Infection Rates and Alkaloid Patterns of Different Grass Species with Systemic Epichloë Endophytes

Abstract

Symbiotic Epichloë species are fungal endophytes of cool-season grasses that can produce alkaloids with toxicity to vertebrates and/or invertebrates. Monitoring infections and presence of alkaloids in grasses infected with Epichloë species can provide an estimate of possible intoxication risks for livestock. We sampled 3,046 individuals of 13 different grass species in three regions on 150 study sites in Germany. We determined infection rates and used PCR to identify Epichloë species diversity based on the presence of different alkaloid biosynthesis genes, then confirmed the possible chemotypes with high-performance liquid chromatography (HPLC)/ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) measurements. Infections of Epichloë spp. were found in Festuca pratensis Huds. (81%), Festuca ovina L. aggregate (agg.) (73%), Lolium perenne L. (15%), Festuca rubra L. (15%) and Dactylis glomerata L. (8%). The other eight grass species did not appear to be infected. For the majority of Epichloë-infected L. perenne samples (98%), the alkaloids lolitrem B and peramine were present, but ergovaline was not detected, which was consistent with the genetic evaluation, as dmaW, the gene encoding the first step of the ergot alkaloid biosynthesis pathway, was absent. Epichloë uncinata in F. pratensis produced anti-insect loline compounds. The Epichloë spp. observed in the F. ovina agg. samples showed the greatest level of diversity, and different intermediates of the indole-diterpene pathway could be detected. Epichloë infection rates alone are insufficient to estimate intoxication risks for livestock, as other factors, like the ability of the endophyte to produce the alkaloids, also need to be assessed. IMPORTANCE Severe problems of livestock intoxication from Epichloë-infected forage grasses have been reported from New Zealand, Australia, and the United States, but much less frequently from Europe, and particularly not from Germany. Nevertheless, it is important to monitor infection rates and alkaloids of grasses with Epichloë fungi to estimate possible intoxication risks. Most studies focus on agricultural grass species like Lolium perenne and Festuca arundinacea, but other cool-season grass species can also be infected. We show that in Germany, infection rates and alkaloids differ between grass species and that some of the alkaloids can be toxic to livestock. Changes in grassland management due to changing climate, especially with a shift toward grasslands dominated with Epichloë-infected species such as Lolium perenne, may result in greater numbers of intoxicated livestock in the near future. We therefore suggest regular monitoring of grass species for infections and alkaloids and call for maintaining heterogenous grasslands for livestock.

Epichloë Endophyte Infection Rates and Alkaloid Content in Commercially Available Grass Seed Mixtures in Europe

“…Climate change could result in environmental conditions that are more conducive to survival of infected grasses, which may increase their distribution to a level that endophyte-infected plants could dominate grasslands. Previous studies on grasslands in Germany and Spain showed that single individuals of cool season grass species infected with Epichloë can contain alkaloid concentrations above the toxicity thresholds for livestock [35,46,55], which for ergovaline are 300-500 ppb for cattle and horses, and 500-800 ppb for sheep, with lolitrem B concentrations toxic at 1800-2000 ppb [48]. In typical meadows, the infected toxic grasses are diluted by a high diversity of other species and low infection frequencies, so have been of little concern for causing toxicity.…”

“…Endophyte infection was performed at the Noble Research Institute, LLC, Ardmore, Oklahoma, USA, using a multiplex PCR with endophyte specific primers on DNA extracted from individual seeds randomly selected from each of the seed mixtures, as described previously [35]. Each seed lot was tested twice.…”

“…In both screens, 22-24 seeds per mixture were tested and the first screen was single blinded. The PCR was performed using only the multiplex 1 primer set [35] with 6 µL instead of 3 µL of DNA, and the number of PCR cycles was increased to 33 cycles as the DNA template concentration was low.…”

“…Epichloë intoxication events are more common in New Zealand, Australia, and the U.S. compared to Europe because overseas grasslands are often dominated by a single non-native grass species with high endophyte infection rates. For example, the Epichloë infection rate of non-native perennial ryegrass is 70% in New Zealand [34] compared to 15% in its native distribution range in Germany [35]. To overcome vertebrate toxicity, Epichloë strains have been found and introduced into grass cultivars that are unable to produce the causative toxins [36,37].…”

“…In other regions of the world, the distribution and toxicity of Epichloë endophytes has received much attention [9], but very limited in European grasslands [35,42,43] and almost none in commercially available cultivars and seed mixtures on the European market [43,44]. The aim of this study is to provide an overview on infection rates of grass seeds with Epichloë spp.…”