SP: UTILIZING HLB TOLERANT CITRUS GERMPLASM AND UNDERSTANDING THEIR ROLE IN MITIGATING HUANGLONGBING

<br>What was accomplished under these goals? 1. A) Characterization of the feeding behavior of the adult Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae) on finger limes and finger lime hybrids using Electropenetrography (EPG): We characterized the feeding behavior of D. citri on finger lime and four finger lime hybrids (Table 1) and compared it with susceptible Valencia plants using EPG techniques. The main objectives of this study were to determine whether the feeding behavior of D. citri is disturbed on these tolerant hosts, specifically, its ability to reach the phloem; better understand CLas inoculation and transmission; confirm whether traits conferring host plant tolerance to D. citri in finger lime are passed down to hybrids. We hypothesized that frequency and duration of phloem salivation (E1) and ingestion (E2) will be smaller in finger lime and hybrid hosts. We also hypothesized that non-probing events (NP) and xylem ingestion (G) will be performed more frequently on finger lime and hybrid plants. Table 1: Hybrid name, crosses, and recording counts used for SAS analysis Plant name Finger lime hybrids cross # EPG recordings Valencia NA 27 FL Finger lime control 19 PFL1-3 C2-5-12 X Finger lime 20 PFL1-11 5-1-99 X Finger lime 22 PRL1-55 5-1-99 X Round lime 19 Two 4-channel AC-DC monitors were used in DC positive voltage. Both instruments were set to apply 150 mV of DC substrate with an input resistor (Ri) level at 109 ohms. A period of 30 min to 1 hour was used from the time psyllids were removed from the colony and placed on the plants. Recordings were done for 24 hours duration. We chose to measure only the original 6 waveforms (Bonani et al., 2010); non-probing (np), potential drop (pd), mesophyll pathway (C), xylem ingestion (G), phloem salivation (E1) and phloem ingestion (E2).We obtained a total of 107 EPG recordings. Out of the 85 variables calculated by the Ebert program, 21 showed significant differences between the hybrids and the control (Valencia). Higher mean duration of non-probing events (walking) in finger lime (almost 3 times higher) compared to Valencia and hybrids. Lower number of pathway events (C) in finger lime (15 times/recording) compared to Valencia (28/recording) Longer duration of xylem ingestion (G) per recording in fingerlime (32%) compared to Valencia (17%) but no difference was found in the number of G. Lower percentage of the total prob was spent in phloem ingestion (E2) in finger lime and the hybrids (9-12%) compared to Valencia (29%). However, the mean duration of phloem salivation was not significantly different among treatments. We are currently in the process of analyzing the data and interpreting the trend to understand the biological mechanisms involved in this feeding interaction. 1. B) Assessment of finger lime hybrids as rootstocks: Valencia graft take ranged from 40% to 100%, depending on the hybrid. The highest graft take percentage was recorded when MFL1-98 was used as rootstock recording 100% with the clean and infected scions.Valencia' grafted onto this rootstock also showed the highest values of the growths parameters (scion height and diameter and rootstock diameter). 'Valencia' grafted onto MFL1-98 showed enhanced growth and chlorophyll pigments compared with 'Valecia' grafted onto swingle. The highest starch content was observed with 'Valencia grafted onto Swingle and PFL1-11 rootstock. 2) Assessment of finger lime hybrids as interstocks: Finger lime hybrid interstock plants had Swingle as the rootstock and 'Valencia' as the scion.Plants were challenge inoculated by CLas-positive ACPs.We monitored ACP population dynamics on these challenge-inoculated interstocked plants. Results are really interesting as two of the interstocks from above with the least CLas titers (UF SUNLIME & MFL1-98) had the most ACP colonization and population increase, indicating these two are providing substantial CLas control despite the increased ACP pressure on them. 3) Understand the role of metabolites in the HLB resistance process. Finger lime hybrids of along with the parents were analysed. Released VOCs (SPME): The released VOCs were collected from the new shoots (where ACP are usually attracted, colonizes and lays eggs) of each eight different parent types and 10 hybrid types by solid phase microextraction (SPME) fibers. The fiber was the "mixed" fiber (Carboxen/DVB/PDMS) which absorbs a wide range of volatiles. Four replicates of each plant type were sampled. The volatile samples were analyzed by GC-MS in scan mode and 72 chromatograms were obtained (18 x 4). 56 VOCs were detected and peak areas were integrated and used to calculate the percent peak area of each chromatogram. The new shoots used for SPME VOCs were removed immediately after taking their released VOCs. These new shoots were weighed, photographed, and stored at stored at -80 °C for further analysis. Peak areas of SPME samples were normalized by fresh weight (FW). Challenges with 'Ca. Liberibacter asiaticus' and ACP Hot ACP inoculation/reproduction:Beginning in January 2023, groups of three of each parent and hybrid with new shoots (good for insect colonization) were taken to the 'Ca. L. asiaticus'-infected psyllid colony. The order of which plants were taken for ACP inoculation was based on which type was flushing. The three plants were placed together inside a medium-sized insect cage and ~60 ACP were released (~20 per plant) and left for one month to colonize the plant and reproduce. Adults were removed after two weeks and eggs and nymphs were left to develop. At the end of one month, the new generation of ACP was collected and counted to enumerate the reproduction. An Excel file is attached and shows the three groups. The last group to flush (and the biggest group) is still in the insect room (the experiment was started at the end of April) and the final insect count is happening this week. There were still nymphs developing on some varieties so I left them longer. Also, Inodora was last to flush and is not very vigorous. Finger lime and many of its hybrids did not support ACP well (PFL2-61, MFL2-108, PFL1-3, MFL-10-12). Graft-inoculation: Three of each parent and hybrid were graft inoculated with 'Ca. L. asiaticus' during the first two weeks of April 2023 using infected plant material. Due to the large number of buds required, various sources were used. For example, budwood from HLB+ SugarBelle was used to graft-inoculate the SugarBelle plants, budwood from HLB+ Valencia was used to graft the OLL-8 sweet orange, and HLB+ C. macrophylla was used for finger lime and many FL hybrids. Most plants received 2-3 grafts depending on how thorny the plant was. For Inodora only 2 plants were grafted because they have been slow-growing and were too small still to graft. A third Inodora will be grafted when one is big enough. All plants will be tested by qPCR in October (6 months after grafting). <br><br><b>Publications</b><br>

<br>What was accomplished under these goals? Objective 1) Identify the most effective rootstocks with Australian lime genetics for HLB resistance to the scion We propagated all rootstocks proposed in this study. A subset of these rootstocks was stick grafted with HLB infected Valencia scion to determine their ability to protect trees from HLB. Trees with clean HLB free Valencia scions were controls. The initial CaLas Ct values was recorded before grafting (22.25 ± 1.11). Trees were evaluated after six months of grafting. There was no change in the chlorophyll content between the treatments. Total phenolic compounds (TPC) content was determined in leaves to evaluate the induced oxidation response in the cultivars. There were significant differences between the Australian lime rootstocks and controls in TPC levels. There was also variation in the CaLas Ct values and trees ranged from 26.9 to 35. It is expected that a better understanding of the Ct values will be obtained in year 2 of this study. Candidate genes responsible for the production of Cys-rich secretory proteins and Pathogenesis-related 1 (PR1-like) proteins were differentially regulated between the treatments and control. Objective 2) Assess the impact of interstocks in protecting scions against HLB Swingle rootstock was used to produce interstocks. All interstock trees were prepared and subsequently budded with Valencia scion. Budding was delayed due to slow growth of some interstock trees. Trees will be challenged with CaLas in the spring 2023. Objective 3) Understand the role of metabolites in the HLB resistance process. A subset population of the Australian lime hybrids along with their parents were produced. Phloem sap composition from these rooted cutting are being analyzed using GC-MS. We are also letting the trees get larger so that other metabolic analysis can be performed on this population. <br><br><b>Publications</b><br>