DLF Trifolium (Denmark)

Abstract Lolium perenne L., is a widely cultivated cool-season grass, is an important turf and forage species worldwide, with moderate salt tolerance. However, soil salinity resulting from poor water management, inadequate drainage of fields and high temperatures significantly impair its yield. This study explores effects of two endophytic fungi on enhancing the growth of non-host L. perenne under salt stress. Two salt-tolerant, plant growth-promoting fungal endophytes, Stereum gausapatum (ENDO_1) and Parasarocladium gamsii (ENDO_2), were inoculated in L. perenne under salt stress conditions (0 mM, 150 mM, and 300 mM NaCl). The plant growth performance, physiological responses, and transcriptomic changes were analyzed in two ryegrass varieties, Indiana and Greensky, to assess the endophytes’ effects on plant resilience and adaptation to salinity stress. The two fungi significantly increased growth and biomass with beneficial physiological effects in L. perenne: ENDO_1 reduced lipid peroxidation and increased total antioxidants, while ENDO_2 increased proline accumulation. Weighted gene correlation network analysis revealed that stress-responsive pathways were differentially regulated in plant organs (leaves and roots) and after fungal inoculation in both varieties. The representative processes included amino acid metabolism, cell wall macromolecule metabolism or biogenesis, organic acids and compatible solutes, plant stress-related, generation of precursor metabolites and energy, transport, and plant nutrition. Salinity-induced growth reductions were ameliorated by fungi leading to greater biomass and increased plant growth compared to non-inoculated plants. Salinity stress-related pathways namely lipid metabolism, carboxylic acid biosynthesis, alpha amino acid catabolism were regulated by endophytes, which can be potential biomarkers used for bioinoculant selection.

Abstract BackgroundIn breeding the aim is to identify and accumulate beneficial variants. However, detection of these variants may be challenging in the presence of extensive genotype x environment interactions (GxE), as variant effects will be conditional on environment.MethodsThe study assesses the performance of264diploid perennial ryegrass accessions in a multi-environment field trial. We investigate the extent of GxE, for yield (total dry matter) and persistence traits, i.e. winter kill and spring cover, under environmental conditions experienced in Nordic and Baltic regions at the limit of the species distribution. Two different approaches to modelling GxE were tested: reaction norm and envirotyping, and the models were validated under three different breeding scenarios.ResultsOur analysis documented the presence of significant GxE interaction for all traits investigated in the study. Validation showed improvements in prediction accuracy when accounting for GxE: up to 4% for yield when predicting in unobserved environments, and up to 22% and 9% for spring cover and winter kill, respectively when predicting unobserved germplasm. Genome-wide-association-studies (GWAS) were utilized to detect genetic variants with marginal effects (environment-independent effect) and conditional effects (environment-dependent effects). Results showed the presence of large-effect genetic variants with marginal effects, in addition to few QTL whose effects were adaptive under specific environmental conditions while neutral or deleterious under different environmental conditions.ConclusionThis study demonstrates the usefulness and limitations of genomic prediction models for predicting GxE in highly diverse samples, and describes the extent of GxE interactions at the limit of species distribution for perennial ryegrass. Finally, our study points toward adaptive variation, which may enhance persistence of perennial ryegrass populations in Nordic and Baltic growing conditions.

Key messageEarly root traits, particularly total root length, are heritable and show positive genetic correlations with biomass yield in perennial ryegrass; incorporating them into breeding programs can enhance genetic gain.Perennial ryegrass (Lolium perenne L.) is an important forage grass widely used in pastures and lawns, valued for its high nutritive value and environmental benefits. Despite its importance, genetic improvements in biomass yield have been slow, mainly due to its outbreeding nature and the challenges of improving multiple traits simultaneously. This study aims to assess the potential advantages of including early root traits in the perennial ryegrass breeding process. Root traits, including total root length (TRL) and root angle (RA) were phenotyped in a greenhouse using rhizoboxes, and genetic correlations with field yield were estimated across three European locations over two years. Bivariate models estimated significant genetic correlations of 0.40 (SE = 0.14) between TRL and field yield, and a weak but positive correlation to RA of 0.15 (SE = 0.14). Heritability estimates were 0.36 for TRL, 0.39 for RA, and 0.31 for field yield across locations. Incorporating root trait data into selection criteria can improve the efficiency of breeding programs, potentially increasing genetic gain by approximately 10%. This results highlight the potential of early root traits to refine selection criteria in perennial ryegrass breeding programs, contributing to higher yield and efficiency.

Tungsten carbide-cobalt (WC-Co) is a composite material consisting of WC as the main phase along with Co as a soft binder metal. It is difficult to process due to its extreme wear resistance and high hardness. Hence, many non-conventional production methods, such as electrical discharge machining and ultrasonic-assisted machining operation, have been found to achieve this. The article is focused on processing WC-Co machining with ultrasonic-assisted grinding (UAG) operation. In this study, WC-25Co material was grinded in different cutting tool angles with Ultrasonic 20 (DMG Mori) machine. Each of cutting tool angle during grinding corresponds distinct operations. Hence, the effect of the ultrasonic factor and cutting direction during grinding on the surface integrity of workpiece was investigated. The ultrasonic oscillation was introduced as on/off in each condition while the remaining parameters were kept constant. After processing, the changes in the specimen surfaces were examined via scanning electron microscopy (SEM) and surface roughness values. As a result, the surface properties of specimens processed with and without ultrasonic effect were compared.

Abstract Joint modeling of correlated multi-environment and multi-harvest data of perennial crop species may offer advantages in prediction schemes and a better understanding of the underlying dynamics in space and time. The goal of the present study was to investigate the relevance of incorporating the longitudinal dimension of within-season multiple harvests of biomass yield and nutritive quality traits of forage perennial ryegrass ( Lolium perenne L.) in a reaction norm model setup that additionally accounts for genotype-environment interactions. Genetic parameters and accuracy of genomic breeding value predictions were investigated by fitting three random regression (random coefficients) linear mixed models (gRRM) using Legendre polynomial functions to the data. All models accounted for heterogeneous residual variance and moving average-based spatial adjustments within environments. The plant material consisted of 381 bi-parental family pools and four check varieties of diploid perennial ryegrass evaluated in eight environments for biomass yield and nutritive quality traits. The longitudinal dimension of the data arose from multiple harvests performed four times annually. The specified design generated a total of 16,384 phenotypic data points for each trait. Genomic DNA sequencing was performed using DNA nanoball-based technology (DNBseq) and yielded 56,645 single nucleotide polymorphisms (SNPs) which were used to calculate the allele frequency-based genomic relationship matrix used in all genomic random regression models. Biomass yield’s estimated additive genetic variance and heritability values were higher in later harvests. The additive genetic correlations were moderate to low in early measurements and peaked at intermediates, with fairly stable values across the environmental gradient, except for the initial harvest data collection. This led to the conclusion that complex genotype-by-environment interaction (G×E) arises from spatial and temporal dimensions in the early season, with lower re-ranking trends thereafter. In general, modeling the temporal dimension with a second-order orthogonal polynomial in the reaction norm mixed model framework improved the accuracy of genomic estimated breeding value prediction for nutritive quality traits, but no gain in prediction accuracy was detected for dry matter yield. This study leverages the flexibility and usefulness of gRRM models for perennial ryegrass research and breeding and can be readily extended to other multi-harvest crops.

Near infrared spectroscopy (NIRS) is an accurate, fast and nondestructive technique whose use in predicting forage quality has become increasingly relevant in recent decades. Epichloë-infected grass varieties are commonly used in areas with high pest pressure due to their better performances compared to endophyte-free varieties. The insect resistance of Epichloë-infected grasses has been associated with four main groups of endophyte secondary metabolites: ergot alkaloids, indole-diterpenes, lolines and peramine. Concentrations of these alkaloids are usually measured with high performance liquid chromatography or gas chromatography analysis, which are accurate methods but relatively expensive and laborious. In this paper, we developed a rapid method based on NIRS to detect and quantify loline alkaloids in wild accessions of Schedonorus pratensis infected with the fungal endophyte Epichloë uncinata. The quantitative NIR equations obtained by modified partial least squares algorithm had coefficients of correlation of 0.90, 0.78, 0.85, 0.90 for N-acetylloline, N-acetylnorloline and N-formylloline and the sum of the three, respectively. The acquired NIR spectra were also used for developing an equation to predict in planta fungal biomass with a coefficient of correlation of 0.75. These results showed that the use of NIRS and chemometrics allows the quantification of loline alkaloids and mycelial biomass in a heterogeneous set of endophyte-infected meadow fescue samples.

Grass endophytes have been shown to confer enhanced environmental resilience to symbiont cultivars with reports of modified growth. If inoculating with an endophyte (E+) made an accession morphologically distinct from its registered endophyte free (E−) accession, there could be protection and ownership issues for testing authorities and breeders. This study investigated if, in official Plant Breeders Rights (PBR) field trials, the morphological characteristics of E+and E− accessions of perennial ryegrass and tall fescue cultivars were sufficiently modified to designate them as mutually distinct and also distinct from their definitive accessions (Def), held by the testing authorities. Testing perennial ryegrass on 17 characters at 2 sites generated 48,960 observations and for tall fescue on 9 characters at 1 site, 12,960 observations (each for 3 accessions of 4 cultivars × 60 plants × 2 growing cycles). Distinctness required a p < 0.01 difference in a single character from the combined over years analysis (COYD). A few significant differences were recorded between E− and E+accessions. Cultivar Carn E+ was smaller than Carn E− for Infloresence Length (p < 0.01) in both years but COYD analysis (p < 0.05) was insufficient to declare distinctiveness. Overall, the number of observed differences between E−/E+ accessions was less or similar to the number expected purely by chance. In contrast, comparisons between Def and E− or E+ accessions showed a number of significant differences that were substantially more numerous than expected by chance. These results showed no conclusive evidence of endophyte inclusion creating false PBR distinctions but unexpectedly, several E− and E+ accessions were distinguished from their official definitive stock.

Brachypodium distachyon is an established model for drought tolerance. We previously identified accessions exhibiting high tolerance, susceptibility and intermediate tolerance to drought; respectively, ABR8, KOZ1 and ABR4. Transcriptomics and metabolomic approaches were used to define tolerance mechanisms. Transcriptional analyses suggested relatively few drought responsive genes in ABR8 compared to KOZ1. Linking these to gene ontology (GO) terms indicated enrichment for “regulated stress response”, “plant cell wall” and “oxidative stress” associated genes. Further, tolerance correlated with pre-existing differences in cell wall-associated gene expression including glycoside hydrolases, pectin methylesterases, expansins and a pectin acetylesterase. Metabolomic assessments of the same samples also indicated few significant changes in ABR8 with drought. Instead, pre-existing differences in the cell wall-associated metabolites correlated with drought tolerance. Although other features, e.g., jasmonate signaling were suggested in our study, cell wall-focused events appeared to be predominant. Our data suggests two different modes through which the cell wall could confer drought tolerance: (i) An active response mode linked to stress induced changes in cell wall features, and (ii) an intrinsic mode where innate differences in cell wall composition and architecture are important. Both modes seem to contribute to ABR8 drought tolerance. Identification of the exact mechanisms through which the cell wall confers drought tolerance will be important in order to inform development of drought tolerant crops.

Hybrid breeding has contributed significantly to the enormous yield increases that many major crops have undergone during the previous century. Its success relies on the exploitation of heterosis, the superior performance of an F1 hybrid compared to its inbred parents. Attempts to implement hybrid breeding in forage grasses, such as perennial ryegrass (Lolium perenne L.), are hampered by its highly effective self-incompatibility system as well as its sensitivity to inbreeding depression. Homozygous inbred lines are therefore difficult to create using the classical method of repeated selfing. Here, we report an efficient method to obtain homozygous genotypes of perennial ryegrass using doubled haploid (DH) induction. By means of anther culture, completely homozygous lines were obtained within one generation cycle. A highly genotype dependent response was observed for traits such as the number of embryos/calli per 100 cultured anthers and the percentages of green and albino plants regenerated. Transgressive segregation, indicative of heritable and polygenic control of the traits, was also found. Our general aim is to develop a molecular marker system to select for high responsiveness and to facilitate the introgression of this trait into advanced breeding germplasm. Segregating mapping populations will be phenotyped during anther culture and genotyped via a genotyping-by-sequencing (GBS) approach. Family-based association mapping will be used to identify marker-trait associations. In this way, an efficient breeding tool to screen germplasm for DH induction capacity will be developed. Our work will significantly accelerate forage grass breeding and constitute the first step towards efficient production of grass hybrids.