The availability of rootstocks highly tolerant or resistant to HLB, and with good horticultural traits, would eliminate this disease as a threat and permit higher crop production at reduced cost. The main goal of this project was to investigate whether we can identify metabolites (small molecules) in citrus rootstocks that are associated with specific rootstock traits, particularly tolerance to HLB, but also tolerance to other stresses and diseases. Identification of metabolites that are associated with specific rootstock traits would aid in the early selection of promising candidate rootstocks prior to long-term field testing and accelerate the release of trees for commercial use. Specific objectives of this project were 1) identify key metabolites that are associated with rootstock traits, 2) investigate the effect of grafting on metabolite profiles, and 3) establish metabolite profiles of trees on different rootstocks in response to HLB.The plant material used in this project consisted of a wide array of rootstocks grown as seedlings or as grafted trees in the greenhouse or in the natural field environment. Metabolite detection and identification was conducted by a commercial service at the West Coast Metabolomics Center, UC Davis, CA, using untargeted gas chromatography and mass spectrometry. Greenhouse and field experiments, sample extraction, and data analyses were conducted by PIs Bowman (USDA) and Albrecht (UF).�Accomplishments: Objective 1. For proof-of-concept, four �standard� rootstocks (Cleopatra, Swingle, Ridge, and sour orange) were included in preliminary and all other studies. Many hundred metabolites were detected in leaves and roots of rootstock seedlings. The majority were present in higher concentrations in the leaves than in the roots, indicating that leaves are metabolically more active. Only one third of all detected metabolites were of known chemical identity; most were unknown. Metabolite profiles corresponded well with the taxonomic relationship of rootstocks. In the roots, several metabolites were identified with differences in concentrations that correspond well with field tolerance of rootstocks to unfavorable soil conditions. Several metabolites were also identified that may be associated with rootstock tolerance to different pathogens and diseases. Results from this study have been submitted for publication. In addition to the four �standard� rootstocks, metabolite profiles of seedlings of other greenhouse-grown rootstock cultivars (Carrizo, US-802, US-812, US-896, US-897, US-942, US-1516) were investigated. This large set of data is still being analyzed.Objective 2. The results from this objective were similar to those from objective 1. Many hundred known and unknown metabolites were identified in grafted greenhouse and field grown citrus trees. Large differences were detected based on rootstock variety that corresponded well with taxonomic relationships. Many root metabolites that varied most significantly among rootstock varieties were the same, whether plants were young and grown in the greenhouse, or older and grown in the field. This proves the consistency of the methodology used in our project, and broad applicability of the approach. The comparison of two different scion cultivars showed large metabolic differences and indicated a considerable influence of the scion on the rootstock. But importantly, metabolite profiles of leaves from the same scion differed based on the rootstock on which it was grafted. This clearly demonstrates that rootstocks can influence the scion metabolically. We also found that many of the root metabolites that showed the largest differences among rootstocks did not show the same differences in the leaves of a grafted scion. This indicates that some metabolites are restricted to the tissue in which they are synthesized or are unable to move across the graft union. Results from a part of this study have been submitted for publication.Objective 3. For this objective we conducted a greenhouse experiment with Valencia trees on 10 different rootstocks (the same mentioned above) and compared the metabolite profiles of leaves and roots from HLB infected and non-infected plants. We detected nearly 600 known and unknown metabolites. In general, concentrations of most leaf metabolites were reduced in infected plants compared with healthy plants. The reverse was observed in roots, where most were increased by infections. The type and extend of metabolic changes were clearly influenced by rootstock. This large set of data is still being analyzed.In conclusion, rootstocks can be differentiated by their metabolic composition and influence on the scion. Several of the most discriminating metabolites may be suitable markers for stress and disease tolerance. Metabolites with the most dramatic differences and changes among rootstocks in healthy and HLB infected plants were in the group of chemically unidentified compounds. This indicates that they are biologically highly significant and provides future opportunities for discovery of new molecules.���