PCR-based identification and phylogeny of species of Ceratocystis sensu stricto

Most species of Ceratocystis sensu stricto are virulent pathogens of a wide variety of plants including forest and fruit trees, sweet potato, pineappl e and sugar cane. Confusion exists regarding the taxonomy of the species in this genus. The aim of this study was to develop a rapid and reliable PCR- based RFLP identification method and to consider phylogenetic relationships among the better-known species of Ceratocystis. A 1.6 kb fragment within t he ribosomal DNA operon was directly amplified from living fungal tissue, w ithout extracting DNA. The amplified fragment included part of the small (S SU) and large (LSU) sub-unit rRNA genes, the 5.8S rRNA gene and the interna l transcribed spacers (ITS) 1 and 2. The PCR fragments were digested with e ighteen restriction enzymes. Four of these (Alu I, DraI, HaeIII and Rsa I) produced RFLPs that separated the species of Ceratocystis. The amplificatio n products from the best-known species were sequenced, and the delimitation of taxa based on this phylogenetic analysis generally agreed with results of previous studies using isozymes and rDNA sequence analysis. This study p rovides an extended understanding of the relationships among species of Cer atocystis and will form a sound foundation for further taxonomic studies of the group.