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

Polymorphism of the nuclear ribosomal DNA intergenic spacer (IGS) of the ec tomycorrhizal basidiomycete Hebeloma cylindrosporum was studied to evaluate whether this sequence could be used in field studies to estimate the diver sity of strains forming mycorrhizas on individual Pinus pinaster root syste ms. This sequence was amplified by PCR from 125 haploid homokaryotic strain s collected in 14 P. pinaster stands along the Atlantic coast of France by using conserved oligonucleotide primers. Restriction enzyme digestion of th e amplified 3.4-kbp-long IGS allowed us to characterize 24 alleles whose fr equencies differed. Nine of these alleles were found only once, whereas abo ut 60% of the strains contained four of the alleles. Local populations coul d be almost as diverse as the entire population along a 150-km stretch of c oastline that was examined; for example, 13 alleles were found in a single forest stand. The IGS from one strain was partially sequenced, and the sequ ence data were used to design oligonucleotides which allowed separate PCR a mplification of three different segments of the IGS. Most polymorphisms obs erved among the full-length IGS regions resulted from polymorphisms in an i nternal 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 ha ploid 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. pi naster mycorrhizal root tips, thus demonstrating the usefulness of this seq uence for studying the below-ground diversity of mycorrhizas formed by gene ts belonging to the same fungal species.