Determining the earliest growth stage to detect the presence of endophytes in tall fescue and perennial ryegrass seedlings using molecular markers

Background:Tall fescue(Festuca arundinacea[Schreb.],Lolium arundinaceum[Schreb.]Darbysh)and perennial ryegrass(Lolium perenne)are important cool-season forage and amenity grasses that have a mutualistic association with an endophytic fungus.Endophytes confer insect and drought resistance to plants but can produce mammalian toxins.Novel endophytes that do not produce mammalian toxins have been introduced to elite cultivars for commercial production.Seed companies need to maintain adequate levels of novel endophytes within the elite forage cultivars.Endophyte detection is performed using immunochemical and molecular techniques because of their speed and reliability.Early detection in seedlings is essential to evaluate the viability of the endophyte within seed lots.Methods:This research aimed to identify the earliest growth stage in which immunochemical and molecular methods can detect viable endophyte in seedlings of tall fescue cultivars BarOptima(e34),Texoma MaxQII(584),and Jesup MaxQ(542),as well as the perennial ryegrass cultivar Remington(NEA2).Results:Immunochemical testing detected endophytes in seedlings 14 days after germination(DAG),but the detection rate increased until 42 DAG in some cultivars tested.The molecular marker Tef1exon detected endophytes at a lower rate than the immunochemical method at 28–42 DAG.However,there was insufficient DNA to detect endophytes in 14 DAG seedlings using markers.Conclusions:We conclude that the most accurate detection of viable endophytes in seedlings was 42 DAG,at which sufficient and consistent endophyte colonization occurred.