The driving force for this project (Hall-15-016) 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 breeders at USDA-ARS-USHRL, Fort Pierce Florida continue producing germplasm that needs to be evaluated. The more rapidly germplasm can be evaluated, the sooner breeders can identify HLB-tolerant germplasm for the Florida citrus industry. The purpose of this project is to support a high-throughput facility to evaluate citrus germplasm for HLB resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. The original inoculation program called for individual plants to be caged with 20 infected psyllids for a two-week infestation, and then housed for six months in a greenhouse with an open infestation of infected psyllids. As indicated below, the open infestation step was abandoned. After the caged inoculation step, plants are moved into a psyllid-free greenhouse and evaluated for growth, HLB symptoms and CLas titer, and finally the plants are transplanted to the field where evaluations of resistance continue and additional inoculations by field psyllids occurs. CRDF funds for the inoculation program 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 technician is assigned ~50% to the program. USDA provides for the program two small air-conditioned greenhouses, two walk-in chambers, and a large conventional greenhouse. Currently 18 individual colonies of infected psyllids are maintained. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected citron plants. Update: As of March 15, 2018, a total of 11,888 plants have passed through inoculation process. A total of 326,295 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. We have abandoned the greenhouse open-infestation step because of continual problems mainly with invasive pests such as thrips, scales and parasitoids. Research indicated that the no-choice inoculation step should usually average around 74% effective and gets plants back to the breeders faster. The plants are subjected to further inoculations in the field. The no-choice inoculation procedure was evaluated monthly for 12 months, and success in getting seedlings infected was evaluated six months after each monthly infestation. The results indicated a 74% average success rate in getting seedlings infected when flush (immature leaves) was present, with success ranging from 40 to 100%. Success was significantly related to how many ACP on a seedling tested positive for CLas, thus greater success rates would be expected using more than 20 ACP per seedling. Based on the results of the research, an infestation shorter than two weeks would be as effective, which would be advantageous for guarding against excessive ACP damage to seedlings if more than 20 per seedling were used. It remains possible that modifications to the no-choice inoculation procedure would increase success rates and reduce variability, for example 25 to 30 ACP per seedling for a 1 week period, perhaps with larger seedlings in larger cages.