Direct PCR of symbiotic fungi using microslides.

Successful DNA isolation is a critical step in molecular studies of symbiotic fungi including plant pathogens, mycorrhizal fungi, endophytic fungi and lichen-forming fungi. Whereas standard isolation protocols are usually the straightforward approach if axenic cultures are available, it is problematic to isolate DNA from fungi that grow in intricate associations with some other organisms. If the symbionts cannot be separated and the isolate contains DNA of different sources, discriminative primers are used during polymerase chain reaction (PCR) to obtain products from the organisms of interest. For example, studies of nuclear small subunit ribosomal (r)DNA from lichen-forming ascomycetes rely on primers that do not amplify the algal symbiont (2,3). Such primers still have to be developed for other genes, and if two closely related fungi occur together, the design of specific primers might be difficult. One strategy in such cases is the careful preparation of small amounts of homogeneous starting material. There are spectacular reports on DNA amplification from single spores (6), but our experience is that this procedure has failed with a variety of organisms, particularly lichens. Previously, a special protocol had been developed for DNA isolation from lichen fruitbodies (4). However, using very small amounts of lichens as starting material can be cumbersome because this includes an increased risk of obtaining odd amplification results due to contaminating organisms. Therefore, it would be ideal to check the material for contaminants under a microscope before PCR. Here, we describe an alternative approach that we often used successfully to amplify DNA from symbiotic fungi. Sections of the organisms were mounted on small glass splinters, and PCR was carried out with fungal-specific primers with this material without previous DNA isolation. Earlier, direct PCR using sections had been carried out with standard microscopic slides (8). In addition to the handling problems that occur when a large number of samples are processed, these techniques require considerable space on a thermal cycler, whereas amplification in our protocol is carried out in tubes conventionally used for PCR. This is possible by using 2× 10-mm large glass slides (or microslides) (5) that were cut from standard coverslips using a diamond cutter. The glass slides were washed in sterile water, 2× for 5 min in 100% EtOH, air-dried and immersed in a gelatin/chromalum solution (0.25% gelatin, 0.025% chromium III potassium sulphate) for 5 s under sterile conditions. Then, the coated microslides were dried on an aluminum foil. To test whether it is possible to selectively amplify material of co-existing fungi, we used a lichen that was infected by another fungus. The parasite Arthonia molendoi develops black fertile structures in the orange, disc-like fruitbodies of its host lichen Xanthoria elegans. An infected fruitbody of Xanthoria was carefully oriented for cryosectioning (-15°C knife temperature; Cryostat; Leitz, Wetzlar, Germany) to obtain a series of parallel, horizontal sections from the apex of the black lichenicolous fruitbody downwards into the infected host. The first few sections with larger amounts of dark pigmented layers of the lichenicolous fruitbodies were discarded. Several sections of 4μm thickness measured 60 × 60 to 750 × 550 μm. In the first of these sections, no material from the host lichen was apparent, whereas subsequent sections contained increasing amounts of material from the host. Each section was placed close to the very bottom of a separate, coated glass slide, which was handled with sterile forceps. The microslides with attached sections were then collected in a Petri dish and dried for 5 min at 45°C in an oven. After drying, the slides were microwaved for 10 min in a conventional microwave oven (800 W maximal power output with 2450 mHz operating frequency), which enhanced the attachment to the slides. A half-closed 500-mL bottle containing 150 mL dis-