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Wheat (Triticum aestivum) is an important food crop grown all over the world, its production is decreasing day after day due to fungal attacks. Present experimental investigation was designed with an aim to synthesize organic fungicide to control these pathogens in future. Therefore, focus of present manuscript was to isolate the fungal strains of Puccinia striiformis tritici causes rust disease of wheat. Morocco as a check was grown in field conditions favorable for fungal growth on 24 November (2020). Experimental plants were inoculated with fungal strains P. striiformis RR01 at stages 55 to 65 according to Zadok scale. Fungal strains (RR-01) were isolated from the experimental crop after the appearance of symptoms of leaf rust disease and were processed further for identification. Identification was done microscopically confirming that the isolated strains belong to Puccinia. Further molecular analysis of RR-01 also confirmed that isolates have close resemblance with the Puccinia striiformis. Identified sequences of the strains were submitted to NCBI which were assigned the accession number for more confirmation dendrogram were also prepared. After complete identification, infected dried leaves were kept in envelopes as rust is biotrophic fungi, while fungal spores were also stored as glycerol stocks. [ABSTRACT FROM AUTHOR]