Genotypic Diversity among Brazilian Isolates of Sclerotium rolfsii

Thirty isolates of Sclerotium rolfsii Sacc. from di€erent hosts and regions of Brazil were studied in relation to morphology, mycelial compatibility, analysis of genomic DNA through random ampli®ed polymorphic DNA (RAPD), variation within the nuclear rDNA [internal transcribed spacers (ITS)] and sequencing of ITS frag- ments. There was considerable variability among iso- lates in relation to the number, size and location of sclerotia on the medium surface. Thirteen mycelial compatibility groups (MCG) were identi®ed among 23 isolates. Seven isolates were only self-compatible. With the exception of group 3, where all the isolates came from soybean, there was no apparent correlation between group and isolate origin. On the basis of RAPD pro®les, 11 haplotypes (A to K) were identi®ed. There was an association between the RAPD groups and MCG. Haplotypes A, B, D, G, I and K belonged to MCG groups 1, 2, 3, 4, 5 and 6, respectively. All other RAPD haplotypes contained incompatible isolates. Polymerase chain reaction (PCR) ampli®cation with primers 4R and 5F ampli®ed two fragments containing ITS1, ITS2 and 5.8 S rDNA sequences, that were present in all isolates, with molecular sizes of 739 and 715 bp. Restriction analysis of PCR products showed that the two fragments had sequence divergency which is referred to as `ITS typesʹ. Four arbitrarily chosen soybean isolates (2, 6, 7 and 23) and two non-soybean isolates (11 and 22) were used to investigate the variation within the ITS sequence and its role in the phylogeny. The strict consensus of nine most-parsimo- nious trees inferred from the data set which included six isolates of S. rolfsii, four of which have two di€erent `ITS typesʹ, showed three well-supported groupings. The neighbour-joining tree inferred from the data set also showed three major clades as did the parsimony tree. The major di€erence was that in the neighbour-joining tree the `ITS typeʹ 11 was resolved and grouped in one clade. These results show that the `ITS typesʹ within isolates are almost always phylogenetically distinct. There was no clear correlation between ITS-based phylogeny and isolate origin.