Background and Objective
Streptomyces are recognized as a rich source of bioactive compounds among actinomycetes. Systematic studies have provided an appropriate framework for identifying species within this genus. In previous studies, isolates of the common scab pathogen from central potato-growing provinces in Iran were not grouped with any of the globally recognized pathogenic Streptomyces species based on 16S rRNA gene sequencing. Although the 16S rRNA gene is widely used in phylogenetic studies of Streptomyces species, its sequence often fails to differentiate species within this genus. In plant-pathogenic Streptomyces, housekeeping genes are used as stable markers for accurate identification and classification of different species. Due to their essential roles in cellular processes, housekeeping genes are rarely subject to natural selection pressures, thereby undergoing fewer changes compared to other genes. Utilizing these genes can facilitate the precise identification of Streptomyces species and reduce diagnostic errors. The present study aimed to investigate the phylogeny of pathogenic Streptomyces isolates representing different provinces of Iran using Multilocus sequence analysis. 
Materials and Methods
Twelve pathogenic Streptomyces isolates were examined based on their morphological characteristics, including the color of aerial and substrate mycelia, colony morphology, pigment production in culture medium, and the shape of spore chains. Pathogenicity was assessed under laboratory conditions using radish seeds and seedlings as well as potato tuber slices. Additionally, the presence of txtAB, tomA, and nec1 genes was investigated in the isolates. Finally, phylogenetic analysis of the representative pathogenic Streptomyces isolates was conducted based on the sequencing of four housekeeping genes: atpD, recA, rpoB, and trpB. For this purpose, multiple sequence alignments were performed, and phylogenetic trees were constructed both individually for each gene and in combination using Multilocus sequence analysis approaches. The trees were generated using the Neighbor-Joining and Maximum Likelihood methods.
Results
Variation in the color of aerial and substrate mycelia was observed among the isolates. Spore chain patterns were spiral in 57% and rectiflexibiles in 43% of the isolates. Isolates SA3 and SFF completely inhibited the germination of radish seeds. Other isolates, including SA4, SA6B, SF6, SKH1, SKH2, and SA1, induced a broader range of symptoms, such as necrosis, stunting, and hypertrophy, excluding seed germination inhibition. Symptoms such as necrosis and pitting in potato disks were observed in isolates SA3, SA4, SFF, SKH2, and SH. In PCR assays, a 700 bp fragment corresponding to the nec1 gene was detected in two of the eight selected isolates including SA6B and SSH1. The txtAB gene (385 bp) was amplified in three isolates: SFF, SA4, and SKH1. The tomA gene (392 bp) was amplified only in isolate SF5. The phylogenetic trees generated using both Neighbor-Joining and Maximum Likelihood algorithms demonstrated consistent interpretations. The position of the Streptomyces isolates from this study was identical in both algorithms when compared with reference strains.
Discussion
Detection of genes associated with the pathogenicity island in Streptomyces isolates using the primers employed in this study was primarily negative. It was revealed that there is no correlation between the presence of pathogenicity-related genes and the pattern of disease symptoms observed in bioassays on radish seedlings and potato slices. Identifying unknown secondary metabolites produced by species of this genus may provide insights into possible links between genotype and pathogenic phenotype. In accordance with the previous phylogenetic revision of Streptomyces species, the clustering pattern in the final concatenated tree, based on four housekeeping genes, was consistent in both the Neighbor-Joining and Maximum Likelihood algorithms. Accordingly, isolates SA1, SKH1, and SH were clustered within the S. rochei species group. Isolates SSH1 and SA6B were grouped with the reference strains S. fimicarius and S. caviscabies. Isolate SA4 was clustered with the reference strain S. anulatus, while isolates SFF and SF5 exhibited greater nucleotide differences and were considered as Streptomyces sp. To validate these results, multilocus sequence analysis (MLSA) is recommended for a larger number of pathogenic isolates collected from various regions of the country.