The effects of monoculture and intercropping on photosynthesis performance correlated with growth of garlic and perennial ryegrass response to different heavy metals.

BACKGROUND: The potential of phytoremediation using garlic monoculture (MC) and intercropping (IC) system with perennial ryegrass to enhance the uptake of cadmium (Cd), chromium (Cr), and lead (Pb) were investigated. RESULTS: Positive correlations were found between MC and IC systems, with varying biomass. Production of perennial ryegrass was affected differently depending on the type of toxic metal present in the soil. Root growth inhibition was more affected than shoot growth inhibition. The total biomass of shoot and root in IC was higher than MC, increasing approximately 3.7 and 2.9 fold compared to MC, attributed to advantages in root IC crop systems. Photosystem II efficiency showed less sensitivity to metal toxicity compared to the control, with a decrease between 10.07-12.03%. Among gas exchange parameters, only Cr significantly affected physiological responses by reducing transpiration by 69.24%, likely due to leaf chlorosis and necrosis. CONCLUSION: This study exhibited the potential of garlic MC and IC with perennial ryegrass in phytoremediation. Although the different metals affect plant growth differently, IC showed advantages over MC in term biomass production.

The growth promotion in endophyte symbiotic plants does not penalise the resistance to herbivores and bacterial microbiota.

A trade-off between growth and defence against biotic stresses is common in plants. Fungal endophytes of the genus Epichloë may relieve this trade-off in their host grasses since they can simultaneously induce plant growth and produce antiherbivore alkaloids that circumvent the need for host defence. The Epichloë ability to decouple the growth-defence trade-off was evaluated by subjecting ryegrass with and without Epichloë endophytes to an exogenous treatment with gibberellin (GA) followed by a challenge with Rhopalosiphum padi aphids. In agreement with the endophyte-mediated trade-off decoupling hypothesis, the GA-derived promotion of plant growth increased the susceptibility to aphids in endophyte-free plants but did not affect the insect resistance in endophyte-symbiotic plants. In line with the unaltered insect resistance, the GA treatment did not reduce the concentration of Epichloë-derived alkaloids. The Epichloë mycelial biomass was transiently increased by the GA treatment but at the expense of hyphal integrity. The response of the phyllosphere bacterial microbiota to both GA treatment and Epichloë was also evaluated. Only Epichloë, and not the GA treatment, altered the composition of the phyllosphere microbiota and the abundance of certain bacterial taxa. Our findings clearly demonstrate that Epichloë does indeed relieve the plant growth-defence trade-off.

Half of the 18O enrichment of leaf sucrose is conserved in leaf cellulose of a C3 grass across atmospheric humidity and CO2 levels.

The 18O enrichment (Δ18O) of cellulose (Δ18OCel) is recognized as a unique archive of past climate and plant function. However, there is still uncertainty regarding the proportion of oxygen in cellulose (pex) that exchanges post-photosynthetically with medium water of cellulose synthesis. Particularly, recent research with C3 grasses demonstrated that the Δ18O of leaf sucrose (Δ18OSuc, the parent substrate for cellulose synthesis) can be much higher than predicted from daytime Δ18O of leaf water (Δ18OLW), which could alter conclusions on photosynthetic versus post-photosynthetic effects on Δ18OCel via pex. Here, we assessed pex in leaves of perennial ryegrass (Lolium perenne) grown at different atmospheric relative humidity (RH) and CO2 levels, by determinations of Δ18OCel in leaves, Δ18OLGDZW (the Δ18O of water in the leaf growth-and-differentiation zone) and both Δ18OSuc and Δ18OLW (adjusted for εbio, the biosynthetic fractionation between water and carbohydrates) as alternative proxies for the substrate for cellulose synthesis. Δ18OLGDZW was always close to irrigation water, and pex was similar (0.53 ± 0.02 SE) across environments when determinations were based on Δ18OSuc. Conversely, pex was erroneously and variably underestimated (range 0.02-0.44) when based on Δ18OLW. The photosynthetic signal fraction in Δ18OCel is much more constant than hitherto assumed, encouraging leaf physiological reconstructions.

Anti-herbivory defences delivered by Epichloë fungal endophytes: a quantitative review of alkaloid concentration variation among hosts and plant parts.

BACKGROUND AND AIMS: In the subfamily Poöideae (Poaceae), certain grass species possess anti-herbivore alkaloids synthesized by fungal endophytes that belong to the genus Epichloë (Clavicipitaceae). The protective role of these symbiotic endophytes can vary, depending on alkaloid concentrations within specific plant-endophyte associations and plant parts. METHODS: We conducted a literature review to identify articles containing alkaloid concentration data for various plant parts in six important pasture species, Lolium arundinaceum, Lolium perenne, Lolium pratense, Lolium multiflorum|Lolium rigidum and Festuca rubra, associated with their common endophytes. We considered the alkaloids lolines (1-aminopyrrolizidines), peramine (pyrrolopyrazines), ergovaline (ergot alkaloids) and lolitrem B (indole-diterpenes). While all these alkaloids have shown bioactivity against insect herbivores, ergovaline and lolitrem B are harmful for mammals. KEY RESULTS: Loline alkaloid levels were higher in the perennial grasses L. pratense and L. arundinaceum compared to the annual species L. multiflorum and L. rigidum, and higher in reproductive tissues than in vegetative structures. This is probably due to the greater biomass accumulation in perennial species that can result in higher endophyte mycelial biomass. Peramine concentrations were higher in L. perenne than in L. arundinaceum and not affected by plant part. This can be attributed to the high within-plant mobility of peramine. Ergovaline and lolitrem B, both hydrophobic compounds, were associated with plant parts where fungal mycelium is usually present, and their concentrations were higher in plant reproductive tissues. Only loline alkaloid data were sufficient for below-ground tissue analyses and concentrations were lower than in above-ground parts. CONCLUSIONS: Our study provides a comprehensive synthesis of fungal alkaloid variation across host grasses and plant parts, essential for understanding the endophyte-conferred defence extent. The patterns can be understood by considering endophyte growth within the plant and alkaloid mobility. Our study identifies research gaps, including the limited documentation of alkaloid presence in roots and the need to investigate the influence of different environmental conditions.

NOL-mediated functional stay-green traits in perennial ryegrass (Lolium perenne L.) involving multifaceted molecular factors and metabolic pathways regulating leaf senescence.

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.

Phosphatidic acid produced by phospholipase D is required for hyphal cell-cell fusion and fungal-plant symbiosis.

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyze the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloë festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne. Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analyzed by light-, confocal- and electron microscopy. Changes in PA distribution were analyzed in E. festucae using a PA biosensor and the impact of these changes on the endocytic recycling and superoxide production investigated. We found that E. festucae PldB, and the N. crassa ortholog, PLA-7, are required for polarized growth and cell fusion and contribute to ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network for hyphal morphogenesis and growth of filamentous fungi.

Getting ready for the ozone battle: Vertically transmitted fungal endophytes have transgenerational positive effects in plants.

Ground-level ozone is a global air pollutant with high toxicity and represents a threat to plants and microorganisms. Although beneficial microorganisms can improve host performance, their role in connecting environmentally induced maternal plant phenotypes to progeny (transgenerational effects [TGE]) is unknown. We evaluated fungal endophyte-mediated consequences of maternal plant exposure to ozone on performance of the progeny under contrasting scenarios of the same factor (high and low) at two stages: seedling and young plant. With no variation in biomass, maternal ozone-induced oxidative damage in the progeny that was lower in endophyte-symbiotic plants. This correlated with an endophyte-mediated higher concentration of proline, a defence compound associated with stress control. Interestingly, ozone-induced TGE was not associated with reductions in plant survival. On the contrary, there was an overall positive effect on seedling survival in the presence of endophytes. The positive effect of maternal ozone increasing young plant survival was irrespective of symbiosis and only expressed under high ozone condition. Our study shows that hereditary microorganisms can modulate the capacity of plants to transgenerationally adjust progeny phenotype to atmospheric change.

Differences in the effects of single and mixed species of AMF on the growth and oxidative stress defense in Lolium perenne exposed to hydrocarbons.

Arbuscular mycorrhizal fungi (AMF) are ubiquitous mutualistic plant symbionts that promote plant growth and protect them from abiotic stresses. Studies on AMF-assisted phytoremediation have shown that AMF can increase plant tolerance to the presence of hydrocarbon contaminants by improving plant nutrition status and mitigating oxidative stress. This work aimed to evaluate the impact of single and mixed-species AMF inocula (Funneliformis caledonium, Diversispora varaderana, Claroideoglomus walkeri), obtained from a contaminated environment, on the growth, oxidative stress (DNA oxidation and lipid peroxidation), and activity of antioxidative enzymes (superoxide dismutase, catalase, peroxidase) in Lolium perenne growing on a substrate contaminated with 0/0-30/120 mg phenol/polynuclear aromatic hydrocarbons (PAHs) kg-1. The assessment of AMF tolerance to the presence of contaminants was based on mycorrhizal root colonization, spore production, the level of oxidative stress, and antioxidative activity in AMF spores. In contrast to the mixed-species AMF inoculum, single AMF species significantly enhanced the growth of host plants cultured on the contaminated substrate. The effect of inoculation on the level of oxidative stress and the activity of antioxidative enzymes in plant tissues differed between the AMF species. Changes in the level of oxidative stress and the activity of antioxidative enzymes in AMF spores in response to contamination also depended on AMF species. Although the concentration of phenol and PAHs had a negative effect on the production of AMF spores, low (5/20 mg phenol/PAHs kg-1) and medium (15/60 mg phenol/PAHs kg-1) substrate contamination stimulated the mycorrhizal colonization of roots. Among the studied AMF species, F. caledonium was the most tolerant to phenol and PAHs and showed the highest potential in plant growth promotion. The results presented in this study might contribute to the development of functionally customized AMF-assisted phytoremediation strategies with indigenous AMF, more effective than commercial AMF inocula, as a result of their selection by the presence of contaminants.

Arbuscular mycorrhizal fungus facilitates ryegrass (Lolium perenne L.) growth and polychlorinated biphenyls degradation in a soil applied with nanoscale zero-valent iron.

Nanoscale zero-valent iron (nZVI) shows an excellent degradation effect on chlorinated contaminants in soil, but poses a threat to plants in combination with phytoremediation. Arbuscular mycorrhizal (AM) fungus can reduce the phyototoxicity of nZVI, but their combined impacts on polychlorinated biphenyls (PCBs) degradation and plant growth remain unclear. Here, a greenhouse pot experiment was conducted to investigate the influences of nZVI and/or Funneliformis caledonium on soil PCB degradation and ryegrass (Lolium perenne L.) antioxidative responses. The amendment of nZVI significantly reduced not only the total and homolog concentrations of PCBs in the soil, but also the ryegrass biomass as well as soil available P and root P concentrations. Moreover, nZVI significantly decreased leaf superoxide disutase (SOD) activity, while tended to decrease the protein content. In contrast, the additional inoculation of F. caledonium significantly increased leaf SOD activity and protein content, while tended to increase the catalase activity and tended to decrease the malondialdehyde content. The additional inoculation of F. caledonium also significantly increased soil alkaline phosphatase activity, and tended to increase root P concentration, but had no significantly effects on soil available P concentration, the biomass and P acquisition of ryegrass, which could be attributed to the fixation of soil available nutrients by nZVI. Additionally, F. caledonium facilitated PCB degradation in the nZVI-applied soil. Thus, AM fungus can alleviate the nZVI-induced phytotoxicity, showing great application potentials in accompany with nZVI for soil remediation.

Physiological and Gene Expression Responses of Six Annual Ryegrass Cultivars to Cobalt, Lead, and Nickel Stresses.

Heavy metals negatively affect soil quality and crop growth. In this study, we compared the tolerance of six ryegrass cultivars to cobalt (Co2+), lead (Pb2+), and nickel (Ni2+) stresses by analyzing their physiological indexes and transcript levels of genes encoding metal transporters. Compared with the other cultivars, the cultivar Lm1 showed higher germination rates and better growth under Co2+, Pb2+, or Ni2+ treatments. After 48 h of Co2+ treatment, the total antioxidant capacity of all six ryegrass cultivars was significantly increased, especially that of Lm1. In contrast, under Pb2+ stress, total antioxidant capacity of five cultivars was significantly decreased, but that of Lm1 was unaffected at 24 h. Staining with Evans blue dye showed that the roots of Lm1 were less injured than were roots of the other five ryegrass cultivars by Co2+, Pb2+, and Ni2+. Lm1 translocated and accumulated lesser Co2+, Pb2+, and Ni2+ than other cultivars. In Lm1, genes encoding heavy metal transporters were differentially expressed between the shoots and roots in response to Co2+, Pb2+, and Ni2+. The aim of these researches could help find potential resource for phytoremediation of heavy metal contamination soil. The identified genes related to resistance will be useful targets for molecular breeding.

Characterization of plant growth-promoting rhizobacteria from perennial ryegrass and genome mining of novel antimicrobial gene clusters.

BACKGROUND: Plant growth-promoting rhizobacteria (PGPR) are good alternatives for chemical fertilizers and pesticides, which cause severe environmental problems worldwide. Even though many studies focus on PGPR, most of them are limited in plant-microbe interaction studies and neglect the pathogens affecting ruminants that consume plants. In this study, we expand the view to the food chain of grass-ruminant-human. We aimed to find biocontrol strains that can antagonize grass pathogens and mammalian pathogens originated from grass, thus protecting this food chain. Furthermore, we deeply mined into bacterial genomes for novel biosynthetic gene clusters (BGCs) that can contribute to biocontrol. RESULTS: We screened 90 bacterial strains from the rhizosphere of healthy Dutch perennial ryegrass and characterized seven strains (B. subtilis subsp. subtilis MG27, B. velezensis MG33 and MG43, B. pumilus MG52 and MG84, B. altitudinis MG75, and B. laterosporus MG64) that showed a stimulatory effect on grass growth and pathogen antagonism on both phytopathogens and mammalian pathogens. Genome-mining of the seven strains discovered abundant BGCs, with some known, but also several potential novel ones. Further analysis revealed potential intact and novel BGCs, including two NRPSs, four NRPS-PKS hybrids, and five bacteriocins. CONCLUSION: Abundant potential novel BGCs were discovered in functional protective isolates, especially in B. pumilus, B. altitudinis and Brevibacillus strains, indicating their great potential for the production of novel secondary metabolites. Our report serves as a basis to further identify and characterize these compounds and study their antagonistic effects against plant and mammalian pathogens.

Effects of shade stress on turfgrasses morphophysiology and rhizosphere soil bacterial communities.

BACKGROUND: The shade represents one of the major environmental limitations for turfgrass growth. Shade influences plant growth and alters plant metabolism, yet little is known about how shade affects the structure of rhizosphere soil microbial communities and the role of soil microorganisms in plant shade responses. In this study, a glasshouse experiment was conducted to examine the impact of shade on the growth and photosynthetic capacity of two contrasting shade-tolerant turfgrasses, shade-tolerant dwarf lilyturf (Ophiopogon japonicus, OJ) and shade-intolerant perennial turf-type ryegrass (Lolium perenne, LP). We also examined soil-plant feedback effects on shade tolerance in the two turfgrass genotypes. The composition of the soil bacterial community was assayed using high-throughput sequencing. RESULTS: OJ maintained higher photosynthetic capacity and root growth than LP under shade stress, thus OJ was found to be more shade-tolerant than LP. Shade-intolerant LP responded better to both shade and soil microbes than shade-tolerant OJ. The shade and live soil decreased LP growth, but increased biomass allocation to shoots in the live soil. The plant shade response index of LP is higher in live soil than sterile soil, driven by weakened soil-plant feedback under shade stress. In contrast, there was no difference in these values for OJ under similar shade and soil treatments. Shade stress had little impact on the diversity of the OJ and the LP bacterial communities, but instead impacted their composition. The OJ soil bacterial communities were mostly composed of Proteobacteria and Acidobacteria. Further pairwise fitting analysis showed that a positive correlation of shade-tolerance in two turfgrasses and their bacterial community compositions. Several soil properties (NO3--N, NH4+-N, AK) showed a tight coupling with several major bacterial communities under shade stress. Moreover, OJ shared core bacterial taxa known to promote plant growth and confer tolerance to shade stress, which suggests common principles underpinning OJ-microbe interactions. CONCLUSION: Soil microorganisms mediate plant responses to shade stress via plant-soil feedback and shade-induced change in the rhizosphere soil bacterial community structure for OJ and LP plants. These findings emphasize the importance of understanding plant-soil interactions and their role in the mechanisms underlying shade tolerance in shade-tolerant turfgrasses.

Natural variation of physiological traits, molecular markers, and chlorophyll catabolic genes associated with heat tolerance in perennial ryegrass accessions.

BACKGROUND: Identification of genetic diversity in heat tolerance and associated traits is of great importance for improving heat tolerance in cool-season grass species. The objectives of this study were to determine genetic variations in heat tolerance associated with phenotypic and physiological traits and to identify molecular markers associated with heat tolerance in a diverse collection of perennial ryegrass (Lolium perenne L.). RESULTS: Plants of 98 accessions were subjected to heat stress (35/30 °C, day/night) or optimal growth temperature (25/20 °C) for 24 d in growth chambers. Overall heat tolerance of those accessions was ranked by principal component analysis (PCA) based on eight phenotypic and physiological traits. Among these traits, electrolyte leakage (EL), chlorophyll content (Chl), relative water content (RWC) had high correlation coefficients (- 0.858, 0.769, and 0.764, respectively) with the PCA ranking of heat tolerance. We also found expression levels of four Chl catabolic genes (CCGs), including LpNYC1, LpNOL, LpSGR, and LpPPH, were significant higher in heat sensitive ryegrass accessions then heat tolerant ones under heat stress. Furthermore, 66 pairs of simple sequence repeat (SSR) markers were used to perform association analysis based on the PCA result. The population structure of ryegrass can be grouped into three clusters, and accessions in cluster C were relatively more heat tolerant than those in cluster A and B. SSR markers significantly associated with above-mentioned traits were identified (R2 > 0.05, p < 0.01)., including two pairs of markers located on chromosome 4 in association with Chl content and another four pairs of markers in association with EL. CONCLUSION: The result not only identified useful physiological parameters, including EL, Chl content, and RWC, and their associated SSR markers for heat-tolerance breeding of perennial ryegrass, but also highlighted the involvement of Chl catabolism in ryegrass heat tolerance. Such knowledge is of significance for heat-tolerance breeding and heat tolerance mechanisms in perennial ryegrass as well as in other cool-season grass species.

Ontogenetic and trans-generational dynamics of a vertically transmitted fungal symbiont in an annual host plant in ozone-polluted settings.

Tropospheric ozone is an abiotic stress of increasing importance in the context of global climate change. This greenhouse gas is a potent phytotoxic molecule with demonstrated negative effects on crop yield and natural ecosystems. Recently, oxidative stress has been proposed as a mechanism that could regulate the interaction between cool-season grasses and Epichloë endophytes. We hypothesized that exposure of Lolium multiflorum plants, hosting endophytes to an ozone-polluted environment at different ontogenetic phases, would impact the trans-generational dynamics of the vertically transmitted fungal symbiont. Here, we found that the ozone-induced stress on the mother plants did not affect the endophyte vertical transmission but it impaired the persistence of the fungus in the seed exposed to artificial ageing. Endophyte longevity in seed was reduced by exposure of the mother plant to ozone. Although ozone exposure did not influence either the endophyte mycelial concentration or their compound defences (loline alkaloids), a positive correlation was observed between host fitness and the concentration of endophyte-derived defence compounds. This suggests that fungal defences in grass seeds were not all produced in situ but remobilized from the vegetative tissues. Our study reveals ozone trans-generational effects on the persistence of a beneficial symbiont in a host grass.

Full-Length Transcriptome Assembly of Italian Ryegrass Root Integrated with RNA-Seq to Identify Genes in Response to Plant Cadmium Stress.

Cadmium (Cd) is a toxic heavy metal element. It is relatively easily absorbed by plants and enters the food chain, resulting in human exposure to Cd. Italian ryegrass (Lolium multiflorum Lam.), an important forage cultivated widely in temperate regions worldwide, has the potential to be used in phytoremediation. However, genes regulating Cd translocation and accumulation in this species are not fully understood. Here, we optimized PacBio ISO-seq and integrated it with RNA-seq to construct a de novo full-length transcriptomic database for an un-sequenced autotetraploid species. With the database, we identified 2367 differentially expressed genes (DEGs) and profiled the molecular regulatory pathways of Italian ryegrass with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis in response to Cd stress. Overexpression of a DEG LmAUX1 in Arabidopsis thaliana significantly enhanced plant Cd concentration. We also unveiled the complexity of alternative splicing (AS) with a genome-free strategy. We reconstructed full-length UniTransModels using the reference transcriptome, and 29.76% of full-length models had more than one isoform. Taken together, the results enhanced our understanding of the genetic diversity and complexity of Italian ryegrass under Cd stress and provided valuable genetic resources for its gene identification and molecular breeding.

Adjustment of Photosynthetic and Antioxidant Activities to Water Deficit Is Crucial in the Drought Tolerance of Lolium multiflorum/Festuca arundinacea Introgression Forms.

Lolium multiflorum/Festuca arundinacea introgression forms have been proved several times to be good models to identify key components of grass metabolism involved in the mechanisms of tolerance to water deficit. Here, for the first time, a relationship between photosynthetic and antioxidant capacities with respect to drought tolerance of these forms was analyzed in detail. Two closely related L. multiflorum/F. arundinacea introgression forms distinct in their ability to re-grow after cessation of prolonged water deficit in the field were selected and subjected to short-term drought in pots to dissect precisely mechanisms of drought tolerance in this group of plants. The studies revealed that the form with higher drought tolerance was characterized by earlier and higher accumulation of abscisic acid, more stable cellular membranes, and more balanced reactive oxygen species metabolism associated with a higher capacity of the antioxidant system under drought conditions. On the other hand, both introgression forms revealed the same levels of stomatal conductance, CO2 assimilation, and consequently, intrinsic water use efficiency under drought and recovery conditions. However, simultaneous higher adjustment of the Calvin cycle to water deficit and reduced CO2 availability, with respect to the accumulation and activity of plastid fructose-1,6-bisphosphate aldolase, were clearly visible in the form with higher drought tolerance.

The LaeA orthologue in Epichloë festucae is required for symbiotic interaction with Lolium perenne.

LaeA is a conserved global regulator of secondary metabolism and development in fungi. It is often required for successful pathogenic interactions. In this study, the laeA homologue in the fungal grass endophyte E. festucae was deleted and functionally characterised in vitro and its role in the mutualistic E. festucae interaction with Lolium perenne (perennial ryegrass) was determined. We showed that laeA in E. festucae is required for normal hyphal morphology, resistance to oxidative stress, and conidiation under nutrient-limited in vitro conditions. In planta studies revealed that laeA is expressed in a tissue-specific manner and is required to form a compatible plant interaction, with the majority of seedlings inoculated with a laeA deletion mutant either dying or being uninfected. In mature infected plants no difference was observed in the number or morphology of endophytic hyphae. However, the number of epiphyllous hyphae were greatly increased. Comparative transcriptomics analyses suggested roles for plant cell wall degradation, fungal cell wall composition, secondary metabolism and small-secreted proteins in Epichloë foliar symbiosis.

Use of identifiability analysis in designing phenotyping experiments for modelling forage production and quality.

Agricultural systems models are complex and tend to be over-parameterized with respect to observational datasets. Practical identifiability analysis based on local sensitivity analysis has proved effective in investigating identifiable parameter sets in environmental models, but has not been applied to agricultural systems models. Here, we demonstrate that identifiability analysis improves experimental design to ensure independent parameter estimation for yield and quality outputs of a complex grassland model. The Pasture Simulation model (PaSim) was used to demonstrate the effectiveness of practical identifiability analysis in designing experiments and measurement protocols within phenotyping experiments with perennial ryegrass. Virtual experiments were designed combining three factors: frequency of measurements, duration of the experiment. and location of trials. Our results demonstrate that (i) PaSim provides sufficient detail in terms of simulating biomass yield and quality of perennial ryegrass for use in breeding, (ii) typical breeding trials are insufficient to parameterize all influential parameters, (iii) the frequency of measurements is more important than the number of growing seasons to improve the identifiability of PaSim parameters, and (iv) identifiability analysis provides a sound approach for optimizing the design of multi-location trials. Practical identifiability analysis can play an important role in ensuring proper exploitation of phenotypic data and cost-effective multi-location experimental designs. Considering the growing importance of simulation models, this study supports the design of experiments and measurement protocols in the phenotyping networks that have recently been organized.

Enhanced Phytoextraction for Co-contaminated Soil with Cd and Pb by Ryegrass (Lolium perenne L.).

A pot experiment was conducted to investigate the effect of three additives - citric acid (CA), polyaspartic acid (PASP), and FeCl3 - on the phytoextraction efficiency of cadmium (Cd) and lead (Pb) by ryegrass (Lolium perenneL.) from artificially contaminated soils with different heavy metal concentrations. The results showed that as the concentration of pollutants increased, the TI (tolerance index) and BCF (bio-concentration factor) of ryegrass gradually increased only when FeCl3 was applied. FeCl3 also exhibited the most significant biomass enhancement and heavy metal accumulation of ryegrass, as well as the highest phytoextraction efficiency in heavily-polluted soils. The overall orders of the optimal phytoextraction efficiency for the three additives in terms of their MER (metal extraction ratio) were: FeCl3 > PASP > CA. Therefore, FeCl3 can be used to improve the Cd and Pb phytoextraction efficiency of ryegrass in heavily-polluted soils.

Integrated omics data of two annual ryegrass (Lolium multiflorum L.) genotypes reveals core metabolic processes under drought stress.

BACKGROUND: Annual ryegrass (Lolium multiflorum L.) is a commercially important, widely distributed forage crop that is used in the production of hay and silage worldwide. Drought has been a severe environmental constraint in its production. Nevertheless, only a handful of studies have examined the impact of short-term drought stress on annual ryegrass. The aim of this study was to explore how stress-induced core metabolic processes enhance drought tolerance, or adaptation to drought, in annual ryegrass. RESULTS: We profiled the transcriptomes, proteomes, and metabolomes of two annual ryegrass genotypes: the drought-resistant genotype "Abundant 10" and drought-susceptible genotype "Adrenalin 11." We identified differentially expressed metabolites and their corresponding proteins and transcripts that are involved in 23 core metabolic processes, in response to short-term drought stress. Protein-gene-metabolite correlation networks were built to reveal the relationships between the expression of transcripts, proteins, and metabolites in drought-resistant annual ryegrass. Furthermore, integrated metabolic pathways were used to observe changes in enzymes corresponding with levels of amino acids, lipids, carbohydrate conjugates, nucleosides, alkaloids and their derivatives, and pyridines and their derivatives. The resulting omics data underscored the significance of 23 core metabolic processes on the enhancement of drought tolerance or adaptation to drought in annual ryegrass. CONCLUSIONS: The regulatory networks were inferred using MCoA and correlation analysis to reveal the relationships among the expression of transcripts, proteins, and metabolites that highlight the corresponding elements of these core metabolic pathways. Our results provide valuable insight into the molecular mechanisms of drought resistance, and represent a promising strategy toward the improvement of drought tolerance in annual ryegrass.