تعیین گروه‌های آناستوموزی جدایه های ایرانی Rhizoctonia solani عامل شانکر ساقه و شوره سیاه سیب‌زمینی با استفاده از آنالیز فیلوژنتیکی توالی نواحی ITS-rDNA

Potato (Solanum tuberosum L.) is one of the most important products that play a vital role in feeding the world, and has the high degree of genetic diversity. Stem canker and black scurf of potato caused by Rhizoctonia solani Kühn is an important and epidemic disease in potato-growing regions worldwide, as well as in Iran. In general, the isolates AG-3, AG-1, AG-2-1, AG-4, AG-5 and AG-9 have been reported as the main isolates causing stem canker and black scurf on potato worldwide; however, AG-3 is considered as the most important isolate causing the disease with high variability. Recently, sequence analyses of ribosomal DNA (rDNA) regions have been employed for the accurate identification of Rhizoctonia species and their anastomosis groups. In this study, 30 isolates of the pathogen were collected from the main potato-growing provinces of Iran, including Isfahan, Ardebil, Fars, Hamedan, Kurdestan and Kerman. Total genomic DNA was extracted from all isolates using the CTAB method. The ITS-rDNA region was used to characterize the isolates using the universal primers ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3'). PCR amplifications of the genes were performed and sequenced. To analyze the relationship of all 30 isolates to known R. solani AGs, the sequences from this study and sequences of 13 reference R. solani AGs (AG-2-1, AG-3, AG-4 and AG-5) were initially aligned using the Clustal W Multiple alignment, checked visually, and improved manually where necessary. The sequence of Athelia rolfsii isolate FSR-052(AY684917) was used as an out-group. Phylogenetic analysis of the rDNA-ITS region was performed with Kimura 2-parameter model in MEGA 7.0. ). Branch support of the trees obtained from the Neighbor-joining analysis was assessed by boot-strapping with 1,000 replications to estimate the reliability of inferred monophyletic groups. All gaps were treated as the missing data. A specific primer for the identification of the anastomosis group AG-3 PT (potato type), was used. The forward primer was as Rs1F2 (5’-TTGGTTGTAGCTGGTCTATTT-3') and the reverse primer was AG-3PR2 (5'-ACACTGAGATCCAGCTAATA-3'). PCR amplification of the ITS-rDNA regions of all 30 isolates produced a 711 bp fragment. Phylogenetic analysis of the ITS-rDNA indicated that the isolates were grouped in a cluster that included reference isolates of R. solani AG-3 with boot strap value of 94%. The result also revealed that the isolates were clustered in a sub-distinct cluster with reference isolates of R. solani AG-3 PT (potato type) with a high bootstrap value of 97%. Additionally, the identification of the isolates was confirmed by positive specific PCR amplification with the 474 bp fragment using the specific primer Rs1F2/AG-3PR2.