. Kfₛ improved in the compacted layer during the second year of the rotation compared with the lettuce control. Subsoiling improved SPR, Kₛ, and Kfₛ in some cases. Once tillage was used to reestablish lettuce in the third year, those effects disappeared quickly (

Summary Epichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with the grass host Lolium perenne. Endophytic hyphae exit the host by an appressorium‐like structure known as an expressorium. In plant‐pathogenic fungi, the tetraspanin Pls1 and the NADPH oxidase component Nox2 are required for appressorium development. Previously we showed that the homologue of Nox2, NoxB, is required for E. festucae expressorium development and establishment of a mutualistic symbiotic interaction with the grass host. Here we used a reverse genetics approach to functionally characterize the role of the E. festucae homologue of Pls1, PlsA. The morphology and growth of ΔplsA in axenic culture was comparable to wild‐type. The tiller length of plants infected with ΔplsA was significantly reduced. Hyphae of ΔplsA had a proliferative pattern of growth within the leaves of L. perenne with increased colonization of the intercellular spaces and the vascular bundles. The ΔplsA mutant was also defective in expressorium development although the phenotype was not as severe as for ΔnoxB, highlighting potentially distinct roles for PlsA and NoxB in signalling through the NoxB complex. Hyphae of ΔplsA proliferate below the cuticle surface but still occasionally form an expressorium‐like structure that enables the mutant hyphae to exit the leaf to grow on the surface. These expressoria still form a septin ring‐like structure at the point of cuticle exit as found in the wild‐type strain. These results establish that E. festucae PlsA has an important, but distinct, role to NoxB in expressorium development and plant symbiosis.

Understanding the characteristics of alien species is a prerequisite for any biological study or anti‐invasion management strategy. Lolium rigidum (Poaceae) is an alien species that has become naturalized on the sandy coasts of Japan; however, it exhibits extensive morphological variation, leading to speculation that several taxa of Lolium have become naturalized. Here, we compared the morphology, reproductive biology and genetic structure of this Lolium species by growing individuals from different locations in the same environment to clarify whether the observed morphological variation is genetically based or is caused by intraspecific variation as a result of environmental differences. Principle component analyses of 11 morphological traits separated the study species into two types. Bagging experiments showed that one type exhibited an outcrossing life history, whereas the other type selfed exclusively. Nuclear DNA microsatellite analyses supported this distinction between these morphological types, with no intermediate individuals being found. The outcrossing type exhibited high genetic diversity, whereas the selfing type exhibited almost no polymorphism, reflecting the differences in their breeding systems. Moreover, both types differed from the economically important outcrossing species Lolium multiflorum and Lolium perenne. These two types of Lolium are expected to have different introduction histories and invasive potential on the coasts of Japan. In conclusion, it is important to distinguish between these types through additional biological studies to design effective control measures.

Background Within its natural habitat, Croton ehrenbergii exhibits an innate defense mechanism that is not seen in other plants; it grows unharmed amidst predators and nature, while other species perish. In light of its capacity for defense, C. ehrenbergii was evaluated to better understand the scope of its phytotoxic and nematicide properties. To assess this, fractions obtained by bipartition of a hydro-alcoholic extract, and l-quebrachitol, the main constituent of C. ehrenbergii, were evaluated on Lactuca sativa (dicotyledon) and Lolium perenne (monocotyledon) seeds. Additionally, bipartition fractions and l-quebrachitol were evaluated on a population of Meloidogyne incognita for their nematicide activity. Results From this phytochemical research, l-quebrachitol (1), phenylalanine (2), trans-4-hydroxy-N-methylproline (3) and the flavonoids: kaempferol (4), tiliroside (5), nicotiflorine (6) and rutin (7) were identified by spectroscopic analysis. Both methanol and hexane fractions from aerial parts of the plant inhibited the germination and elongation of roots and stems in L. sativa, but not in Lolium perenne, showing that these fractions mostly inhibit the dicotyledon species used in this research. l-Quebrachitol showed slightly higher seed germination inhibition for Lolium perenne in comparison with L. sativa. Three of the fractions evaluated showed nematicide activity against Meloidogyne incognita larvae (J2) at the 48 h benchmark, compared with carbofuran. l-Quebrachitol shows higher mortality after 48 h exposure at a lower concentration than carbofuran. Conclusion A variety of compounds were isolated from this research, some were common within the Croton genus (4-7), whereas others were not (1-3). This is the first phytochemical, phytotoxic and nematicide report on C. ehrenbergii. Methanol and hexane fractions from the aerial parts of C. ehrenbergii and l-quebrachitol could be used as alternative natural herbicides, predominantly against dicotyledon weed seeds. All fractions had a good mortality percentage against J2 Meloidogyne incognita larvae. © 2019 Society of Chemical Industry.

Grassed waterways are used to mitigate the offsite transport of sediment generated by soil erosion. This study used a novel trait‐based ranking approach as a method to screen potential candidate grass monocultures and mixes based on their theoretical performance in reducing (1) detachment via rainsplash, (2) detachment via scouring due to concentrated flow and (3) sediment transport and deposition processes. Selected grass species were grown under simulated UK summer and autumn establishment conditions under three different replicated rainfall scenarios: drought, normal rainfall and excess rainfall. The grass species used were the novel hybrid species Festulolium cv Prior (Fest_1), Festulolium Bx511 (Fest_2) and a conventional mixture of Lolium perenne and Festuca rubra (Conv). Monocultures and mixtures of these species were studied. Plant traits pertinent to control of soil erosion by water were measured. Above ground traits included plant height, percentage ground cover, above ground biomass, stem diameter, stem area density and number of tillers. Below ground traits included total root length, root total surface area, below ground biomass, root diameter and % fine roots ≤0.25 mm. For summer conditions, the species treatments which had the highest overall soil erosion mitigation potential were Conv, Fest_1 + 2 + Conv and Fest_2. For autumn conditions, the best treatments were Fest_1 + 2, Fest_1 + 2 + Conv and Conv. The Fest 1 + 2 + conv had more desirable traits for erosion control than mono Festulolium treatments for the autumn conditions. The conventional mixture had more desirable traits for erosion control than mono Festulolium treatments in both climate scenarios. The results indicate that the trait‐based ranking approach utilised in this study can be used to inform rapid screening of candidate grass species for soil erosion control.

Anaerobic digestate applied to land is a source of readily available nutrients, yet there is a paucity of knowledge regarding effects on grassland. To address this, we investigated the viability of using digestate as an alternative to mineral fertilizer for Lolium perenne L. grassland maintenance. We present findings of two independent field‐trials, where food‐waste digestate was applied over two growing seasons at two rates (100 and 200 kg N ha−1 year−1) and compared to mineral fertilizer (N:P:K‐12:4:6 at 100 kg N ha−1 year−1) and control (no additions) plots. L. perenne nutrition (N, P and K), chlorophyll and sward composition were assessed in the summer and autumn to observe treatment and seasonal effects. The sward benefited from digestate application in the summer with reduced occurrence of dead L. perenne. Both the digestate and mineral fertilizer shifted the sward composition similarly and in favor of Poa annua L. in summer and L. perenne in autumn, with reduced broad‐leaved weeds and bare soil coverage regardless of season. Quantities of foliar N and K uptake were similar between the digestate and mineral fertilizer; however, the highest rate of digestate application was required to supply similar quantities of P to the grass compared to the mineral fertilizer. Grass chlorophyll was not adversely affected by the high ammonium‐N in the digestate. These broadly positive results for digestate present opportunities for the development of digestate use as a fertilizer on amenity grassland such as outfields in sports facilities, parks, and road verges as well as showing potential for supplementing the fertility of pasture systems.

Increasing herbage dry‐matter (DM) production and utilization on‐farm improves efficiency and sustainability. The objective of this study was to investigate the effect of perennial ryegrass, Lolium perenne L., ploidy and white clover, Trifolium repens L., inclusion on herbage DM production and utilization in an intensive animal grazing system, with high nitrogen (N) inputs (250 kg N ha⁻¹) and a high stocking rate (2.75 livestock units ha⁻¹). The study was a 2 × 2 factorial design, consisting of diploid and tetraploid cultivars sown as grass‐only and grass‐white clover to give four sward treatments (diploid‐only, tetraploid‐only, diploid‐white clover, tetraploid‐white clover). These were evaluated at each grazing occasion (8–10 per year) for 3 years (2014–2016). Tetraploid‐white and grass‐white clover swards had a lower sward density (−9 and −8 kg DM cm⁻¹, respectively) and perennial ryegrass tiller density (−820 and −1,436 tillers m⁻², respectively) leading to lower postgrazing sward heights (−0.15 and −0.41 cm, respectively), and maintained a superior nutritive value when compared with diploid‐only and grass‐only swards respectively. White clover inclusion resulted in higher productivity characterized by increased herbage DM production (+1,468 kg DM ha⁻¹), improved nutritive value and increased utilization across the three grazing seasons. Varying sward composition with perennial ryegrass ploidy led to improved nutritive value and favourable grazing characteristics in tetraploid swards.

The objectives of this research were to establish a practicable phenotyping platform for assessing the drought stress response of perennial ryegrass (Lolium perenne L.; Lp), and to use this platform for evaluating the variation for drought tolerance among a panel of 39 diverse Lp populations. A moderate-to-strong correlation was assessed between the performance of plants grown in a hydroponics system, where the stress was generated by the addition of polyethylene glycol (PEG), and those grown in the field in a rainout shelter. Following the application of drought stress, tetraploid Lp populations, along with a small number of reference Festulolium and Festuca sp. accessions, were able to develop more shoot and root dry matter than diploid Lp populations. The onset of drought symptoms was also delayed within these accessions and the plants recovered better once drought had been relieved. Although most of the diploid Lp populations were drought susceptible, there was a considerable accession-to-accession variation for performance under drought stress conditions. Measuring biomass production and post-drought recovery in rainout shelter experiments in combination with the assessment of root biomass accumulation in PEG-supplemented hydroponics represented a viable means of screening Lp germplasm for drought tolerance.

Perennial ryegrass (Lolium perenne L.) is grown as a biennial when produced for seed in Minnesota; however, producers desire multiyear seed production. One approach to achieving multiyear seed production could be through improved management practices. In particular, plant growth regulators (PGRs) and fall residue management could help overcome several obstacles hindering multiyear seed production in northern climates. We assessed the impact of fall residue management practices and various PGRs after first-year harvest on winter survival and second-year seed yield of perennial ryegrass grown in northern Minnesota. The experiment was conducted for three trial years from 2013 to 2016 in second-year perennial ryegrass seed production swards. Three fall residue management treatments and five fall-applied PGR treatments were evaluated in a split-plot design. Fall-applied PGRs had no effect on second-year seed yield and minimal effects on winter survival. Managing residue by open-burning consistently resulted in significantly higher second-year seed yields than in the no residue removal control (1064 vs. 680 kg ha⁻¹, respectively). Open-burning and baling and mowing residue resulted in similar seed yields in 2 out of 3 yr, and both increased yields compared with no residue removal. Winter survival was only moderately affected by residue management, was highly dependent on year, and had an impact on mean seed yields across treatments. This indicates that winter survival and fall residue management are both important in maintaining multiyear seed yield in Minnesota.

Background A novel subcutaneous allergen immunotherapy formulation (gpASIT+™) containing Lolium perenne peptides (LPP) and having a short up-dosing phase has been developed to treat grass pollen–induced seasonal allergic rhinoconjunctivitis. We investigated peptide immunotherapy containing the hydrolysate from perennial ryegrass allergens for the optimum dose in terms of clinical efficacy, immunogenicity and safety. Methods This prospective, double-blind, placebo-controlled, phase IIb, parallel, four-arm, dose-finding study randomized 198 grass pollen–allergic adults to receive placebo or cumulative doses of 70, 170 or 370 μg LPP. All patients received weekly subcutaneous injections, with the active treatment groups reaching assigned doses within 2, 3 and 4 weeks, respectively. Efficacy was assessed by comparing conjunctival provocation test (CPT) reactions at baseline, after 4 weeks and after completion. Grass pollen–specific immunoglobulins were analysed before and after treatment. Results Conjunctival provocation test (CPT) response thresholds improved from baseline to V7 by at least one concentration step in 51.2% (170 μg; P = .023), 46.3% (370 μg), and 38.6% (70 μg) of patients receiving LPP vs 25.6% of patients receiving placebo (modified per-protocol set). Also, 39% of patients in the 170-μg group became nonreactive to CPT vs 18% in the placebo group. Facilitated allergen-binding assays revealed a highly significant (P < .001) dose-dependent reduction in IgE allergen binding across all treatment groups (70 μg: 17.1%; 170 μg: 18.8%; 370 μg: 26.4%). Specific IgG4 levels increased to 1.6-fold (70 μg), 3.1-fold (170 μg) and 3.9-fold (370 μg) (mPP). Conclusion Three-week immunotherapy with 170 μg LPP reduced CPT reactivity significantly and increased protective specific antibodies.

Interactions between sward diversity and forage selectivity of cattle and sheep managed within mixed grazing systems are not fully understood. The objective of this study was to investigate the effect of either mono‐ or co‐grazing of sheep and cattle on swards differing in botanical composition (either diverse or grass‐dominated) on the intake choices of six target forage species. Jacobs' selection index (JSI) was employed to quantify the preference for single target species in relation to their proportion in the sward. Results revealed distinct intake preferences of sheep and cattle; as expected, sheep were more selective than cattle. To a lesser extent, the sward composition had an effect on intake preferences: cattle responded to botanical composition by changing their intake preferences, but to a lesser extent than sheep. Phleum pratense was the most preferred target forage species (JSI = 0.62). Lolium perenne, Taraxacum sect. Ruderale and Trifolium repens were also highly preferred regardless of sward composition or type of grazing (JSI = 0.47, 0.32 and 0.27 respectively). Dactylis glomerata and Festuca pratensis tended to increase in abundance after grazing events in both diverse and grass‐dominated swards. Co‐grazing of cattle and sheep facilitated a more homogeneous consumption of the target forage species evaluated.

Summary The application of genomics in crops has the ability to significantly improve genetic gain for agriculture. Many marker-dense tools have been developed, but few have seen broad adoption in plant genomics due to issues of significant variations of genome size, levels of ploidy, single nucleotide polymorphism (SNP) frequency and reproductive habit. When combined with limited breeding activities, small research communities and scant sequence resources, the suitability of popular systems is often suboptimal and routinely fails to effectively balance cost-effectiveness and sample throughput. Genotyping-by-sequencing (GBS) encompasses a range of protocols including resequencing of the transcriptome. This study describes a skim GBS-transcriptomics (GBS-t) approach developed to be broadly applicable, cost-effective and high-throughput while still assaying a significant number of SNP loci. A range of crop species with differing levels of ploidy and degree of inbreeding/outbreeding were chosen, including perennial ryegrass, a diploid outbreeding forage grass; phalaris, a putative segmental allotetraploid outbreeding forage grass; lentil, a diploid inbreeding grain legume; and canola, an allotetraploid partially outbreeding oilseed. GBS-t was validated as a simple and largely automated, cost-effective method which generates sufficient SNPs (from 89 738 to 231 977) with acceptable levels of missing data and even genome coverage from c. 3 million sequence reads per sample. GBS-t is therefore a broadly applicable system suitable for many crops, offering advantages over other systems. The correct choice of subsequent sequence analysis software is important, and the bioinformatics process should be iterative and tailored to the specific challenges posed by ploidy variation and extent of heterozygosity.

We aimed to evaluate the effect of soya bean oil (SBO) supplementation with different forages on in vitro gas production kinetics, methane (CH4) emissions and potentially digestible neutral detergent fibre (pdNDF) digestibility (IVpdNDFD). Samples of whole-crop maize silage (MS; Zea mays), sugarcane (SC; Saccharum sp.), perennial ryegrass (RG; Lolium perenne), guinea grass (GG; Panicum maximum) and palisadegrass (PG; Brachiaria brizantha) were incubated with three concentrations of SBO (0, 30 and 60 g/kg of dry matter). The interaction between forage species and SBO inclusion affected molar proportion of acetate, acetate-to-propionate ratio, asymptotic gas and IVpdNDFD. Acetate-to-propionate ratio numerically decreased from 3.56 to 3.44 and 3.77 to 3.56 for MS and SC respectively. Soya bean oil inclusion at 60 g/kg DM decreased 21.2% and 12.9% of IVpdNDFD for MS and SC respectively. Soya bean oil increase did not affect IVpdNDFD for RG, GG and PG. Soya bean oil inclusion decreased quadratically the asymptotic gas (294–265 ml/g OM) for MS and increased linearly (275–283 ml/g OM) for GG. Lower and greater CH4 production was observed for RG and SC respectively. Methane production decreased quadratically by SBO increase. Soya bean oil inclusion linearly decreased CH4 concentration in total gas at 48 hr of incubation from 133 to 128 ml/L. In conclusion, MS and SC are more sensitive to adverse effects of SBO supplementation from 30 to 60 g/kg DM on rumen fermentation when compared to RG, GG and PG.

With a globally strong interest in bio-based products such as fuels and chemicals, a feasible source of protein for the industry with positive economic impacts could be from leaves. However, more knowledge is needed on how to improve the content of extractable protein. Grasses and legumes have a high content of protein with a favourable amino acid composition. The extractable true protein was estimated at two harvest dates in leaf and stem of the legume species white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and lucerne (Medicago sativa L.) and the grass species perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinacea L.) using the Cornell Net Carbohydrate and Protein System. Extractable true protein was more concentrated in the leaf than in the stem of all the species. Easily extractable true protein in the leaf constituted 63%–68% and 54% of crude protein in legumes and grasses respectively. If the cell wall-bound true protein in the leaf can be extracted, concentration of extractable true protein may increase by 7%–14% and 21%–26% of crude protein in legume and grass respectively. Legume leaf could be superior to grass leaf for protein production in a biorefinery due to higher extractable true protein both on a mass basis and per hectare. In white clover leaf, a significant decline in concentration of extractable true protein with maturity was simultaneous with a substantial increase in extractable true protein per hectare.

Perennial ryegrass (Lolium perenne) is an important source of protein and energy for dairy cattle. To improve the protein/energy ratio of this forage, focus is now on improving its cell wall digestibility. The in vitro assessment of the digestible fraction of the neutral detergent fibre (NDFD) is a superior method for determining the cell wall digestibility, but requires the use of ruminal fluid, which has a highly variable composition and is often not readily available. As lignin is considered the main cell wall component that impedes NDFD, we investigated whether this “subtrait” could serve as alternative breeding selection criterion to improve NDFD. Therefore, we assessed the accuracy of two lignin quantification methods: van Soest (ADL) and Klason lignin (KL). We also considered KL estimates corrected for the solubilized lignin (total lignin or TL) and non-solubilized protein (TL'). Although the latter is considered the truest possible lignin content, it was not always the most correlated to NDFD, due to the limited accuracy of protein quantification on lignin residue. TL is most correlated to NDFD and we therefore recommend it for use in conventional breeding if NDFD determination is not a possibility. However, NDFD is still a superior selection criterion, as it combines the effect of several subtraits and not just lignin. For marker–trait association studies, a more accurate estimate of lignin content is more important than a high correlation with NDFD, but also here, TL performs best.

Near-infrared reflectance spectroscopy (NIRS) has been widely used in forage quality control because it is faster, cleaner and less expensive than conventional chemical procedures. In Lolium perenne (perennial ryegrass), one of the most important forage grasses, the infection by asymptomatic Epichloe fungal endophytes alters the plant nutritional quality due to the production of alkaloids. In this research, we developed a rapid method based on NIRS to detect and quantify endophyte alkaloids (peramine, lolitrem B and ergovaline) using a heterogeneous set of L. perenne plants obtained from wild grasslands and cultivars.; Results: NIR spectra from dried grass samples were recorded and classified according to the absence or presence of alkaloids, based on reference methods. The best discriminant equations for detection of alkaloids classified correctly 94.4%, 87.5% and 92.9% of plants containing peramine, lolitrem B and ergovaline, respectively. The quantitative NIR equations obtained by modified partial least squares (MPLS) algorithm had coefficients of correlation of 0.93, 0.41, and 0.76 for peramine, lolitrem B and ergovaline respectively.; Conclusion: NIRS is a suitable tool for qualitative analysis of endophyte alkaloids in grasses and for the accurate quantification of peramine and ergovaline. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

The water-soluble carbohydrate relative to crude protein (WSC:CP) ratio in perennial ryegrass (Lolium perenne L.) has been postulated as a useful indicator of nitrogen (N) use efficiency in grazing dairy cows. A mini-sward study was conducted to quantify the effects of defoliation frequency based on leaf stage on the CP fractions and the WSC:CP ratio in perennial ryegrass. Defoliation frequency corresponded to the time taken by ryegrass to develop 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 leaves per tiller. Plant growth dynamics and plant nutritive parameters related to N use efficiency were measured. The results showed that perennial ryegrass plants that were defoliated more frequently had reduced herbage mass accumulation, leaf elongation rate and total lamina length than plants defoliated less frequently in both growth cycles. Extending the defoliation frequency from the one- to five-leaf stage resulted in a decline in WSC content, an increase in CP and buffer soluble CP and a decline in the WSC:CP ratio. Given the defoliation frequencies tested here, we concluded that plants defoliated too frequently have a potential risk of low N efficiency use by grazing animals, due to a low WSC:CP ratio. The best nutritional quality balance occurs when plants were defoliated at the 3.0-leaf stage during the first growth cycle and at the 2.0-leaf stage during the second growth cycle.

Premise of the study Genetic resources for forage crops often consist of seeds of specific species and cultivars in cold storage for future use in breeding and selection programs. Temperate grasses such as Lolium perenne, used worldwide for forage and turf, produce seeds commonly infected by hyphae of an endophytic fungus (Epichloe festucae var. lolii). This research determined whether endophytes could persist and infect seedlings of L. perenne emerging from seeds stored for over two decades. Methods Endophyte-infected seeds (>90% infected) of four cultivars were obtained in 1994 and stored dry in plastic bags at 4°C. Seed germination was tested after 12 yr (for two cultivars) and after 18 and 22 yr (for all cultivars). Seedling leaf sheaths were excised, stained, and examined at 400× for endophytic hyphae to quantify infection frequency (% plants infected) and intensity (mean number of endophytic hyphae per field of view). Key results Seed germination after 22 yr depended on cultivar, ranging from 53 to 78%. Between 58 and 73% of plants grown from seeds stored for 22 yr still contained viable endophytic hyphae. Infection intensity remained at original levels for 18 yr in one cultivar; however, in all cultivars, infection intensity declined significantly between 18 and 22 yr. Conclusions Persistence of the grass seed-endophyte symbiosis for over 20 yr surpasses all prior records of endophyte longevity within stored seeds. Storage of germplasm of cool-season grass cultivars that contain potentially beneficial fungal endophytes should be possible for several decades under dry, cold conditions.

Ruminant urine deposition onto pasture creates hot-spots where emissions of nitrous oxide (N₂O) are produced by aerobic and anaerobic microbial pathways. However, limited measurements of in situ soil oxygen (O₂)–N₂O relationships hinder the prediction of N₂O emissions from urine-affected soil. This study tested whether soil O₂ concentration or relative diffusivity of O₂ (Dₚ/DO) could explain N₂O emissions from urine patches. Using a randomized plot design, N₂O emissions were measured daily from a perennial ryegrass (Lolium perenne L.) pasture for 56 d following bovine (Bos taurus) urine deposition to an imperfectly drained silty loam soil. Soil O₂, volumetric water content, pH, conductivity, and extractable N and C were measured in urine-amended and non-amended soil. Values of water-filled pore space (WFPS) and DP/DO were modeled. When data from treatments were pooled together, daily mean DP/DO explained 73% of the total variance in mean daily N₂O flux, compared with 65

Three grazing experiments were carried out in late spring (early lactation), summer (mid-lactation) and autumn (late lactation) to compare the effects of perennial ryegrass cultivar or grass species, sown in binary or multispecies mixtures, on milk yield and nitrogen excretion of dairy cows. Replicated groups of multiparous Holstein Friesian × Jersey cows were offered either a control or high-sugar perennial ryegrass (Lolium perenne) or tall fescue (Festuca arundinacea) base grass in a binary mixture with white clover (Trifolium repens) or in a multispecies mixture with additional legumes, bromegrass (Bromus willdenowii) and forbs. During each 9-day experiment, botanical composition, milk production and faecal and urine composition were measured. Milk solid (MS) yield for the control ryegrass, high-sugar ryegrass and tall fescue grass types averaged, respectively, 1.53, 1.64 and 1.70 kg MS cow−1 day−1 for a binary mixture sward, compared with 1.65, 1.54 and 1.53 kg MS cow−1 day−1 for a multispecies sward. Legume content influenced milk production more than the number of species present in a mixture. There was lower urine N concentration from a multispecies sward compared with a binary mixture. Urine N concentration of cows grazing the control ryegrass, high-sugar ryegrass and tall fescue grass types averaged, respectively, 4.6, 5.3 and 6.8 g N L−1 for a binary mixture, compared with 4.1, 3.9 and 3.9 g N L−1 for a multispecies mixture. Feeding dairy cows on multispecies swards containing forbs presents an opportunity to reduce N losses without compromising milk yield.

Grass–legume mixtures have proven successful at improving the sustainability of grasslands. Their ability to suppress unsown species is particularly important for sainfoin (Onobrychis viciifolia), a forage crop with valuable bioactive compounds, yet low competitiveness. In this 3-year study, the suitability of six partner species cultivated in mixtures with sainfoin at different annual numbers of cuts and partner sowing densities were evaluated. Mixture yields outperformed average monoculture yields by 31%, reduced the ratio of unsown species by 65% and increased the symbiotic N2 fixation up to 158 kg ha−1. A key finding of the study was the highly differential patterns of development of the partner grass species over the 3 years. These patterns of development had a large effect on the persistency of the proportion of sainfoin in the mixtures, which varied in the third year between 17% (when grown with Dactylis glomerata) and 38% (with Lolium perenne). These findings contribute to the further development of sustainable grass–legume systems, as evidence for the successful cultivation of sainfoin as an alternative legume species can help diversification, and knowledge of mixture development supports more persistent legume proportions.

Brassinosteroids are steroidal phytohormones with a significant role in the amelioration of various stresses. The objective of this study was to determine the effect of foliar application of 24-epibrassinolide (EBL) on growth and quality of perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinaceae Schreb.) grown under salt stress. The plants were grown under non saline or saline conditions (6 dS m−1), and were sprayed with 0.00 and 0.15 mg L−1 EBL. Salt stress significantly reduced the shoot growth and turfgrass quality. However, EBL alleviated detrimental effects of the salt stress by enhancing turfgrass quality (7–26%), and increasing shoot growth and clipping yield. The EBL application under salt stress alleviated loss in clipping yield by 35 and 12%; and reduced leaf firing, through weeks 2–6 post-application, by 75–40 and 50–20% for perennial ryegrass and tall fescue, respectively. The EBL may find additional use in stress management of turfgrass areas.