Analysis, determination and cultivation of endophytic fungi associated with the orchid Spiranthes spiralis.

Mycorrhizas can sustain plants in most habitats through mutualistic interactions with fungi. The presence of these in internalized location can be due to true symbiotic relationships or to different levels of endophytism. In the case of orchids the fungal assistance is crucial throughout the seedling stage and only established plants begin to return organic carbon to the symbiont. Fungi from orchids are often difficult to isolate and their cultivation depends on seasonal and physiological conditions. The submediterranean species Spiranthes spiralis (L.) Chevall. is the latest-blooming native orchid in western Europe. Its rosettes appear in summer to die off in the following spring, while stalks flower in September (Willems and Dorland, 2000). The putative mycorrhizal fungi were isolated in culture from the surfacesterilized Spiranthes spiralis roots and their taxonomical identity was assessed by molecular techniques upon amplification of diagnostic ribosomal DNA regions. In parallel, DNA was isolated directly from the root tissues for comparison. Selective PCR amplification using ITS1-ITS4, ITS1F-ITS4 primers was carried out. Additionally, using fluorescence and confocal microscopy on acridine orange-stained freehand sections we observed a diffuse cortical colonization by intracellular hyphae. Their further ultrastructural details were resolved by electron microscopy.