Species belonging to the Festuca-Lolium complex are often naturally infected with endophytic fungi of genus Epichloë . Recent studies on endophytes have shown the beneficial roles of host-endophyte associations as protection against insect herbivores in agriculturally important grasses. However, large-scale screenings are crucial to identify animal friendly strains suitable for agricultural use. In this study we analyzed collected populations of meadow fescue ( Schedonorus pratensis ) from 135 different locations across Europe, 255 accessions from the United States Department of Agriculture and 96 accessions from The Nordic Genetic Resource Centre. The analysis also included representatives of S. arundinaceus , S. giganteus , and Lolium perenne . All plants were screened for the presence of Epichloë endophytes, resulting in a nursery of about 2500 infected plants from 176 different locations. Genetic diversity was investigated on 250 isolates using a microsatellite-based PCR fingerprinting assay at 7 loci, 5 of which were uncharacterized for these species. Phylogenetic and principal components analysis showed a strong interspecific genetic differentiation among isolates, and, with E. uncinata isolates, a small but significant correlation between genetic diversity and geographical effect ( r = 0.227) was detected. Concentrations of loline alkaloids were measured in 218 infected meadow fescue plants. Average amount of total loline and the proportions of the single loline alkaloids differed significantly among endophyte haplotypes ( P < 0.005). This study provides insight into endophyte genetic diversity and geographic variation in Europe and a reference database of allele sizes for fast discrimination of isolates. We also discuss the possibility of multiple hybridization events as a source of genetic and alkaloid variation observed in E. uncinata .

Many efforts have been made to select and isolate naturally occurring animal-friendly Epichloë strains for later reinfection into elite cultivars. Often this process involves large-scale screening of Epichloë-infected wild grass populations where strains are characterized and alkaloids measured. Here, we describe for the first time the use of genotyping-by-sequencing (GBS) on a collection of 217 Epichloë-infected grasses (7 S. arundinaceum, 4 L. perenne, and 206 S. pratensis). This genotyping strategy is cheaper than complete genome sequencing, is suitable for a large number of individuals, and, when applied to endophyte-infected grasses, conveniently genotypes both organisms. In total, 6273 single nucleotide polymorphisms (SNPs) in the endophyte data set and 38 323 SNPs in the host data set were obtained. Our findings reveal a composite structure with three distinct endophyte clusters unrelated to the three main S. pratensis gene pools that have most likely spread from different glacial refugia in Eurasia. All three gene pools can establish symbiosis with E. uncinata. A comparison of the endophyte clusters with microsatellite-based fingerprinting of the same samples allows a quick test to discriminate between these clusters using two simple sequence repeats (SSRs). Concentrations of loline alkaloids and mycelial biomass are correlated and differ significantly among the plant and endophyte subpopulations; one endophyte strain has higher levels of lolines than others, and one specific host genotype is particularly suitable to host E. uncinata. These findings pave the way for targeted artificial inoculations of specific host-endophyte combinations to boost loline production in the symbiota and for genome association studies with the aim of isolating genes involved in the compatibility between meadow fescue and E. uncinata.

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.