In this project we are profiling the new scions and rootstocks for their tolerance to citrus greening pathogen by studying the metabolite content using gas-chromatography mass spectrometry (GC-MS), along with biological assays such as challenging new varieties with psyllids and HLB.Since our last report, we began sampling the new rootstocks and scions that we have propagated thus far. The analyses for these samples include stored volatiles and leaf polar metabolites.Objective(s) pursued: 1. To understand the mechanism behind the tolerance of different varieties toward HLB. The comparison between the varietal responses will allow us to determine the mechanism of tolerance to CLas. 2. To understand the role of rootstocks in citrus tolerance to HLB. The comparison between rootstock metabolites will allow us to determine the best scion/rootstock combination for tolerating CLas. Methods: This quarter we sampled the mature leaves from the new scions and rootstocks, and did two different extractions – 1) hexane for leaf volatiles such as limonene, linalool and caryophyllene; and 2) methanol- chloroform-water for non-volatile metabolites such as sugars, amino acids and organic acids. Progress on Objectives: Scion evaluations `Marathon’ mandarin – we were only able to obtain one tree from a nursery, so we made cuttings for the mist bed. Although some of the cuttings did grow roots, when transplanted, the cuttings have not grown well. Our trial showed clearly that compared with many other citrus varieties with which we have experience with vegetative propagation, `Marathon’ is much less easily propagated. The source tree is growing fairly well and for this reason, we anticipate completing the metabolite work using only one biological sample, but 5 or more technical replicates.For Lucky, Sugar Belle, Nava x Osceola, and Grapefruit 914:1. Stored leaf volatiles (hexane extracts): 20 samples were run on the GC-MS (4 varieties x 5 biological samples), resulting in good chromatograms with approximately 50 volatile organic compounds identified. The peaks are being integrated and quantified currently.2. Polar leaf metabolites (TMS) 20 samples were run on the GC-MS (4 varieties x 5 biological samples), the chromatograms look good, with approximately 60 peaks. Integration and data analysis is in progress. Rootstock evaluationsFor the eight rootstocks we propagated and that are growing well: UFR-1, -2, -4, -5, -15 and -17; 46 x 20-04-6; 46 x 20-04-29:1. Stored leaf volatiles (hexane extracts): 40 samples were run on the GC-MS (8 varieties x 5 biological samples), resulting in good chromatograms. The integration and data analysis has not been started yet.2. Polar leaf metabolites (TMS) – 40 samples were run on the GC-MS (8 varieties x 5 biological samples), and the data analysis is waiting to be analyzed.Nematode susceptibility – Because the evaluation of new rootstocks should include tolerance to underground pests and pathogens such as Phytophthera and nematodes – we planted 6 of each of the 7 UFR rootstock seedlings in sandy soil for evaluation to burning nematodes. The soil was inoculated with burning nematodes and we are beginning our evaluations for resistance to nematodes. Our initial findings are that all of the UFR rootstocks have stunted growth, and damage in the root cortex with subsequent yellowing foliage compared to control plants.UFR-6 is not growing well, when it looks good we will analyze this one. Next quarter work1. We will complete the GC-MS data analysis for the first two sets of scions and rootstocks (8 rootstocks, four scions x two methods).2. `Marathon’ mandarin may be ready for GC-MS analysis.3. We should obtain more scions for evaluation. We will receive more rootstock seeds from the USDA.4. We will begin the biological evaluation of three scions (Lucky, and its parents, Nava x Osceola, and Sugar Belle) by challenging with ACPs to inoculate with HLB, as well as complete host preference studies.5. We will sample the foliage of the 6 UFR rootstocks under nematode pressure and their healthy controls for volatile and non-volatile metabolite analyses.