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Loose smut has a wide distribution and can occur anywhere wheat is cultivated and produced. It is caused by the fungus Ustilago tritici. Mild symptoms may be present prior to heading, including yellowish leaf streaks and stiff, dark green leaves. Spores are not enclosed by the seed coat, so are quickly dispersed by rain or wind after emergence. These spores infect other wheat plants at flowering, causing seed infection. Wheat (Triticum aestivum L.) is an important crop and staple food of human population throughout the World. It is widely cultivated in Pakistan and in most of the other agricultural countries like China, India and Bangladesh. Therefore, an experimental study was designed to cultivate Galaxy-2013 for the isolation of pathogenic strains of tritici having potential to cause loose smut of wheat, after natural attack during cropping year 2019-2020. Main purpose of study was to isolate samples of Ustilago, which would be used for the testing of anti-fungal products for future studies during cropping years (2020-2022) after complete identification, isolated strains (RS01) were identified as Ustilago tritici and an accession number (OP164708_RS01) was obtained after blast the obtained sequences at NCBI, which were further confirmed by phylogenetic analysis. Therefore, it was expected that some wheat varieties those are vulnerable to the attack of Ustilago required careful cultivation by adapting suitable strategies to protect crops especially wheat from certain disease to enhance food requirement in the country. [ABSTRACT FROM AUTHOR]