Phylogenetic analysis of Phytophthora species based on ITS1 and ITS2 sequences of the ribosomal RNA gene repeat

Abstract

The internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal RNA gene repeat from Phytophthora species were amplified using the polymerase chain reaction and sequenced. Sequences from P. cambivora, P. cinnamomi, P. citricola, P. cryptogea, P. drechsleri, P. fragariae var. fragariae, P. fragariae var. rubi, P. megasperma var. megasperma and P. nicotianae were compared with published sequences and phylogenetic trees were produced. The resultant grouping of species generally agreed with groupings established using classical morphological criteria, primarily sporangial morphology. Amongst species with non-papillate sporangia two clades were evident, one consisting of P. fragariae, P. cambivora and P. cinnamomi and the other of P. megasperma, P. drechsleri and P. cryptogea. The latter three were placed in the tree between the non-papillate groups and the papillate and semi-papillate groups which formed three distinct clades. One group comprised P. citricola, P. citrophthora and P. capsici, another P. megakarya and P. palmivora and a third P. pseudotsugae, P. cactorum, P. idaei, P. nicotianae and P. infestans. More isolates of P. megasperma, P. drechsleri and P. cryptogea will need to be examined to settle more precisely the relationship of these species to the others. PCR amplification of ITS sequences using freeze-thawed mycelial scrapings from pure cultures growing on agar followed by digestion with restriction enzymes is a quick and easy way to compare and identify isolates without the need for laborious DNA extraction procedures. With improved technology, rapid automatic sequencing of PCR-amplified ITS regions is now possible and yields detailed information of relationships within the genus as well as allowing the design of species-specific PCR primers for diagnostic purposes.