This quarter:This quarter , the colony of CLas infected psyllids supplied a total of 5,045 ACPs used for (1) evaluation of rootstock breeding material, (2) transgenic events evaluation, (3) applied research for CLas control in citrus performed by USDA and University researchers; and (4) monitoring the colony quality.The Bowman lab continues the experiments with grafted Valencia trees on groups of rootstocks which were inoculated with ACP during the previous quarter (Janyuary to March 2021). Each experiment compares replications on nine different rootstocks. This quarter 63 trees were inoculated with 1,280 ACP from the positive ACP 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.) expressing antimicrobial peptides and defensive proteins targeting CLas, as well as natural insecticide peptides to control ACP. Six detached leaf assays experiments, involving individual 240 leaves, were inoculated using 2,400 CLas infected ACPs in this quarter. Transgenic material tested in DLAs were Carrizo and Hamlin plants expressing ONYX peptide under constitutive and phloem specific promoter. A total of 48 independent events were tested alongside WT controls. The leaves (midribs) and ACPs are being processed and submitted to qPCR for CLas titer after each DLA to better understand the effect of the transgenic peptide in bacteria control and transmission. These trials have being very useful in terms of providing information that allow to select the best transgenic events (ones causing high ACP mortality and/or low CLas transmission to plant) for propagation and further evaluation at greenhouse environment. We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leaves. Research involving evaluation of the microbiome of ACPs fed on transgenic causing high insect mortality was conducted this quarter using 230 ACPs fed in a set of 22 transgenic leaves. In addition 775 CLas+ ACP were provided to researcher collaborators (600 ACPs for Dr. YongPing Duan of USDA and 175 for Florida International University, for Jessica Dominguez, a Ph.D. student, who is developing a thesis in alternative compounds to control CLas bacteria). Also, to monitor colony quality, 360 ACPs were used for Clas detection by qPCR this quarter. In this quarter, seven new colony cages (with 250 ACPs/cage) were set up to renew and support the demand of the hot ACPs for future use. 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% (I don’t think Anna Sara has 50% of her time to take care of the colonies) to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Previous quarter:United States Department of Agriculture scientists Kim Bowman, Ed Stover and YongPing Duan have all run experiments totalling 7,280 ACPs. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR.