In the current project we are establishing the volatile and non-volatile polar metabolite profiles of new scions and rootstocks and evaluating them for their tolerance to HLB by challenges with psyllids and HLB.Progress on Objectives: This reporting period we focused on data from the study involving a new mandarin hybrid we call Lucky (Sugar Belle x Nova×Osceola) and the two parental varieties by challenging with ACP and taking leaf samples after infestation.Objective 1.1. To understand the mechanism behind the tolerance of new varieties toward HLB. The comparison between the varietal responses will allow us to determine the response to CLas-infected ACP infestation. Subsequent PCR testing will help determine susceptibility to HLB.a. For the hybrid Lucky (its parents are Sugar Belle and Nova × Osceola), we successfully repeated the challenge with CLas-infected ACPs ending in September. Leaf samples were taken initially (before infestation with ACP) and then 3 days, 1 month, 2 months and 3 months after ACPs were introduced into the cages. We will determine the biochemical response of Lucky to ACP infestation and its relative tolerance to CLas over time, with PCR confirmation of HLB status scheduled for Mar 2022 (9 months). The chromatograms have now been integrated and a summary is provided here:b. VOC profile summary: At this time we have finished the preliminary integration and have a general idea of the VOC profile of each of the three varieties. Overall, we detected 54 volatile compounds among the leaf extracts of the three varieties. `Lucky’ and Nova produced sabinene and beta-ocimene as their major monoterpenes, whereas in Sugar Belle (SB) the major monoterpenes are beta-ocimene and gamma-terpinene. Linalool appears at similar levels in all three, and limonene is relatively low in all three (compared to sweet orange varieties, where it is the dominant monoterpene). The dominant sesquiterpenes are caryophyllene and beta- and gamma-elemene.c. The majority of the compounds detected were in common between the three varieties, but a few were unique to each variety. As reported previously, the characteristic VOCs for SB included thymol and thymol methyl ester. Neither `Lucky’ nor Nova produced thymol, but Nova produced traces of the thymol methyl ester. In addition, we detected the monoterpenes para-cymenene and para-methatriene in SB, which was not found in the hybrid `Lucky’ offspring or Nova. Overall, the VOC profile of `Lucky’ showed contributions from both parents but did not express all of the possible compounds (44/54). There are many that are found uniquely or in two of the three varieties. These include para-cymene, para-menthatriene, and thymol (in SB only), thymol methyl ester (SB, Nova), germacrene C (Nova only), beta- and gamma-elemene (SB, Nova), 7-epi-thujene (Nova, Lucky), beta-cubebene (Lucky), 4-epicubedol (new, in Nova), and beta-sesquiphellandrene (Nova, Lucky).d. We will continue the data analysis to determine the effect of the infestation on the VOC profile of the three varieties over time. We expect to see changes in the VOC profile with increasing time after the infestation. e. Most importantly, survival of ACPs on the three varieties was not good again, as was found previously in our January 2021 experiment, which was attributed to cold weather. The second biology experiment was carried out in a protected growth room between July and August, 2021 and showed that ACP survives and reproduces poorly on the two parents. The ACP count was the highest on the hybrid Lucky. Therefore, we can suggest a correlation may exist between the lack of inheriting thymol and several other compounds from its parents and the higher ACP counts on Lucky, so maybe it is not very Lucky after all.Objective 2.To support objective 2 (understanding the role of rootstocks in citrus tolerance to HLB), we have two remaining experiments. 1. USDA rootstocks. We collected leaves from all of the USDA rootstocks grown from seeds since last year (US-802, 812, 897, 942, 1283, 1284, 1516) for volatile metabolite profiling. These samples are in the freezer and await processing and analysis. In addition, we have scheduled challenges with ACP and HLB graft-inoculations in the early spring of 2022 to determine if any of these are tolerant toward HLB.2. Grapefruit varieties on different rootstocks from the CUPS. The volatile data was reported in the previous reporting period. However, the data for the non-volatile metabolites are still being analyzed at this time.