Fungal composition of lichen thalli assessed by single strand conformation polymorphism

Fungi that are unrelated to the mycobiont species frequently colonize lichens. Some of these fungal colonists are described lichenicolous fungi, lichen parasites and pathogens that produce recognizable morphological characters, while others apparently produce no noticeable structures. Here we apply the single strand conformation polymorphism (SSCP) technique to directly assess the abundance of different fungi in lichens. Twenty-eight lichen thalli were chosen, some with and some without externally visible symptoms of parasite infection, and these were subjected to total DNA extraction. PCR was conducted with fungal-specific primers for the ITS region of ribosomal DNA. Single strands of the products were separated on native acrylamide gels. The majority of lichen specimens, both infected and those without symptoms, displayed more than one band in the stained gels. In one case, 14 bands were detected using SSCP. Some of these bands apparently represent other neighbouring lichens in the habitat, but many are apparently non-lichen-forming. Since few lichen-associated fungi have been cultured and sequenced, it is difficult to know if SSCP bands represent obligate lichenicolous fungi, other asymptomatic lichen parasites, or fungi not obligately associated with lichens, but our results indicate that large numbers of non-lichen-forming fungi commonly co-occur with lichens in nature. For specimens of the filamentous lichens Cystocoleus ebeneus and Racodium rupestre we used cloned sequences to compare the number of sequences obtained by the SSCP method to the number obtained by direct sequencing of thallus extracts, and we generally found that more sequences could be detected by SSCP than could be seen by direct sequencing.