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The Nuclear Ribosomal DNA Intergenic Spacer as a Target Sequence To Study Intraspecific Diversity of the Ectomycorrhizal Basidiomycete Hebeloma cylindrosporum Directly on Pinus Root Systems

Abstract : Polymorphism of the nuclear ribosomal DNA intergenic spacer (IGS) of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was studied to evaluate whether this sequence could be used in field studies to estimate the diversity of strains forming mycorrhizas on individual Pinus pinaster root systems. This sequence was amplified by PCR from 125 haploid homokaryotic strains collected in 14 P. pinaster stands along the Atlantic coast of France by using conserved oligonucleotide primers. Restriction enzyme digestion of the amplified 3.4-kbp-long IGS allowed us to characterize 24 alleles whose frequencies differed. Nine of these alleles were found only once, whereas about 60% of the strains contained four of the alleles. Local populations could be almost as diverse as the entire population along a 150-km stretch of coastline that was examined; for example, 13 alleles were found in a single forest stand. The IGS from one strain was partially sequenced, and the sequence data were used to design oligonucleotides which allowed separate PCR amplification of three different segments of the IGS. Most polymorphisms observed among the full-length IGS regions resulted from polymorphisms in an internal ca. 1,500-bp-long sequence characterized by length variations that may have resulted from variable numbers of a T 2 AG 3 motif. This internal polymorphic sequence could not be amplified from the genomes of nine other Hebeloma species. Analysis of this internal sequence amplified from the haploid progenies of 10 fruiting bodies collected in a 70-m 2 area resulted in identification of six allelic forms and seven distinct diplotypes out of the 21 possible different combinations. Moreover, optimization of the PCR conditions resulted in amplification of this sequence from more than 80% of the DNA samples extracted from individual H. cylindrosporum infected P. pinaster mycorrhizal root tips, thus demonstrating the usefulness of this sequence for studying the below-ground diversity of mycorrhizas formed by genets belonging to the same fungal species. In most terrestrial ecosystems, the root system of a single plant may host a variety of symbionts. Thus, different fungal ectomycorrhizal (ECM) species may contribute, according to their relative abundance on the root system and to their physiological properties, to improving the growth and fitness of a common host tree. These multipartner symbioses are not stable but evolve during the life of the host plant, as illustrated by results based on samples of basidiocarps of different ECM species found in the vicinity of individual trees (23). It was recently found that results of ECM community studies based on basidiocarp records (above-ground view) may be misleading and do not necessarily reflect the frequency and abundance of individual ECM species on root systems (below-ground view) (13, 24). At the intraspecific level, genetically distinct mycelia of the same ECM species can also coexist on the root system of a single tree. This was demonstrated after coinoculation of Pinus banksiana seedlings with different strains of Laccaria bicolor (9) and was deduced from the results of field studies which showed that up to nine different genets of the agaric Hebeloma cylindrosporum can be found in a 1-m 2 patch of ground under a Pinus pinaster tree (14). Population studies of the latter ECM species were conducted after basidiocarp samples were obtained in the same forest stands in successive fruiting seasons, and the results revealed that there were high levels of genetic diversity within local populations. At two of the three sites sampled, there were never more than two basidiocarps that had the same genotype (i.e., emerged from the same below-ground mycelium). Moreover, none of the genets identified during the first year of the study was found 3 years later. Such observations made at the intraspecific level pose questions similar to the questions already addressed at the community level concerning the below-ground distribution of individual genets. To answer such questions, a powerful method is needed to discriminate between different below-ground individuals. One of the most accurate methods for identifying the fungal sym-biont present in a single, field-collected, mycorrhizal root tip is PCR-restriction fragment length polymorphism (RFLP) analysis of polymorphic DNA sequences. In ECM community studies , one of the most-studied DNA sequences is the PCR-amplified nuclear ribosomal internal transcribed spacer (ITS) sequence. ITS sequences can easily be amplified from minute amounts of DNA with PCR primers (34) and are conserved at the species level; however, there are enough differences be-* Corresponding author. Mailing address:
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Alice Guidot, Erica Lumini, Jean-Claude Debaud, Roland Marmeisse. The Nuclear Ribosomal DNA Intergenic Spacer as a Target Sequence To Study Intraspecific Diversity of the Ectomycorrhizal Basidiomycete Hebeloma cylindrosporum Directly on Pinus Root Systems. Applied and Environmental Microbiology, American Society for Microbiology, 1998, 65, pp.903- 909. ⟨hal-02936799⟩

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