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Actinomycetes are well-known microorganism as secondary metabolites producers which serve as antibiotics. In this study, sugar cane (Saccharum officinarum) rhizosphere soil samples were collected from Piyungan, Bantul, Yogyakarta. The aims of this study were to obtain Actinomycetes isolates from sugar cane (Saccharum officinarum) rhizosphere which potentially produce antibiotics and to analyse the Thin Layer Chromatography (TLC) profile of ethyl acetate extract of active Actinomycetes isolates. The method used in primary screening to evaluate Actinomycetes antibacterial activity from the sugar cane rhizosphere are agar block method and cup-plate method, and the extract activity assay used Kirby-Bauer method. Determination of antibiotic activity was observed by measuring the diameter of inhibition zone. Extract component was analyzed by Thin Layer Chromatography (TLC). The results of this research was found 39 isolates of Actinomycetes classified into 7 groups based on color grouping. The result of agar block test was revealed that among 39 isolates, four isolates (10.25%) were able to inhibit Staphylococcus aureus. They were Te.234, Te.235, Te.236, and Te.325 isolates. Inhibition zone of crude extracts from these isolates against Staphylococcus aureus were Te.234=7.67 mm, Te.235=3.75 mm, Te.236=12.42 mm, and Te.325=19.16 mm. However, none of these isolates were able to inhibit Escherichia coli. Results of TLC profile showed that the suspected compounds contained in ethyl acetate extract of culture broth are alkaloid, polypeptide, steroid, alcohol, phenol and ketone/aldehyde compounds. Based on the results, it can be concluded that Actinomycetes can be isolated from sugar cane rhizosphere which has potential as an antibiotic producer by narrow spectrum to inhibit Gram-positive bacteria such as Staphylococcus aureus. Keywords—actinomycetes, isolation, rhizosphere, antibiotic, sugar cane, TLC profile

Isolation of Actinomycetes from Sugarcane (Saccharum officinarum) Rhizosphere and the Ability to Produce Antibiotic.