Restriction fragment length polymorphism (RFLP) analysis was performed on the internal transcribed spacer regions of 204 Sporothrix schenckii isolates and on one strain each of the related fungi, S. schenckii var. luriei, S. curviconia, S. inflata and Ceratocystis stenoceras. S. schenckii isolates, which have been collected from around the world, have already been typed according to their mitochondrial DNA (mtDNA), and are kept in the Department of Dermatology, Kanazawa Medical University, Japan.
Approximately 600bp of the internal transcribed spacer region 1 (ITS1) of their nuclear ribosomal RNA gene (rDNA), 5.8S rDNA and ITS2 was amplified by PCR. From ITS-RFLP analysis of the PCR products, S. schenckii isolates comprised 4 types, rDNA types I-IV. The rDNA type I-III strains corresponded to the Group A strains (mtDNA types 1-3, 11, 14-19, 22 and 23), while the rDNA type IV strains corresponded to the Group B strains (mtDNA types 4-10, 12, 13, 20 and 21), as previously categorized according to their mtDNA-RFLP. The ITS-RFLP patterns of the above 4 related fungi all differed from those of the 4 rDNA types of S. schenckii.
Furthermore, only 22 (3.5%) out of a sequence of about 620 bases of the ITS regions of the rDNA differed among representatives of the mtDNA types 1-5, 7, 11, 14-19, 22 and 23. This difference in the ITS region is smaller than the 10% difference among isolates when estimated by mtDNA-RFLP. From the phylogenetic tree based on the base sequences, rDNA type I-III strains belong to Group I, while rDNA type IV strains belong to Group II which correspond with Groups A and B based on their mtDNA. The Group I strains are predominant in South America and Africa, while Group II are predominant in Australia and Asia.
ITS-RFLP analysis is better than mtDNA-RFLP in allowing faster discrimination and identification, and for its ability to divide the 4 types into groups, which is useful in clinical diagnosis and epidemiological investigations of S. schenckii.