Determining the Earliest Growth Stage to Detect the Presence of Endophytes in Tall Fescue and Perennial Ryegrass Seedlings Using Molecular Markers

Abstract

Abstract Background Tall fescue and perennial ryegrass are important cool-season forage and amenity grasses. Both types of grass have a mutualistic association with an endophytic fungus. Novel endophytes, non-toxic to livestock, have been bred in association with elite cultivars of the two species. It is critically important for producers to ensure that their seeds have an adequate level of endophyte-infection. There are different ways to test for endophyte presence and toxicity. Immunoblot tests have been commonly used but molecular markers have been developed that can be used as well. The goal of this research was to identify the earliest growth stage endophyte presence in tall fescue and perennial ryegrass can be successfully detected following seed germination using molecular markers as compared to immunoblot tests. We also sought to test an in-house primer, lolF, against a housekeeping gene marker, tef1exon, for endophyte detection. Results We found that molecular marker detection is limited at 14 DAG as there are not enough mycelia in the leaf tissue for DNA extraction and PCR to be carried out. Molecular markers can be reliably used at 28 DAG and later, whereas immunoblot testing can be done as early as 14 DAG. We find that at the time points when they can be reliably tested, molecular markers are comparable to the immunoblot assay in identifying endophyte presence. We also found that lolF is equal in endophyte detection to tef1exon in tall fescue but is not effective in perennial ryegrass. Conclusions Immunoblot assays identify endophyte presence at the earliest time point. Molecular markers can be reliably used at 28 days post germination and later. These findings provide researchers and producers with useful information that help them decide when and how to test their populations for endophytes.