Application of PCR-RFLP technique to species identification and phylogenetic analysis of Streptomyces associated with potato scab in Brazil based on partial atpD gene sequences

Abstract

In the present study the atpD gene was evaluated as an alternative molecular marker for differentiation of Streptomyces species causing potato scab and to establish the phylogenetic relationships within this group. PCR-RFLP experiments using HaeIII and CfoI restriction enzymes allowed differentiation of most Streptomyces species, except the genomospecies S. reticuliscabiei and S. turgidiscabies which showed identical profiles between them. Phylogenetic relationships within Streptomyces spp. were also examined by comparing partial atpD gene sequences (858 nt) and the strains were separated in three main groups: Group I, supported by bootstrap values of 79%, comprised of S. scabiei, S. europaeiscabiei, S. stelliscabiei, S. reticuliscabiei, S. turgidiscabies and S. acidiscabies; Group II, supported by bootstrap values of 100%, comprised of S. sampsonii; and Group III supported by bootstrap values of 100%, comprised of S. caviscabies, S. setonii, S. luridiscabiei and S. puniciscabiei. The atpD gene sequence similarity observed among the species in this study ranged from 89.2% to 100%. Twenty-nine Streptomyces isolates obtained from potato growing areas in Brazil were analyzed by PCR-RFLP of atpD gene technique and identified as S. scabiei (15 strains), S. caviscabies/ S. setonii (12) and S. sampsonii (2). Using this tool S. europaeiscabiei strains were also detected from import material from the Netherlands. The presence of genes associated with the S. turgidiscabies pathogenicity island (PAI) was also determined and potato tuber slice assays were performed to confirm the pathogenicity of the strains. The data obtained herein indicated that the PCR-RFLP of atpD gene technique can be used for diagnosis and identification of phytopathogenic Streptomyces strains, representing a rapid and inexpensive tool. Moreover, this gene can be useful for phylogenetic analysis of Streptomyces spp. associated with potato scab.