This quarter: The Bowman lab has established three new experiments with grafted Valencia trees on groups of rootstocks which were inoculated with ACP during the quarter. Each experiment compares replications on nine different rootstocks and made use of 2,520 ACP from the positive colonies. Periodic evaluation of tree health, growth, and CLas titer via PCR were conducted on trees from rootstock experiments that were inoculated during the previous months following a set schedule. Periodic colony checks were conducted by PCR to maintain CLas positive colonies. The Stover lab conducted weekly detached leaf assays (DLAs) challenging transgenic citrus with CLas inoculated by infected ACP in the lab, which is used to identify best performing transgenic events (transgenics varying by position of transgene insertion etc.). Eight detached leaf assays were run using 500 ACP each with a total of 4,000 ACP used in this quarter. Transgenic material tested in DLAs were of three constructs ( ONYX, 35s-BT and Scamp-ST), with seven events per construct equaling 56 individual plants tested alongside WT controls. The balance of Covid-19 restricted lab hours was spent processing the leaf and ACP samples in preparation for CLas qPCR. We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leaves In addition 200 CLas+ ACP were provided to Dean Gabriel of UF and 600 were provided to YongPing Duan of USDA. Project rationale and focus: The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Previous quarter: Dean Gabriel of UF, and USDA scientists Kim Bowman, Ed Stover and G avin Poole have all run experiments totalling ~2000 ACP. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR.