Fourth quarter 2021- 100% done Project rationale and focus: The driving force for this three-year project is the need to evaluate citrus germplasm for resistance to HLB, including germplasm transformed to produce 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 for the last 3 years. 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 also assigned ~30% of her time to the program in order to maintain colonies (including watering, setting up new cages, terminating old cages, cleaning growth chambers and cages). USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. This quarter: Colonies of CLas infected psyllids supplied a total of 3,770 ACPs used for (1) transgenic events evaluation, (2) applied research for CLas control in citrus performed by USDA and University researchers; and (3) monitoring the colony quality by qPCR.The Stover lab conducted 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. Five detached leaf assays experiments, involving individual 228 leaves, were inoculated using 2,280 CLas infected ACPs in this quarter. Transgenic material tested in DLAs were Carrizo plants expressing ONYX peptide and chimeric AMP TS, both under SCAmp-P3 phloem specific promoter. A total of 38 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, with some ONYX events killing around 80% of the psyllids with reproducible results. Research involving evaluation of the microbiome of ACPs fed on transgenic causing high insect mortality was conducted this quarter using 440 ACPs fed in a set of 44 transgenic leaves. A research paper has been prepared (Rapid in vivo screening for huanglongbing resistance in genetically modified citrus by detached leaf assay- J.Krystel, M. Grando, Q. Shi, E. Cochrane, E. Stover) in order to report important modifications implemented into the DLAs using Clas+ ACPs to evaluate transgenic plants and investigate the mode of actions of peptide in controlling the psyllids. In addition, 1,080 CLas+ ACP were provided to researcher collaborators:780 for Florida International University, for Jessica Dominguez, a Ph.D. student, who is developing a thesis in alternative compounds to control CLas bacteria and 300 ACP were furnished to Dr. Randy Niedz (USDA Fort Pierce) for activities in a HLB NIFA project. Periodic colony checks were conducted by PCR to maintain CLas positive colonies. This quarter 410 ACPs were used for Clas detection by qPCR to monitor colony quality. Also, six new colony cages were set up this quarter to renew and support the demand of the hot ACPs. For that 24 HLB positive plants were infested with 2.500 ACPs. Previous quarter: United States Department of Agriculture scientists Kim Bowman, Ed Stover, Michelle Heck, Randy Niedz, and researcher collaborators have all run experiments totaling 6,840 ACPs. Samples have all been collected on-time from ongoing experiments. ACP mortality were computed and statistically analyzed. Surviving ACPs and leaf midribs were collected and processed for future qPCR analysis to access the bacteria CLas titer).