Nested-PCR for Detecting Terfezia claveryi in Roots of ‎Helianthemum Species in Field and Greenhouse Conditions

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 Roots of Helianthemum species were collected from various rangeland sites in Fars, and other provinces in Iran. The partial small subunits of ribosomal DNA genes were amplified with the genomic DNA extracted from their roots by nested polymerase chain reaction (PCR) using the universal fungal primer pair ITS1/ITS4 and specific primer pair FTC/RTC, which was designed based on internal transcribed spacer 1, 2 and 5.8S gene of rDNA sequences of Terfezia claveryi. The nested-PCR was sensitive enough to re-amplify the direct-PCR product, resulting in a DNA fragment of 500 bp. The efficacy of the nested-PCR showed that it could re-amplify the direct-PCR product and detect 2fg genomic DNA. Restriction fragment length polymorphism (RFLP) was analyzed using the two restriction enzymes Hinf I and Alu I. Nucleotides sequence analysis revealed that the sequences from infected Helianthemum species were close to those of T. claveryi. With the nested PCR method, H. lipii and H. salicifolium were confirmed as host plants of T. claveryi in greenhouse inoculated plants and also in the rangelands of different areas in Fars and other provinces inIran.