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Two actinobacterial strains, designated NEAU-383T and NEAU-Y11T, exhibiting antifungal activity, were isolated from the rhizosphere soil of plants in Harbin, Heilongjiang Province, and Guangzhou, Guangdong Province, respectively. Both strains formed short, wrinkled, spiral spore chains on aerial hyphae. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains NEAU-383T and NEAU-Y11T were most closely related to Streptomyces iranensis HM 35T, Streptomyces rapamycinicus NRRL B-5491T and Streptomyces coffeae CA1R205T. However, digital DNA–DNA hybridization values between the two strains and their close relatives (27.1% between the two strains; 27.5–59.0% for NEAU-383T; 27.0–39.8% for NEAU-Y11T) fell below the 70% species delineation threshold. The genomic G+C contents were 71.0 mol% for NEAU-383T and 70.5 mol% for NEAU-Y11T. Both strains contained glucose and ribose as whole-cell sugars. The predominant menaquinones were MK-9(H6) and MK-9(H8), with NEAU-Y11T additionally containing MK-9(H4). The major fatty acids (>10%) in both strains included iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and C16 : 0, whilst iso-C17 : 0 was also a major component in NEAU-383T. Both strains contained phosphatidylethanolamine and hydroxy-phosphatidylethanolamine as polar lipids. Additionally, NEAU-383T also possessed diphosphatidylglycerol, phosphatidylinositol, an unidentified ninhydride-positive lipid and five unidentified lipids, whereas NEAU-Y11T additionally contained two unidentified ninhydride-positive lipids and five unidentified lipids. Genomic analysis revealed that both strains possess abundant secondary metabolite biosynthesis gene clusters, including those for antibiotics, siderophores, polyketides and terpenoids, along with metabolic pathways associated with environmental adaptation. These findings provide insight into the potent antimicrobial activity exhibited by both strains. Based on genotypic, phenotypic and chemotaxonomic analyses, strains NEAU-383T and NEAU-Y11T represent two novel species within the genus Streptomyces, for which the names Streptomyces lycopersici (type strain NEAU-383T=JCM 36736T=MCCC 1K08878T) and Streptomyces cucumeris (type strain NEAU-Y11T=JCM 36418T=MCCC 1K08681T) are proposed.