Objective 1. Identify key metabolites that are associated with rootstock traits. Summary of accomplishments: Rootstock seedlings were grown in the USDA greenhouses and included standard rootstock cultivars and rootstocks developed by the USDA breeding program and in great demand by the industry. To establish metabolite profiles on a set of cultivars with known horticultural traits, we used the four rootstocks Cleopatra, Swingle, Sour orange, and Ridge Pineapple, and assessed the metabolite profiles of leaves and roots at two different seedling stages under greenhouse conditions. This allows us to characterize rootstocks based on their metabolite composition, and to assess the stability of metabolite profiles at different developmental stages. For a subset of the data, analysis has been completed and several compounds have been identified that may be associated with rootstock traits. A manuscript has been prepared and submitted to the journal Plant Science. In addition to these four standard cultivars, other rootstock seedlings were sampled and leaf and root extracts were submitted for GC-TOF MS analysis at the West Coast Metabolomics Center (WCMC), UC-Davis. Data were received in March 2017 and are currently being processed and analyzed by the team. Preliminary results of a selected set of the most recent data were presented at the IRCHLB in Orlando in March 2017. Objective 2. Investigate the effect of grafting on metabolite profiles. Summary of accomplishments: The four standard rootstocks (Cleopatra, Swingle, Ridge, and Sour orange) and other rootstocks developed by the USDA breeding program were analyzed as grafted trees in combination with Valencia and other scions. Trees were grown in the greenhouse or under field conditions at different locations. Comparison of the metabolite profiles of roots and leaves in these grafted trees with profiles obtained for leaves and roots of rootstock seedlings allows us to analyze rootstock and scion interactions, and evaluate how they affect tolerance to HLB and other biotic or abiotic stresses. The different field conditions under which the plants are grown will also aid in identifying environmental effects on metabolite profiles. The latest data set from this part of the study was received in March 2017 and is currently being processed and analyzed. Analysis of a subset of the data has been completed and we are in the process of preparing a manuscript for publication. Objective 3. Establish metabolite profiles of trees on different rootstocks in response to HLB. Summary of accomplishments: A paper was published last year describing our initial research on metabolic profiles of rootstocks with different response to HLB (U. Albrecht, O. Fiehn, K.D. Bowman. 2016. Metabolic variations in different citrus rootstock cultivars associated with different responses to huanglongbing. Plant Physiology and Biochemistry 107:33-44). In addition, an experiment was initiated that included many hundred grafted trees grown in the USDA greenhouses. These trees are composed of Valencia grafted on a diverse array of standard and USDA rootstock cultivars. One set of trees was inoculated with Las using graft-inoculation; a second set of trees was mock-inoculated using the same procedure. Trees were analyzed by PCR for presence of Las in leaf and in root tissue at regular time intervals. A subset of trees was selected, and leaf and root tissues of these plants were collected for metabolite analysis. These samples have been extracted and were sent to WCMS in April 2017. Some of the selected plants are being used for continuing infection and metabolic studies. Experimental design, data collected, analysis, results, and interpretation are too complex to present here. Additional information is available on request.