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Molecular identification of ericoid mycorrhizal fungi Monreal, Marcia Amelia


Molecular techniques facilitated the identification of ericoid mycorrhizal fungi associated with an ericaceous plant, salal (Gaultheria shallon Purch), dominant in some reforestation sites in the Canadian West Coast. The polymerase chain reaction (PCR) was used to amplify the DNA coding for the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal repeat of 28 fungal isolates. Restriction fragment length polymorphism (RFLP) patterns of these isolates obtained using a set of four restriction enzymes were compared and a synoptic key that differentiates these isolates into 14 groups was created. A fungal specific primer (ITS1-F) was used to amplify DNA of salal mycorrhizae obtained under axenic conditions with specific combinations of salal plants and fungal inoculum. Comparison among RFLP patterns of the fungal isolates with salal mycorrhizae showed that the fungal specific primer (ITS1-F) allowed the preferential amplification of the fungal component of the mycorrhizal association. RFLPs of fungal isolates and salal mycorrhizae were identical. Mycorrhizal root fragments from a salal plant were collected at a reforestation site on Vancouver Island. From one set of root fragments, DNA was extracted and amplified by PCR. Subsequent RFLP patterns obtained indicated the presence of a selection of various fungi. A second set of 5 m m mycorrhizal roots fragments were plated on Petri dishes and 20 fungal isolates were obtained. The in vitro capacity to form mycorrhizae with salal was tested for all isolates and only five were confirmed mycorrhizal with salal. Known RFLP patterns were detected from two new isolates: one matched the RFLP pattern produced by Oidiodendron species and the other matched a nonsporulating fungus (Unknown 2) described from previous work. The RFLP patterns of the other 18 newly isolated sterile fungi, including three isolates that formed in vitro mycorrhizae with salal, were different from those of all known ericoid mycorrhizal fungi. The fifteen new isolates that did not form ericoid mycorrhizae in vitro corresponded with seven RFLP patterns and their roles in association with salal roots is unknown. To create a method that would allow the direct assesment of the presence of known ericoid mycorrhizal fungi in salal roots collected from the field, the internal transcribed spacer ITS2 region and 3' end of the 5.8S rRNA gene (400 base pairs) of 24 fungal isolates were amplified and sequenced. Sequence data analysis segregated the mycorrhizal isolates into two main groups, one including species of Oidiodendron, and the second including Hymenoscyphus and allied taxa. Accordingly, three different specific primers were designed, the first to amplify Oidiodendron species, and the second and third, to identify clusters of isolates from the Hymenoscyphus group. Tests performed using the new primers with fungal DNA mixtures of known mycorrhizal isolates and nonmycorrhizal fungal isolates detected only the fungal DNA.

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