Large Scale Screening of Epichloë Endophytes Infecting Schedonorus pratensis and Other Forage Grasses Reveals a Relation Between Microsatellite-Based Haplotypes and Loline Alkaloid Levels

Abstract

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 .

Determination of Loline Alkaloids and Mycelial Biomass in Endophyte-Infected Schedonorus pratensis by Near-Infrared Spectroscopy and Chemometrics

“…The measurement of the exact concentration of each of the three loline alkaloids might be interesting for some studies, but in terms of insect resistance, the most studied trait is the total amount of lolines [28,49] which can deter insects from feeding on the host or have insecticide effects. Large-scale screenings are used to isolate strains producing high levels of loline alkaloids to artificially inoculate them in elite grass varieties [36]. In this respect NIRS can be used for a fast and relatively cheap preliminary screening to isolate the most performing strains, where the high levels of lolines are only slightly affected from the SECV.…”

“…The samples for this study were a selection of 216 meadow fescue plants infected with Epichloë uncinata previously characterized by Cagnano et al [36]. This dataset included 135 plants collected in wild and semi-wild meadows in different countries in Europe, 65 plants from accessions requested to The Nordic Genetic Resource Centre (NordGen) and 19 plants from accessions requested to the United States Department of Agriculture (USDA).…”

“…Non-infected plants did not produce the alkaloids. Results of the chemical measurement have been described in Cagnano et al [36]. Briefly, there was a wide variation of the total concentration of lolines, calculated as sum of the concentrations of the single compounds (NAL, NANL and NFL), spanning from barely detectable traces (<25 mg•kg −1 ) up to 5629 mg•kg −1 ( Table 1).…”

Impact of endophyte inoculation on the morphological identity of cultivars of Lolium perenne (L) and Festuca arundinacea (Schreb.)

“…This was done by agrinostic immunoblot SKU Number: Endo797-3 http://www.agrinostics.com/shop/. As per this previously published detection method 22 , the presence of Epichloë endophytes was assessed in two tiller sections from each plant, 6 weeks after transplanting, using the immunoblot assay according to the manufacturer's description. The presence of endophyte was tested with an in planta assay using microsatellite markers B10 and B11 23 on up to 200 tillers per accession.…”

The Impact of Alkaloid-Producing Epichloë Endophyte on Forage Ryegrass Breeding: A New Zealand Perspective

“…A recent study (Barenbrug NZ 2020, unpublished) using a non-lolitrem-producing strain nea3, was shown to produce high levels of paxilline and terpendole C greater than observed in SE strain and under hot dry summer conditions with ‘rank’ feed this was sufficient to cause ryegrass staggers, even though no lolitrem B was detectable. Analysis of E. uncinata haplotypes for loline content identified similar differences in the three forms of loline (NAL, NANL, and NFL) between different haplotypes [ 54 ] Analysis of six endophytes by Young 2013 looked at presence vs. absence of the Eas gene cluster and whether ergot alkaloids were produced or not [ 55 ]. The complex nature of ergot production in the Claviceps [ 48 ], between strain variation of the genes, expression profiles, and ergot bioactivity suggest that a greater level of characterisation is needed than is currently undertaken before a new endophyte strain is advanced for commercial exploitation.…”