Effective protoplasting of Neotyphodium endophytes in forage grasses

Due to the characterization of the secondary metabolites they produce, Neotyphodium endophytes have become a major agronomic concern in the forage grass industries. Protoplast fusion has been successfully carried out in many different categories of fungi. This being the case, we wish to explore its practicality with Neotyphodium mycosymbionts. Protoplasts of Neotyphodium lolii have been formed in an effort to pursue nuclear fusion and recombination with protoplasts of Neotyphodium coenophialum. In order to facilitate work with this generally slow growing fungus a unique PDA based media system is proposed in effort to increase endophyte growth rate. Visualization of endophytes to verify infection is done quite regularly throughout the world. Due to unknown reasons however, the conventional phenolic based aniline blue stain is not sufficient to visualize the mycelia within the leaf sheaths of our grass in the greenhouse in Utah. Visualization of the endophyte in Utah is demonstrated using a non-phenolic based aniline blue stain and using the non-phenolic stain rose bengal.