Streptomyces carneohygroscopicus SH 121 isolated during selecting biocontrol agent of citrus diseases exhibits strong inhibition towards Gram positive bacteria and many pathogenic fungi in plant.Establishment of a highly efficient genetic manipulation system is critically required for functionally analysing genes and genetic engineering in Streptomyces.In this study,the effects of heat treatment,pregermination time,donor hosts,ratio of donor and recipient and MgCl2 concentration in medium on conjugation efficiency were studied by single factor experiments.Under heat treatment at 50℃ for 10 min,pregermination at 37℃ for 1 h,using Escherichia coli DCDA/pUZ8002 as donor host,with the ratio of donor to recipient being 100 to 1,supplementing MISP4 medium with 40 mmol/L MgCl2,a highly efficient intergeneric conjugation method for SH121 was developed with highest efficiency of 1.03×10-3 conjugants/ recipient.The integrative plasmid pSET152 and the plasmid pYZ09 for gene knockout was successfully transferred intoSH121 with the method newly established. It is indicated that this genetic manipulation system could be used to discover bioactive natural products and study their biosynthetic gene clusters in SH121.