SUMMARYThere is a clear requirement to develop sensitive methods for detecting defined isolates of ectomycorrhizal fungi within the complex microbial communities of natural ecosystems and reforestation sites. We present a method that permits the rapid identification of an ectomycorrhizal isolate using enzymatic amplification (polymerase chain reaction) of DNA extracted either from pure cultures or ectomycorrhizas. A set of oligonucleotide primers capable of amplifying full-length nuclear 17S and 25S ribosomal RNA genes, together with the ribosomal internal transcribed spacer and intergenic spacer, have been designed and could be used for amplifying target sequences from a wide range of ectomycorrhizal genera. Length polymorphism in the amplified rDNA and restriction endonuclease analysis of nearly 6-0 kbp of amplified rDNA provided useful criteria for the rapid typing of isolates from different genera and species. Restriction endonuclease analysis of amplified DNA from 26 isolates representing four species oi Laccaria (L. bicolor, L. laccata, L. proximo, L. tortilis) yielded up to 20 scored RFLPs and revealed interspecific and intraspecific polymorphism. Most of the polymorphisms were located within the regions corresponding to the internal transcribed spacer and intergenic spacer. The degree of variation observed was sufificient to discriminate several isolates from the same species. Genetic variation was correlated to some extent with geographical origin of the isolates. However, RFLPs of the rRNA genes cannot unambiguously discriminate all selected isolates within Laccaria species, requiring the development of additional DNA probes. Alone, or in combination with other DNA probes, the amplified rDNA genes may serve in the determination of pure fungal cultures and in the characterization of genetic variation of field ectomycorrhizal populations.

No abstract

SUMMARYSitka spruce mycorrhizas, macroscopically identified as being formed by Tylospora fibrillosa Donk, were sampled from a young and an old plantation and the mycobionts were isolated into pure culture. DNA was extracted and fragments of the ribosomal DNA (rDNA) were amplified using the polymerase chain reaction (PCR). The primers were directed to conserved regions of fungal rDXA and hybridize to a wide range of fungi. One amplified region includes the internal spacer (ITS) region which has a low degree of conservation. The ITS amplification products, which were approximately 600 base pairs (bp), were digested with a variety of restriction endonucleases in order to detect restriction fragment length polymorphisms (RFLPs). The RFLPs clearly separated T. fibrillosa from other ectomycorrhizal species but there were only minor differences between the T. fibrillosa isolates. PCR amplification of the ITS region, digestion with the endonuclease Hin/ I and examination of the RFLPs produced proved to be a rapid method by which to distinguish T. fibrillosa from a large number of other basidiomycetes. The method was also applied to DNA extracted from single mycorrhiza! root tips. The lntergenic spacer region (IGS) of the rDNA is more variahle than the ITS region in several fungal species. The 5' end of the 25S and the intergenic region between the 25S and the 5S genes were amplified and analyzed as above. Polymorphisms between T. fibrillosa isolates within this region were limited and RFLPs were not useful in discriminating between isolates, suggesting a low genetic variability in this species.

The large‐scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR‐amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large‐scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated‐Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large‐scale microbial application.

No abstract