Recently we have made significant progress in the study of antibiotic resistance in Liberibacter using L. crescens as a model. A former postdoc in the lab, Kin Lai, was able to obtain five spontaneous mutants of L. crescens that were resistant to streptomycin at a frequency of about 1 in 100 million cells (the expected frequency). He was never able to generate spontaneous mutants of L. crescens to oxytetracycline. Kin left the lab and I gave the project to a very talented undergaraduate, Alexa Cohn. Alexa repeated the work of Kin and found the same results. Also like Kin, Alexa was unable to obtain any spontaneous resistant to oxytetracyline even after screening 10e13 cells. But Alexa took the work one step further and discovered something very interesting. She screened for mutants that were simultaneously resistant to streptomycin and oxytetracycline. She obtained two colonies that were resistant to both antimicrobials. Alexa then discovered that all eleven of our streptomycin-resistant mutants were also resistant to both antimicrobials. We are not in the process of getting sufficient DNA form each mutant to sequence each genome to identify the sites of mutation in each strain and then re-create those mutations in wild-type L. crescens using CRISPR technology. This result may have significant management implications. Two antimicrobial products are available to treat citrus for HLB. One contains only oxytetracycline. The other contains oxytetracycline and streptomycin. Our results suggest that treatment with only oxytetracycline is not likely to generate resistance very soon. In contrast, treatment with both antimicrobials may generate resistance to both antimicrobials quickly. We need to work with growers over the next six months to test this notion in the field. This can be done easily once we identify the source of the resistance mutation, which is likely within the rps12 gene. That will allow us to do rapid screening for resistance in the field by qPCR. Our culturing work has also suggested a means by which citrus greening disease might be controlled by a non-antimicrobial means. We have discovered that the preferred carbon source for L. crescens is either alpha-ketoglutarate or citric acid. Citric acid is a common constituent of citrus phloem while alpha-ketoglutarate is not. We are designing experiments to test the hypothesis that if we can nutritionally prevent phloem loading of citric acid, we can then starve Liberibacter in the phloem. We are first testing this nutritional approach in a greenhouse experiment that begins December 12, 2016. This experiment will require two months of nutritional treatment of the saplings followed by another month to extract the phloem, measure organic acids, and interpret the results. We still need a culture of Ca. L. asiaticus (CLas). We have a greatly simplified defined medium for L. crescens that allows the organism to grow much faster than on BM-7, the complex, undefined medium. The necessary changes to this medium are now being made that should encourage CLas growth. Testing of these media will be started within two weeks.