VARIATION IN THE ITS AND IGS REGIONS OF RIBOSOMAL DNA AMONG THE BIOLOGICAL SPECIES OF EUROPEAN ARMILLARIA

Variation within the internal transcribed spacer (ITS) and the interge nic spacer (IGS) of the ribosomal RNA gene of isolates representing se ven European species of Armillaria was examined by PCR, coupled with R FLP analysis and partial sequencing of the ITS region. The amplified-I TS region was about 840 bp long and uniform in length among the Europe an isolates, while the amplified-IGS region showed four different leng ths which corresponded to A. mellea, A. tabescens, A. ectypa and a gro up including the four other species. A. borealis, A. ostoyae, A. cepis tipes and A. gallica. Restriction digestions of the ITS and IGS region s by Alu I and Rsa I, respectively, gave rise to the same four polymor phic groups. However, A. borealis and A. ostoyae were easily separated from A. cepistipes and A. gallica by digestion of the two rDNA spacer s with Hinf I and Taq I. Nde II digests of the amplified ITS could dis tinguish A. borealis from A. ostoyae and each of the seven species wer e separated by Alu I digestion of the IGS region. A North American A. mellea isolate, partly examined in this work, was found to be differen t from the European A. mellea isolates, while there was a close simila rity between the European A. ostoyae and an isolate of the same specie s isolated from the U.S.A. Cluster analysis based on the presence or a bsence of comigrating restriction fragments indicated more than 80% si milarity between A. borealis and A. ostoyae and between A. cepistipes and A. gallica, while A. mellea, A. tabescens and A. ectypa were found in separate clusters exhibiting, respectively, about 40, 38 and 32% a verage similarity with the other species. Although little intraspecifi c variation was observed in many species, A. gallica and A. cepistipes were found to be heterogeneous. In view of recent results suggesting several groups in A. borealis and A. cepistipes, the Alu I restriction patterns obtained in this work would identify the former species as t ype B, characterized by an Abi I pattern different from that of A. ost oyae, and the latter species as composed of one isolate (C4) of type B and three isolates (CI, C2 and C3) of type A. Nucleotide sequences of the rDNA internally transcribed spacer I (ITS1) of one isolate of eac h species showed that A. mellea and A. ectypa differed from the other species by several insertions and point mutations giving rise to a lev el of similarity ranging from 66 to 79%. This DNA region was highly co nserved within the other species which revealed a similarity of 97 to 100%. These results demonstrate that analysis of rDNA spacers provides appropriate data to circumscribe taxonomic entities within the Armill aria complex and that the phylogenic relationships among species deduc ed from the present study are consistent with previous analyses based on pairing tests as well as morphological and physiological characters .