Histological Methods to Detect Early-stage Plant Defense Responses during Artificial Inoculation of Lolium perenne with Epichloë festucae

Epichloë species form agriculturally important symbioses with many cool season grasses. To study these symbioses, such as the interaction of Epichloë festucae with perennial ryegrass (Lolium perenne), host plants can be infected by artificial inoculation of etiolated seedlings. This inoculation is performed by placing mycelium into an incision in the meristem, as previously described by Latch and Christensen (1985). In recent years, this method has been broadly used to study this interaction at the molecular level using different Epichloë festucae mutants that can cause incompatible interactions. We have developed and adapted methods to study four of the most important host plant responses to infection, including cell death, callose deposition, lignin production, and hydrogen peroxide (H2O2) production, which are useful in defining the host response to infection at a very early time point.