Technical Abstract: Asiatic citrus canker (Acc) (causal organism Xanthomonas citri subspc. citri (Xcc) is threatening sustainability of the Florida citrus industry. Resistant cultivars, whether developed through conventional breeding or genetic transformation, will be he best solution for dealint with Acc. In Florida, prior to 2005, regulatory constraints made experiments with Xcc very difficult to conduct. Greenhouse experiments with Xcc are now permitted, expanding opportunities for resistance screening. We evaluated whole-plant spray inoculation (WPSI) with Xcc for resistance screening of citrus germplasm. WPSI requires minimal effort and numerous hybrids can be evaluated concurrently. Our initial WPSI trial included 68 genotypes of citrus, citrus hybrids and citrus relatives; a wide range of disease expression was observed. We are currently using WPSI to screen transgenic citrus to determine if non-tissue-specific expression of antimicrobial peptides provides resistance to Xcc. Although WPSI is an effective screening method, it requires inoculation of whole plants and takes at least one month after inoculation to obtain results. Detached shoot inoculation methods show promise for screening without exposing source plants to pathogens. We have developed an RT-PCR method for quantification of Xcc in plant tissue and results are highly correlated with plate count data; RT-PCR is much more efficient than plate counting and is not affected by the presence of non-target bacteria. RT-PCR evaluations have revealed several aspects of the citrus/Xcc pathosystem. For example Xcc growth is more rapid in leaves of grapefruit than in kumquat, suggesting that kumquat resistance to Xcc may occur prior to hypersensitivity. A disadvantage of the RT-PCR method is that dead cells cannot be distinguished from live cells. We are currently evaluating the use of ethidium monoazide as a means for overcoming this limitation.