A Staining Method for Systemic Endophytic Fungi in Plants

Native desert plants are extensively colonized by dark septate fungal endophytes (DSE). These are characterized by stained or pigmented hyphae and microsclerotia growing inter- and intra-cellularly within the root cortex. A dual-staining method was developed using trypan blue that targets fungal chitin and sudan IV that targets internal fungal lipid bodies. Roots and leaves of dominant grasses and shrubs of arid southwestern rangeland were dual-stained to determine the nature and extent of colonization by DSE fungi. Fungal structures ranged from lipid bearing protoplasts, hyaline, stained and melanized hyphae and microsclerotia. They inhabit the apoplastic spaces of roots and shoots. Predominant fungal structures are protoplasts in physiologically active tissues that are identified with stained, associated lipid bodies. This method shows colonization by DSE fungi is more extensive than was previouisly throught.