1, IC TOMMERUP2, NL BOUGHER2 and PA O'BRIEN1
1 School of Biological Sciences and Biotechnology, Murdoch University, South Street, Murdoch, Western Australia, 6150; 2Forestry and Forest Products, CSIRO, PO Wembley, Western Australia, 6014
Background and Objectives
Identification of fungi inhabiting plants, whether symbiotic or parasitic, has been facilitated by the use of PCR-RFLP . This technique relies on matching an unknown organism to an identified one through similarities in DNA sequence, as determined by PCR amplification followed by digestion with restriction enzymes, avoiding the need for fungal isolation and culture. This approach is of particular benefit when working with obligatory biotrophs and other hard-to-grow species, as well as those which only occasionally produce identifying features such as basidiocarps and spores.
Ectomycorrhizal fungi, in general, fall into both these categories. Ectomycorrhizal fungi have been recognized as significant components in forest ecology, providing trees and shrubs with minerals in exchange for sugars and accelerating the rate of litter decomposition and nutrient recycling. They are involved in protecting roots from disease and enhancing disease resistance through nutritional factors and, perhaps, altered plant hormones. In comparison with forests of the Northern Hemisphere, eucalyptus forests in Australia possess a great diversity of ectomycorrhizal fungi. As well as supporting plant growth, the basidiocarps are an important food source for native marsupials. Further knowledge of these organisms is necessary to improve conservation and sustainable timber production in forests. The aim of this project is to compare the presence and abundance of ectomycorrhizal species on root tips in two adjacent blocks of Eucalyptus marginata (jarrah) forest in Western Australia. One block has been burned every 5-6 years for the past 60 years, while the other has remained unburned during that time.
Materials and methods
Basidiocarps of ectomycorrhizal fungi were collected, identified and DNA was extracted from them. The nuclear ribosomal DNA ITS region was amplified in a PCR reaction using primers specific for higher basidiomycetes. The PCR product was digested with restriction enzymes and the fragments sized after polyacrylamide gel electrophoresis. Ectomycorrhizal root tips were collected from 70 soil samples from each site, DNA extracted and the fungus identified by matching the patterns of restriction fragments with those of the identified basidiocarps.
Results and conclusions
Primers amplified DNA from all but one of the 60 basidiomycete species tested, with no product from plant, bacterial or ascomycete DNA. Digestion with two or three restriction enzymes gave a uniquely distinguishable pattern of fragments for each species. PCR-RFLP analysis of the root tips resulted in a further 100 patterns that are as yet unmatched to described taxa. That a total of at least 150 species occur in 3 ha illustrates the great diversity of Australian ectomycorrhizal fungi. The results reveal very clear differences in the relative abundance of fungal species on each of the forest blocks, emphasizing rather than contradicting the differences shown by sampling of basidiocarps.
1. Gardes M, Bruns TD, 1996. In JP Clapp (ed.) Methods in Molecular Biology, Vol. 50: Species Diagnostic Protocols: PCR and Other Nucleic Acid Methods. Totowa, NJ: Humana Press Inc., pp. 177-186.