In this project we are profiling the new scions and rootstocks for their tolerance to HLB by studying the metabolite content by GC-MS, and challenging new varieties with psyllids and HLB.Progress on Objectives: Objective 1.1. 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. This quarter we focused in three areas: 1) Marathon Mandarin analyses; 2) CUPS Mandarins, 3) new Valencia varieties.a. For the evaluation of the new mandarin hybrid Marathon, the leaf samples for analysis of volatiles and polar metabolites have been run on the GC-MS and integrated. The analysis found eucalyptol in Marathon, which we have not detected before in other citrus varieties. However, overall profile strongly suggests that Marathon is unfortunately susceptible and does not behave like Sugar belle. Challenging with CLas by grafting confirmed that. b. For the leaf samples from inside the CUPS taken in March 2021, we collected all of the varieties of Mandarins or mandarin hybrids that were available. The VOC analysis is completed for these. Findings: From the VOC analysis we found two distinct chemotaxonomic groups – those that produce thymol and those that don’t. Previously we found that leaves of Sugar Belle, a mandarin hybrid considered tolerant to HLB, contained thymol and its methyl ester, and leaves of most sweet orange (considered HLB susceptible) do not. The group that contained thymol and/or its methyl ester included Sugar Belle, Dancy tangerine (a parent of Sugar Belle), Minneola (also a parent of Sugar Belle), BB4-8-20, and UF711. We associated thymol with tolerance to HLB based on its well-known antimicrobial properties.The thymol non-producers were UF950 clementine, Murcott, Bingo, Early Pride, UF411, and Kinnow. These thymol non-producers also did not produce gamma-terpinene, and had high levels of sabinene. We will look at the ratios of thymol and other blends of VOCs to detect any trends that might indicate HLB tolerance. We expect that robust data analysis will help separate the varietal differences and identify key volatiles involved in ACP deterrence or action against CLas bacteria. We hope to be able to predict or screen for HLB tolerance based on the chemometric profiles of the new varieties if we can develop an accurate model.c. For Lucky and its parents Sugar Belle and Nava × Osceola, we repeated this experiment in June using CLas-infected ACPs. We will measure the response to ACP infestation and the acquisition of CLas over time. New work:d. We obtained Valencia and two new varieties from Southern Citrus (5 each of Valencia, Valquarius, and Vernia) for a small study. So far the VOC profile of the three varieties are nearly identical. We will look at non-volatile metabolites next and perform some ACP challenges. Objective 2.2. 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 combinations for tolerating CLas. a. The rootstock seeds from the USDA (US-802, 812, 897, 942, 1283, 1284, 1516) for metabolite profiling and HLB/nematode screening are about three months old and ready to be moved outside to encourage growth.b. We continue evaluating the previous rootstocks for growth habits and HLB tolerance.We greatly appreciate the Schumann Lab for granting access to the CUPS at CREC for sampling for this part of the study. They are presently one of the few sources of healthy mature citrus trees.