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Rhizosphere microorganisms are crucial for enhancing plant stress resistance. Current studies have shown that Arbuscular mycorrhizal fungi (AMF) can facilitate vegetation recovery in heavy metal-contaminated soils through interactions with rhizosphere microbiota. However, the mechanisms by which AMF influences rhizosphere microbiota and plant growth under cadmium (Cd) stress remain unclear. In this study, Lolium perenne L. was inoculated with AMF ( Rhizophagus irregularis ) and grown in soils supplemented with Cd (0 mg kg -1 , Cd0; 100 mg kg -1 , Cd100). Plant biomass, antioxidant enzyme activities, peroxide content, Cd uptake, and rhizosphere bacterial community composition were evaluated. AMF inoculation reduced Cd influx in aboveground tissues, enhanced nutrient availability in the rhizosphere, and mitigated Cd biotoxicity. Additionally, AMF inoculation improved the scavenging efficiency of reactive oxygen species and alleviated oxidative stress in L. perenne , thereby mitigating biomass reduction. Moreover, AMF treatment increased leaf and root biomass by 342.94% and 41.31%, respectively. Furthermore, under the same Cd concentration, AMF inoculation increased bacterial diversity (as measured by the Shannon index) and reduced bacterial enrichment (as indicated by the ACE index). AMF promoted the enrichment of certain bacterial genera (e.g., Proteobacteria and Actinobacteria ) in the Cd100 group. These findings suggest that AMF regulated the composition of the rhizosphere bacterial community and promoted the growth of potentially beneficial microorganisms, thereby enhancing the resistance of L. perenne to Cd stress. Cd contamination in soil severely limits plant growth and threatens ecosystem stability, highlighting the need to understand how AMF and rhizosphere microbes can enhance Cd tolerance in L. perenne . Therefore, inoculating plants with AMF is a promising strategy for enhancing their adaptability to Cd-contaminated soils.

We conducted a four-year cutting experiment on herbage yield, with three years focused on testing the effect of perennial ryegrass (PR) cultivars 'Elena DS', 'Raminta', and 'Verseka', along with a cultivar mixture and compositions with white clover (WC) and red clover (RC) diversity, on crude protein (CP), modified acid detergent fibre (MADF), neutral detergent fibre (NDF), water-soluble carbohydrates (WSC), and dry matter digestibility (DMD) content. PR cultivars and cultivar mixtures were sown alone (N 150 ), and the PR 'Elena DS' and PR cultivar mixtures were also sown with each WC, or WC and RC (N 0 ). The average four-year herbage productivity was the lowest in PR 'Elena DS' the and cultivar mixture/WC, followed by all of the pure PR swards, and the highest in PR 'Elena DS' and the cultivar mixture/WC+RC; however, the PR 'Elena DS'/WC+RC mixture had the highest legume proportion and CP content. There was less NDF but also WSC in swards with legumes, but higher CP content than in pure PR swards; however, the highest CP content was in mixtures with RC. Among the grasses, PR 'Verseka' had lower NDF contents and a higher WSC than PR 'Elena DS', 'Raminta', and the cultivar mixture. Overall, this study revealed significant differences in the productivity of PR cultivars with a potential yield difference of up to 1.7 t ha -1 .

In traditional ryegrass/white clover ( Lolium perenne L./ Trifolium repens L.) pastoral systems, forage herbs such as chicory ( Cichorium intybus L.) present an opportunity to fill feed deficits during late spring and summer. Although multiple research publications have evaluated the efficacy of chicory for enhancing milk production and milk fatty acid (FA) profile, no publication has quantitatively synthesised the body of research. This systematic review and meta-analysis examined the effect of chicory on milk production and composition, as well as on the milk fatty acid composition of dairy cattle. A total of 29 comparisons from 15 unique research publications involving 597 dairy cattle were used to develop a dataset for analysis. Three-level random-effect and robust variance estimator models were used to account for the hierarchical structure of the data and the dependency of effect sizes within publications. Chicory inclusion increased milk yield when compared to grass-based diets {weighted mean difference (WMD) = 1.07 (95% CI 0.54-1.60) kg/cow/d, p < 0.001}, but it provided a similar milk yield when compared to other forages such as legumes and herbs {dicots; WMD = -0.30, (95% CI -89-0.29) kg/cow/day, p = 0.312}. Increases in milk yield were congruent with differences in DM intake ( p = 0.09) and ME intakes ( p = 0.003), being similar in chicory-fed and dicot-fed cows but higher than grass-fed cows. Chicory feeding's effect on milk solids was twice as high during mid lactation {154 days in milk; WMD = 0.13, (95% 0.081-0.175) kg/cow/day, p < 0.001} as during late lactation {219 days in milk; WMD = 0.06, (95% 0.003-0.13) kg/cow/day, p = 0.041}. In line with milk yield, greater and more significant effect sizes were found for alpha linolenic acid {ALA; WMD = 0.20 (95% CI 0.06-0.35) g/100 g FA, p = 0.011} when chicory was compared to grass species only. Comparing chicory with dicots suggests that chicory inclusion did not impact ALA concentrations {WMD = 0.001 (95% CI -0.02-0.2) g/100 g FA, p = 0.99}. There were no differences in conjugated linoleic acid concentration in the milk of cows fed chicory or control diets. The study provides empirical evidence of chicory's efficacy for improved milk production and milk fatty acid composition.

Water-use efficiency (WUE) is an under-researched but very important drought tolerance trait in forage breeding. This research estimated quantitative genetic parameters of morpho-physiological traits linked to agronomic water-use efficiency (WUE A ) and its proxy measures based on δ 13 C (WUE i ) or gas exchange (evapotranspiration, WUE AET , or stomatal conductance WUE ASC ) of genotypes from half-sib families of Lolium perenne L. (PRG) in a simulated summer drought cycle. Principal component analysis (PCA) of trait data distinguished a group of PRG genotypes where high WUE A and dry matter yield was associated with deep rooting, leaf hydration at more negative leaf osmotic and water potential, and reduced soil moisture depletion. Plants with this trait association sustained net assimilation and postdefoliation regrowth in drought. However, WUEi, WUE ASC , and WUE AET were poorly correlated with most traits of interest at p  < .05. Another PCA revealed a weak association between WUE A and its proxy measures under conditions tested. Quantitative genetic parameters including high estimates of narrow-sense heritability ( h n 2 > 0.7 ; p < .05 ) of WUE A and related traits emphasized the genetic potential of the key trait combination for selecting PRG for improved drought tolerance. Research findings highlight the relative importance of WUE A and its proxy measures in the broad definition of PRG drought tolerance for breeding purposes., Competing Interests: No potential conflict of interest was reported by the authors., (© 2023 The Authors. Plant‐Environment Interactions published by New Phytologist Foundation and John Wiley & Sons Ltd.)

The contamination of soil and water by metals such as mercury (Hg) and cadmium (Cd) has been increasing in recent years, because of anthropogenic activities such as mining and agriculture, respectively. In this work, the changes in the rhizosphere microbiome of Lolium perenne L. during the phytoremediation of soils contaminated with Hg and Cd were evaluated. For this, two soil types were sampled, one inoculated with mycorrhizae and one without. The soils were contaminated with Hg and Cd, and L. perenne seeds were sown and harvested after 30 days. To assess changes in the microbiome, DNA isolation tests were performed, for which samples were subjected to two-step PCR amplification with specific 16S rDNA V3-V4 primers (337F and 805R). With mycorrhizae, changes had been found in the absorption processes of metals and a new distribution. While with respect to microorganisms, families such as the Enterobacteriaceae have been shown to have biosorption and efflux effects on metals such as Hg and Cd. Mycorrhizae then improve the efficiency of removal and allow the plant to better distribute the absorbed concentrations. Overall, L. perenne is a species with a high potential for phytoremediation of Cd- and Hg-contaminated soils in the tropics. Inoculation with mycorrhizae modifies the phytoremediation mechanisms of the plant and the composition of microorganisms in the rhizosphere. Mycorrhizal inoculation and changes in the microbiome were associated with increased plant tolerance to Cd and Hg. Microorganism-assisted phytoremediation is an appropriate alternative for L. perenne., (© 2023. The Author(s).)

With developments in industry, petroleum and heavy metal pollution are increasingly affecting soil, significantly harming the environment, biosecurity, and human health. Therefore, the remediation of contaminated soil is becoming increasingly important. In this study, ryegrass (Lolium perenne L.) was planted in petroleum-heavy metal co-contaminated soil with the application of nitrogen and phosphorus fertilizers. Three treatments were set up: uncontaminated soil + ryegrass (SH); petroleum-heavy metal co-contaminated soil + ryegrass (SPGH); and petroleum-heavy metal co-contaminated soil + ryegrass + nitrogen and phosphorus fertilizer (SPGH + NP). The results showed that the petroleum-heavy metal co-contamination promoted increases in the root length, surface area, volume, and diameter of ryegrass roots, increasing the below-ground biomass and decreasing the photosynthetic pigment content in the early stages of the experiment. The ratios of chlorophyll a/b and chlorophyll/carotenoid also increased. However, the application of fertilizer reduced the length, surface area, volume, and diameter of ryegrass roots in the co-contaminated soil, and the below-ground biomass decreased while the above-ground biomass increased. Furthermore, the photosynthetic pigment content was significantly higher than that in the unfertilized treatment and the chlorophyll a/b ratio decreased while the chlorophyll/carotenoid ratio increased. Therefore, fertilizers could alleviate the toxic effects of petroleum-heavy metal combined pollution on ryegrass roots and promote the synthesis of chlorophyll and other pigments, thus reducing the inhibitory effect of petroleum-heavy metal combined pollution on ryegrass growth and facilitating the remediation of the polluted soil., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Perennial ryegrass ( Lolium perenne L.) is an important turfgrass and gramineous forage widely grown in temperate regions around the world. However, its perennial nature leads to the inevitable exposure of perennial ryegrass to various environmental stresses on a seasonal basis and from year to year. Like other plants, perennial ryegrass has evolved sophisticated mechanisms to make appropriate adjustments in growth and development in order to adapt to the stress environment at both the physiological and molecular levels. A thorough understanding of the mechanisms of perennial ryegrass response to abiotic stresses is crucial for obtaining superior stress-tolerant varieties through molecular breeding. Over the past decades, studies of perennial ryegrass at the molecular and genetic levels have revealed a lot of useful information to understand the mechanisms of perennial ryegrass adaptation to an adverse environment. Unfortunately, molecular mechanisms by which perennial ryegrass adapts to abiotic stresses have not been reviewed thus far. In this review, we summarize the recent works on the genetic and molecular mechanisms of perennial ryegrass response to the major abiotic stresses (i.e., drought, salinity, and extreme temperatures) and discuss new directions for future studies. Such knowledge will provide valuable information for molecular breeding in perennial ryegrass to improve stress resistance and promote the sustainability of agriculture and the environment.

Methyl jasmonate (MeJA) plays a role in improving plant stress tolerance. The molecular mechanisms associated with heat tolerance mediated by MeJA are not fully understood in perennial grass species. The study was designed to explore transcriptomic mechanisms underlying heat tolerance by exogenous MeJA in perennial ryegrass (Lolium perenne L.) using RNA-seq. Transcriptomic profiling was performed on plants under normal temperature (CK), high temperature for 12 h (H), MeJA pretreatment (T), MeJA pretreatment + H (T-H), respectively. The analysis of differentially expressed genes (DEGs) showed that H resulted in the most DEGs and T had the least, compared with CK. Among them, the DEGs related to the response to oxygen-containing compound was higher in CKvsH, while many genes related to photosynthetic system were down-regulated. The DEGs related to plastid components was higher in CKvsT. GO and KEGG analysis showed that exogenous application of MeJA enriched photosynthesis related pathways under heat stress. Exogenous MeJA significantly increased the expression of genes involved in chlorophyll (Chl) biosynthesis and antioxidant metabolism, and decreased the expression of Chl degradation genes, as well as the expression of heat shock transcription factor - heat shock protein (HSF-HSP) network under heat stress. The results indicated that exogenous application of MeJA improved the heat tolerance of perennial ryegrass by mediating expression of genes in different pathways, such as Chl biosynthesis and degradation, antioxidant enzyme system, HSF-HSP network and JAs biosynthesis., (© 2022. The Author(s).)

Various adaptive mechanisms can ensure that seedlings are established at the most favourable time and place. These mechanisms include seed dormancy i.e., incapacity to germinate in any environment without a specific environmental trigger and inhibition i.e., incapacity to germinate in an unfavourable environment (water availability, temperature: thermoinhibition and light). The objective of this research was to study in the temperate range for germination of forage and turf grass species perennial ryegrass, if the thermal requirements for germination are under genetic controlled and could be selectively bred. Two divergent selections of three cycles were realized on a natural population: one to select for the capacity to germinate at 10°C vs. the impossibility to germinate at 10°C, and one to select for the capacity to germinate at 32°C vs. the impossibility to germinate at 32°C. Seeds of all the lots obtained from the two divergent selections were then germinated at constant temperatures from 5 to 35°C to evaluate their germination ability. Concerning the positive selection, the first cycle of positive selection at 10°C was highly efficient with a very strong increase in the germination percentage. However, afterward no selection effect was observed during the next two cycles of positive selection. By contrast, the positive selection at 32°C was efficient during all cycles with a linear increase of the percentage of germination at 32°C. Concerning the negative selection, we observed only a large positive effect of the first cycle of selection at 10°C. These findings demonstrate that seed thermoinhibition at 10 and 32°C observed in a natural population of perennial ryegrass has a genetic basis and a single recessive gene seems to be involved at 10°C., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ghaleb, Barre, Teulat, Ahmed and Escobar-Gutiérrez.)

Perennial ryegrass is an important forage crop in dairy farming, either for grazing or haying purposes. To further optimise the forage use, this study focused on understanding forage digestibility in the two most important cuts of perennial ryegrass, the spring cut at heading and the autumn cut. In a highly diverse collection of 592 Lolium perenne genotypes, the organic matter digestibility (OMD) and underlying traits such as cell wall digestibility (NDFD) and cell wall components (cellulose, hemicellulose, and lignin) were investigated for 2 years. A high genotype × season interaction was found for OMD and NDFD, indicating differences in genetic control of these forage quality traits in spring versus autumn. OMD could be explained by both the quantity of cell wall content (NDF) and the quality of the cell wall content (NDFD). The variability in NDFD in spring was mainly explained by differences in hemicellulose. A 1% increase of the hemicellulose content in the cell wall (HC.NDF) resulted in an increase of 0.81% of NDFD. In autumn, it was mainly explained by the lignin content in the cell wall (ADL.NDF). A 0.1% decrease of ADL.NDF resulted in an increase of 0.41% of NDFD. The seasonal traits were highly heritable and showed a higher variation in autumn versus spring, indicating the potential to select for forage quality in the autumn cut. In a candidate gene association mapping approach, in which 503 genes involved in cell wall biogenesis, plant architecture, and phytohormone biosynthesis and signalling, identified significant quantitative trait loci (QTLs) which could explain from 29 to 52% of the phenotypic variance in the forage quality traits OMD and NDFD, with small effects of each marker taken individually (ranging from 1 to 7%). No identical QTLs were identified between seasons, but within a season, some QTLs were in common between digestibility traits and cell wall composition traits confirming the importance of hemicellulose concentration for spring digestibility and lignin concentration in NDF for autumn digestibility., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Colas, Barre, van Parijs, Wolters, Quitté, Ruttink, Roldán-Ruiz, Escobar Gutiérrez and Muylle.)

This paper describes the antigerminant capacity of water extracts of silver fir needles created by means of hydrodynamic cavitation processes. Fir needles (2 kg fresh weight) collected in the winter were blended and crushed in ice, poured in water only (120 L) and processed in a controlled hydrodynamic cavitation device based on a fixed Venturi-shaped reactor. The A. alba water extract (AWE), comprising an oil-in-water emulsion of silver fir needles' essential oil (100% AWE), was diluted in distilled water to 75% and 50% AWE, and all aqueous solutions were tested as antigerminant against four weeds and four horticultural species and compared to control (distilled water). This study shows the effective inhibitory effect of pure AWE on germination, which mainly contains limonene (15.99 ng/mL) and α-pinene (11.87 ng/mL). Seeds showed delayed germination and inhibition but also a reduction in radicle elongation in AWE treatments as compared to control. This combined effect was particularly evident in three weeds ( C. canadensis , C. album and A. retrofllexus ) while horticultural species showed mainly effects on the radicle elongation as found in L. sativa , P. crispum and S. lycospermum , which showed on average 58%, 32% and 28%, respectively, shorter radicles than in the control. P. sativum was not affected by AWE, thus raising the hypothesis that seed characteristics and nutrition reserve might play a role in the resistance to terpenes inhibitory effect.

The accumulation of heavy metals in crops is largely dependent on the availability of heavy metals in soils. Due to the differences of soil types and pollution characteristics, there is no widely recognized method for the determination of the bio-available heavy metals in soils such as Pb and Cd. In order to screen and establish suitable methods, the extractable abilities of five extractants (CaCl 2 , NH 4 OAc, HCl, EDTA, and DTPA) and Gradient Diffusion Film Technology (DGT) on four typical farmland soils with very different properties in Chongqing (acid purple soil, neutral purple soil, calcareous yellow soil and calcareous purple soil) were systematically compared. Simultaneously, pot experiments were carried out with Lolium perenne L. as an indicator plant to explore the relationship between the extractable soil Pb and Cd and their accumulation in plants. The feasibility of the tested methods were evaluated based on the extractability and co-relationships. The results showed that the extractability of the tested extractants for soil Pb and Cd varied a lot. HCl showed highest extractability on Pb in acid purple soil and calcareous yellow soil, while EDTA did the best in neutral purple soil and calcareous purple soil; HCl showed best extractability in all soils except calcareous purple soil, where EDTA was the best. Considering the correlation between the extraction amount and uptake by plant, all the extraction methods could be applied for the evaluation of the bioavailability of soils Pb and Cd except CaCl 2 for Pb due to its weak extractability for Pb for a specific soil type. For the comparison of heavy bioavailability in different soil types, EDTA-extractable Pb and DGT-extractable Cd were recommended due to their well co-relationships between extractable amount in soil and uptake by plant ( Lolium perenne L.) as indicated by the correlation coefficients of 0.941 and 0.919, respectively. HCl was relatively suitable as Cd extractant compared to others if DGT method could not be used.

Perennial ryegrass ( Lolium perenne ) is the most cultivated cool-season grass worldwide with crucial roles in carbon fixation, turfgrass applications, and fodder for livestock. Lolium perenne forms a mutualism with the strictly vertically transmitted fungal endophyte, Epichloë festucae var lolii . The fungus produces alkaloids that protect the grass from herbivory, as well as conferring protection from drought and nutrient stress. The rising concentration of atmospheric CO 2 , a proximate cause of climatic change, is known to have many direct and indirect effects on plant growth. There is keen interest in how the nature of this plant-fungal interaction will change with climate change. Lolium perenne is an obligately outcrossing species, meaning that the genetic profile of the host is constantly being reshuffled. Meanwhile, the fungus is asexual implying both a relatively constant genetic profile and the potential for incompatible grass-fungus pairings. In this study, we used a single cultivar, "Alto", of L. perenne . Each plant was infected with one of four strains of the endophyte: AR1, AR37, NEA2, and Lp19 (the "common strain"). We outcrossed the Alto mothers with pollen from a number of individuals from different ryegrass cultivars to create more genetic diversity in the hosts. We collected seed such that we had replicate maternal half-sib families. Seed from each family was randomly allocated into the two levels of the CO 2 treatment, 400 and 800 ppm. Elevated CO 2 resulted in an c . 18% increase in plant biomass. AR37 produced higher fungal concentrations than other strains; NEA2 produced the lowest fungal concentrations. We did not find evidence of genetic incompatibility between the host plants and the fungal strains. We conducted untargeted metabolomics and quantitative proteomics to investigate the grass-fungus interactions between and within family and treatment groups. We identified a number of changes in both the proteome and metabalome. Taken together, our data set provides new understanding into the intricacy of the interaction between endophyte and host from multiple molecular levels and suggests opportunity to promote plant robustness and survivability in rising CO 2 environmental conditions through application of bioprotective epichloid strains.

Global warming is predicted to impact many agricultural areas, which will suffer from reduced water availability. Due to precipitation changes, mild summer droughts are expected to become more frequent, even in temperate regions. For perennial ryegrass ( Lolium perenne L.), an important forage grass of the Poaceae family, leaf growth is a crucial factor determining biomass accumulation and hence forage yield. Although leaf elongation has been shown to be temperature-dependent under normal conditions, the genetic regulation of leaf growth under water deficit in perennial ryegrass is poorly understood. Herein, we evaluated the response to water deprivation in a diverse panel of perennial ryegrass genotypes, employing a high-precision phenotyping platform. The study revealed phenotypic variation for growth-related traits and significant ( P < 0.05) differences in leaf growth under normal conditions within the subgroups of turf and forage type cultivars. The phenotypic data was combined with genotypic variants identified using genotyping-by-sequencing to conduct a genome-wide association study (GWAS). Using GWAS, we identified DNA polymorphisms significantly associated with leaf growth reduction under water deprivation. These polymorphisms were adjacent to genes predicted to encode for phytochrome B and a MYB41 transcription factor. The result obtained in the present study will increase our understanding on the complex molecular mechanisms involved in plant growth under water deficit. Moreover, the single nucleotide polymorphism (SNP) markers identified will serve as a valuable resource in future breeding programs to select for enhanced biomass formation under mild summer drought conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Jaškūnė, Aleliūnas, Statkevičiūtė, Kemešytė, Studer and Yates.)

The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë -infected perennial ryegrass ( Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography-mass spectrometry (LC-MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC-MS (for peramine). This work provides the first highly sensitive quantitative LC-MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The successful introduction of isopentenyl transferase (IPT) gene into perennial ryegrass, cultivars Numan and Grassland using Agrobacterium tumefaciens via three explants (callus, seed and meristem tip) under three individual experiment was evaluated. In the first experiment, the calli were inoculated with LBA4404 Agrobacterium strain under vacuum, heat and in combination of both at 42 °C for 5 min followed by vacuum treatment (390 mm Hg pressure) for 15 min. Sonication-assisted Agrobacterium-mediated transformation (SAAT) was applied for seed and meristem tip transformation of perennial ryegrass for the first time. Results showed positive effects of heat treatment on transformation efficiency during Agro-infection in both cultivars. However, heat shock treatment was more effective in 'Grassland' than 'Numan' (14.2% vs 9.2%). In addition, high transformation efficiency of about 46.65% and 29.15% was observed using meristem tip explants of 'Grassland' and 'Numan' based on IPT and RD29A positive PCR results, respectively. Seed transformation efficiency in 'Grassland' and 'Numan' under SAAT method reached to 37.5% and 16.65%, respectively. Results of these experiments revealed that LBA4404 strain was more efficient than GV3101 in transformation of both perennial ryegrass cultivars. The DNA-blot analysis confirmed that a single T-DNA copy of the IPT gene was integrated into the genomic DNA of the positive transgenic T0 plants which obtained from callus and meristem tip explants of 'Grassland' after heat and SAAT treatment, respectively. Because monocots are not the host of Agrobacterium tumefaciens, this novel protocol can be used in further experiments on genetic transformation of perennial ryegrass cultivars.

Alkaloid concentration of perennial ryegrass herbage is affected by endophyte strain and host plant genotype. However, previous studies suggest that associations between host and endophyte also depends on environmental conditions, especially those affecting nutrient reserves and that water-soluble carbohydrate (WSC) concentration of perennial ryegrass plants may influence grass-endophyte associations. In this study a single transgenic event, with altered expression of fructosyltransferase genes to produce high WSC and biomass, has been crossed into a range of cultivar backgrounds with varying Epichloë endophyte strains. The effect of the association between the transgenic trait and alkaloid production was assessed and compared with transgene free control populations. In the vast-majority of comparisons there was no significant difference between alkaloid concentrations of transgenic and non-transgenic plants within the same cultivar and endophyte backgrounds. There was no significant difference between GOI+ (gene of interest positive) and GOI- (gene of interest negative) populations in Janthritrem response. Peramine concentration was not different between GOI+ and GOI- for 10 of the 12 endophytes-cultivar combinations. Cultivar Trojan infected with NEA6 and Alto with SE (standard endophyte) exhibited higher peramine and lolitrem B (only for Alto SE) concentration, in the control GOI- compared with GOI+. Similarly, cultivar Trojan infected with NEA6 and Alto with NEA3 presented higher ergovaline concentration in GOI-. Differences in alkaloid concentration may be attributable to an indirect effect in the modulation of fungal biomass. These results conclude that the presence of this transgenic insertion, does not alter the risk (toxicity) of the endophyte-grass associations. Endophyte-host interactions are complex and further research into associations with high WSC plant should be performed in a case by case basis.

In this study, the effects of inoculating arbuscular mycorrhizal fungi ( Glomus mosseae ) on the growth, chlorophyll content, photosynthetic gas exchange parameters, and chlorophyll fluorescence characteristics of Lolium perenne L. in cadmium (Cd) contaminated soil were investigated. The results showed that the root vigor of L. perenne declined, while the chlorophyll content significantly decreased with the increase of Cd content, especially the chlorophyll a content in leaves. The photosynthetic carbon assimilation capacity and PSII activity of L. perenne leaves were also significantly inhibited by Cd stress, especially the electron transfer at the receptor side of PSII, which was more sensitive to Cd stress. The infection level of G. mosseae on L. perenne roots was relatively high and inoculation with G. mosseae increased the mycorrhizal infection rate of L. perenne roots up to 50-70%. Due to the impact of the mycorrhizal infection, the Cd content in L. perenne roots was significantly increased compared to non-inoculated treatment; however, the Cd content in the aboveground part of L. perenne was not significantly different compared to the non-inoculated treatment. After inoculation with G. mosseae , the root vigor of L. perenne increased to some extent, alleviating the chlorophyll degradation in L. perenne leaves under Cd contaminated soil. Infection with G. mosseae can improve the stoma limitation of L. perenne leaves in Cd contaminated soil and increase the non-stomatal factors including the tolerance of its photosynthetic apparatus to Cd, to improve photosynthetic capacity. G. mosseae infection can improve the photosynthetic electron transport capacity of PSII in L. perenne leaves under Cd stress and promotes the activity of the oxygen-evolving complex to different degrees at the donor side of PSII and the electron transport capacity from Q A to Q B on the receptor side of PSII. Thus, this guarantees that L. perenne leaves inoculated with G. mosseae in Cd contaminated soil have relatively higher PSII activity. Therefore, inoculation with G. mosseae can improve the capacity of Cd tolerance of L. perenne with regard to various aspects, such as morphological characteristics and photosynthetic functions, and reduce the toxicity of Cd on L. perenne .

Cadmium (Cd) pollution is becoming increasingly prevalent, posing a global environmental hazard due to its negative effects on plants growth and human health. Phytoremediation is a green technology that involves uptake of Cd from the soil by a combination of plants and associated microbes. The objective of this study was to investigate the role of Aspergillus aculeatus in perennial ryegrass Cd tolerance. This fungus produced indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylate deaminase. Physiological traits including growth rate, turf quality and chlorophyll content were measured to evaluate the physiological responses of perennial ryegrass to Cd stress. These physiological traits were improved after inoculated with A. aculeatus . Inoculation of A. aculeatus actively reduced DTPA-Cd concentration in the soil and Cd translocation to plant shoots. Chlorophyll a fluorescence transient and the C/N ratio in shoots were elevated by A. aculeatus , which implied that the fungus could protect the photosystem II against Cd stress and increase the photosynthetic efficiency. These results suggested that A. aculeatus is beneficial in improving Cd tolerance of perennial ryegrass and reducing Cd-induced injuries, thus, it has promising potential for application of phytostabilization in Cd contaminated soil.

Background: Pyrrolizidine alkaloids (PAs) are a class of secondary metabolites that function as feeding deterrents in a range of different plant species. In perennial ryegrass (Lolium perenne L.) the only PAs that have been identified are the thesinine-rhamnoside group, which displays significant genetic variation. Homospermidine synthase (HSS) has evolved from deoxyhypusine synthase (DHS) and catalyses the first step in the PA pathway, making it a key candidate for the investigation of genes influencing observed PA trait variation., Results: During PCR amplification and sequence analysis of DHS we identified two putative HSS genes in perennial ryegrass. One of the genes (LpHSS1) was absent in some perennial ryegrass plants. Thesinine-rhamnoside levels were measured using liquid chromatography coupled with mass spectrometry in a diverse association mapping population, consisting of 693 plants free of fungal endophytic symbionts. Association tests that accounted for population structure identified a significant association of absence of the LpHSS1 gene with lower levels of thesinine-rhamnoside PAs. HSS-like gene sequences were identified for other grass species of the Poaceae, including tall fescue, wheat, maize and sorghum., Conclusion: HSS is situated at the crucial first step in the PA pathway making it an important candidate gene for investigation of involvement in PA phenotypic variation. In this study, PA level in perennial ryegrass was strongly associated with the presence or absence of the LpHSS1 gene. A genetic marker, developed for the presence/absence of LpHSS1, may be used for marker-assisted breeding to either lower or increase PAs in breeding populations of perennial or Italian ryegrass to investigate a potential role in the deterrence of herbivore pests. The presence of HSS-like genes in several other Poaceae species suggests that PA biosynthesis may occur in plant family members beyond perennial ryegrass and tall fescue and identifies a potential route for manipulating PA levels.

The objective of this study was to examine the effect of wintering pregnant, nonlactating dairy cows outdoors on either kale or grass, fed in 1 [11 kg dry matter (DM) of kale or grass + 3 kg DM of baled barley straw offered in the morning] or 2 allocations (5.5 kg DM of kale or grass grazed + 1.5 kg DM of barley straw offered morning and afternoon) per day. The body condition score (BCS) gain over the 47-d winter feeding period was higher for grass-fed (0.5 BCS units) than kale-fed cows (0.3 BCS units), but was unaffected by feeding frequency. Forage DM utilization was higher for kale-fed (97%) than grass-fed cows (76%), leading to higher estimated dry matter intake (DMI) in kale-fed (10.7 kg of DM/cow per day) than grass-fed cows (7.7 kg of DM/cow per day). Forage DM utilization and estimated DMI were not affected by feeding frequency. Prehension bite rate was greater for grass-fed (37.3 bites/min) than kale-fed cows (7.6 bites/min), but more mastication bites were required for kale-fed cows. Cumulative DMI after 2, 3, and 6 h was greater in cows allocated forage once than twice a day and for kale than grass after 3 and 6 h. Mean eating time was greater for cows offered forage once (477 min) than twice (414 min) per day. In conclusion, increasing feeding frequency from once to twice per day decreased the intake rate within the first 6 h after allocation, but did not affect total daily DMI, DM utilization or BCS gain. Thus, moving cows more frequently would not have any significant advantage. It may increase labor requirements, thereby creating a more challenging wintering management than feeding once per day., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Copper mine tailings pose many threats to the surrounding environment and human health, and thus, their remediation is fundamental. Coal spoil is the waste by-product of coal mining and characterized by low levels of metals, high content of organic matter, and many essential microelements. This study was designed to evaluate the role of coal spoil on heavy uptake and physiological responses of Lolium perenne L. grown in copper mine tailings amended with coal spoil at rates of 0, 0.5, 1, 5, 10, and 20%. The results showed that applying coal spoil to copper mine tailings decreased the diethylenetriaminepentaacetic acid (DTPA)-extractable Cd, Cu, Pb, and Zn contents in tailings and reduced those metal contents in both roots and shoots of the plant. However, application of coal spoil increased the DTPA-extractable Cr concentration in tailings and also increased Cr uptake and accumulation by Lolium perenne L. The statistical analysis of physiological parameters indicated that chlorophyll and carotenoid increased at the lower amendments of coal spoil followed by a decrease compared to their respective controls. Protein content was enhanced at all the coal spoil amendments. When treated with coal spoil, the activities of superoxide dismutases (SOD), peroxidase (POD), and catalase (CAT) responded differently. CAT activity was inhibited, but POD activity was increased with increasing amendment ratio of coal spoil. SOD activity increased up to 1% coal spoil followed by a decrease. Overall, the addition of coal spoil decreased the oxidative stress in Lolium perenne L., reflected by the reduction in malondialdehyde (MDA) contents in the plant. It is concluded that coal spoil has the potential to stabilize most metals studied in copper mine tailings and ameliorate the harmful effects in Lolium perenne L. through changing the physiological attributes of the plant grown in copper mine tailings.

Considering the serious pollution of heavy metal-chromium (Cr) in soil, there is an urgent need for effective selection of Cr-tolerant plant species. In order to gain fundamental insights into the tolerance and accumulation capabilities of Lolium perenne L. and Pharibitis purpurea (L.) Voigt under Cr stress, a pot experiment was conducted to investigate their growth, physiology and accumulation characteristics under Cr(Ⅲ) and Cr(Ⅵ) stress. The results showed the growth parameters could intuitively reflect the toxicity levels of Cr for plants. For instance, a low-level Cr(Ⅲ) (<250 mg·kg -1 ) in soil was good for plant growth as indicated by the significant elevation of plant height, root length and biomass in L. perenne ( P <0.05). However, Cr(Ⅵ) at all concentrations (≥25 mg·kg -1 ) in the soil inhibited the growth of both plant species, and the root length was particularly sensitive to the toxicity of Cr. The physiological parameters of plant represented both the toxicity of Cr and the tolerance of plants under Cr stress. A decrease of root activity and an increase of malonaldehyde content were observed under Cr stress, which indicated the physiological metabolism of plants was disturbed. In the presence of both Cr species, the proline content increased, which served as an indicator for both high Cr toxicity and increase of osmotic balance in plants. A rise in SOD and POD activity reflected the defense ability of plants against oxidative stress caused by Cr. In addition, the Cr-accumulation related parameters were the major standards for tolerant species selection. The Cr(Ⅵ) accumulation capacities of both plant species were greater than their Cr(Ⅲ) accumulation capacities. The maximum accumulation amounts of L. perenne and P. purpurea reached 957.4 mg·kg -1 and 743.3 mg·kg -1 in roots and 394.7 mg·kg -1 and 340.4 mg·kg -1 in shoots, respectively. In comparison with P. purpurea , L. perenne displayed a stronger Cr accumulation capacity in roots with a maximum bioaccumulation factor of 15.55. However, the transport ability of P. purpurea was superior to L. perenne . All of the parameters demonstrated that both L. perenne and P. purpurea could be used as alternative plants for phytoremediation of Cr-contaminated soil.

In urban areas with high fallout of airborne particles, metal uptake by plants mainly occurs by foliar pathways and can strongly impact crop quality. However, there is a lack of knowledge on metal localization and speciation in plants after pollution exposure, especially in the case of foliar uptake. In this study, two contrasting crops, lettuce (Lactuca sativa L.) and rye-grass (Lolium perenne L.), were exposed to Pb-rich particles emitted by a Pb-recycling factory via either atmospheric or soil application. Pb accumulation in plant leaves was observed for both ways of exposure. The mechanisms involved in Pb uptake were investigated using a combination of microscopic and spectroscopic techniques (electron microscopy, laser ablation, Raman microspectroscopy, and X-ray absorption spectroscopy). The results show that Pb localization and speciation are strongly influenced by the type of exposure (root or shoot pathway) and the plant species. Foliar exposure is the main pathway of uptake, involving the highest concentrations in plant tissues. Under atmospheric fallouts, Pb-rich particles were strongly adsorbed on the leaf surface of both plant species. In lettuce, stomata contained Pb-rich particles in their apertures, with some deformations of guard cells. In addition to PbO and PbSO4, chemical forms that were also observed in pristine particles, new species were identified: organic compounds (minimum 20%) and hexagonal platy crystals of PbCO3. In rye-grass, the changes in Pb speciation were even more egregious: Pb-cell wall and Pb-organic acid complexes were the major species observed. For root exposure, identified here as a minor pathway of Pb transfer compared to foliar uptake, another secondary species, pyromorphite, was identified in rye-grass leaves. Finally, combining bulk and spatially resolved spectroscopic techniques permitted both the overall speciation and the minor but possibly highly reactive lead species to be determined in order to better assess the health risks involved., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A detailed study was carried out to obtain information on the relative rates of NH 4 + -N uptake and assimilation by perennial ryegrass (Lolium perenne L.), when grown in soil and supplied with different ratios of NH 3 - -N uptake and assimilation by perennial ryegrass (Lolium perenne L.), when grown in soil and supplied with different ratios of NH 4 + and NO 3 - -N. Following two preliminary defoliations, plants were supplied with 15 N-(differentially) labelled NH 4 + and NO 3 - -N in three different ratios (20∶80, 50∶50 and 80∶20), followed by sequential destructive harvests of shoots and roots taken at four points during a 35-d regrowth period. When supplied with equimolar concentrations of NH 4 + and NO 3 - -N, perennial ryegrass absorbed both forms of nitrogen at almost identical rates; and even when the two N forms were supplied in widely disproportionate concentrations, both forms appear to have been absorbed at equal rates, at least until the supply of the minor nitrogen component in each treatment was almost exhausted, i.e. by day 3 of regrowth. It is suggested that this matching of NH 4 + and NO 3 - -N absorption rates by plants maximizes the advantages and minimizes the disadvantages associated with the exclusive use of either form of nitrogen, and thereby optimizes the potential for vegetative and reproductive regeneration. Fifteen days after the application of 15 N-labelled fertilizer, shoot and root yields differed little between treatments. However, plants grown with NH 4 + and NO 3 - -N at a ratio of 50∶50 had accumulated significantly more root (soluble) carbohydrate than those in the other two treatments. Consequently, when subjected to defoliation at day 15, these former plants were clearly in the best position to re-mobilize root carbohydrate and initiate shoot regeneration. However, further work is needed in order to determine whether or not this finding holds true for plants subjected to a multiple (bi- to tri-weekly) defoliation regime, e.g. in a grazing situation.

Apparent K m and V max for net NO S″ uptake and short-term translocation patterns of recently absorbed N were compared in a stay-green mutant and wild-type selection line of Lolium perenne L. by means of a series of depletion studies using 18 NO 3 , performed over 12 d under conditions of progressively increasing N deprivation. In view of the greater retention of N in senescent leaves of the stay-green phenotype, it was predicted that NO S″ uptake would be up-regulated relative to the normal line, and that a proportionally higher fraction of recently absorbed N would be allocated to young leaves. It was shown that the stay-green trait had significant phenotypic consequences for plant N relations, with higher 'sink strength' of shoots for recently absorbed N, and higher V max for NO 3 uptake compared with those of normal plants. The stay-green mutation had no effect on the K m of the nitrate uptake system. Although the N-use efficiency might he expected to be lower in stay-green than in normal plants, there were no differences in rates of dry matter production.