Progress 09/15/23 to 09/14/24
Outputs
Target Audience:The project's outreach efforts disseminate information about strategies to control HLB, as well as updates on the project's advancements, to various stakeholders such as farmers, industry representatives, government regulators at state and federal levels, as well as students and the general public Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Several postdoctoral scholars are participating in the project. Additionally, graduate students are also conducting research on different aspects of this project. How have the results been disseminated to communities of interest?We routinely disseminate the outcome of our research to interested stakeholders through in-person meetings and presentations. What do you plan to do during the next reporting period to accomplish the goals?We will continue to monitor CLas and ACP populations on all our rootstock, 'inarched' and 'interstocked' trees and we will provide update in next reporting cycle. The ACP feeding behavior that has been observed to bedifferent on hybrids and fingerlimes compared to Valencia plants will be confirmed. We will fully characvterise the leaf metabolites (TMS/GC-MS) from the the 85 samples and identify the compounds, integrate the chromatograms and perform data analysis.
Impacts
What was accomplished under these goals?
Summary of Study on Feeding Behavior and Disease Resistance of Diaphorina citri on Finger Limes and Hybrids This study investigates the feeding behavior of the adult Asian citrus psyllid (Diaphorina citri) on finger limes (Citrus australasica) and their hybrids using Electropenetrography (EPG) to explore potential mechanisms of tolerance to Huanglongbing (HLB) in these plants. It hypothesizes that differences in the feeding behavior of the psyllid on these plants may influence the acquisition and transmission of the HLB bacterium, Candidatus Liberibacter asiaticus (CLas), potentially explaining the tolerance observed in finger limes and their hybrids. EPG Analysis and Feeding Behavior EPG is a technique that records insect feeding behavior by detecting electrical signals produced when the psyllid's stylet pierces plant tissue. This study focused on the following varieties of plants: Valencia (Control) Finger lime Hybrid 3 (H-03) Hybrid 55 (H-55) Hybrid 11 (H-11) Over 107 recordings of 24 hours each were made using AC-DC monitors. Results from EPG recordings highlighted significant differences in feeding behavior: Longer Non-feeding Periods: Psyllids spent significantly more time not feeding (almost an hour more) on finger limes and hybrids compared to Valencia. Delayed Phloem Ingestion: The first phloem ingestion (E2 waveform) was delayed by 6-9 hours on hybrids and finger limes compared to Valencia. Reduced Phloem Ingestion: There were fewer instances of phloem ingestion (E2) on hybrids and finger limes, with only 9-11% of probing time spent on phloem ingestion, compared to 29% on Valencia. These findings suggest that psyllids face difficulty in identifying and accepting finger limes and hybrids as suitable hosts, which may interfere with their ability to acquire and inoculate CLas. Inarching and Interstock Experiments Two experiments were conducted to assess the potential of finger limes as rootstocks in preventing CLas transmission. Inarching Experiment: 65 finger lime seedlings were inarched with CLas-infected Valencia plants. Results indicated no significant reduction in CLas titer across various rootstocks, and the plants remained CLas-positive with no difference between inarched and control plants. Interstock Experiment: A total of 109 Valencia plants were interstocked with different finger lime accessions. Two interstock types, UF SL (Sunlime) and R1T98, showed significantly lower CLas accumulation despite high psyllid colonization, indicating some resistance mechanisms in these hybrids. Graft Compatibility and Performance The compatibility of different finger lime hybrids as rootstocks for Citrus sinensis (Valencia) was also studied. Hybrids MFL1-98 and PFL2-61 showed high graft success and plant vigor, while others, such as O+M2-75 and KB08, exhibited poor compatibility, especially when infected budwoods were used. CLas Infection and Physiological Responses Quantitative PCR (qPCR) revealed significant variations in CLas titers among rootstocks. At 6 months after infection, Ct values ranged from 21.1 to 35.5, with higher values indicating lower bacterial titers. Infected plants exhibited physiological and biochemical responses indicative of stress, including: Decreased Chlorophyll: Infected plants had lower chlorophyll a and b concentrations. Increased Carotenoids: Higher carotenoid levels were observed, reflecting stress responses. Starch Depletion: Infected plants showed reduced starch content, indicating impaired carbohydrate storage. Moreover, higher levels of total phenolic compounds (TPC) and flavonoids (TFC) were observed in infected plants, which are believed to be part of the plant's defense mechanisms. Gene Expression Analysis Gene expression profiles revealed varied responses to CLas infection. Genes associated with carbohydrate metabolism (such as beta-amylase) and cell wall synthesis (such as 1,3-Beta-Glucan Synthase) were upregulated in certain hybrids, indicating active defensive responses. Stress-related genes like Glutathione S-Transferases and Catalase showed differential expression, highlighting the plants' adaptation to oxidative stress caused by infection. Chemical Analysis of Volatiles Chemical analysis of volatile compounds from finger limes and hybrids was performed using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). Results showed distinct differences in stored volatiles and pigments between hybrids and parent plants, with good separation between finger lime and other hybrids. The overall concentration of volatiles was lower than expected from mature leaves, but notable variations were found between parent varieties and hybrids. Conclusion The study suggests that the different feeding behaviors of the Asian citrus psyllid on finger limes and their hybrids, as well as variations in plant responses to CLas infection, contribute to the tolerance mechanisms in these plants. Despite the increased psyllid colonization, certain hybrids, such as UF SL (Sunlime) and R1T98, exhibited significantly lower CLas titers, pointing to their potential in managing HLB. Additionally, chemical and gene expression analyses indicate that these plants mount strong defense mechanisms in response to pathogen stress, making them promising candidates for future rootstocks and disease management strategies in citrus farming.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Kellee Britt-Ugartemendia, Kristian Stevens, Maher Al Rwahnih, Amit Levy, and Ozgur Batuman (2024). RNA-Sequencing-Based Virus Discovery in Florida Citrus Pests. Plant Health, APS Annual Meeting, July 27-31, Memphis, TN
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Lauren Fessler, Kellee Britt-Ugartemendia, Ana Redondo, Selin Boga, Jean-Yves Berisse, Fetchina Gereus, and Ozgur Batuman (2024). Screening antibiotics for phytotoxicity & potential to control Candidatus Liberibacter asiaticus, bacterial agent of Huanglongbing, in Florida citrus. Plant Health, APS Annual Meeting, July 27-31, Memphis, TN
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Ana Redondo, Sanju Kunwar, Denise Manker, Meagan Iott, Thomas Knobloch, Stephane Brunet, Jeremy Dufour, and Ozgur Batuman (2024). Defending Citrus Orchards: Novel Approaches to HLB and Canker Management Using New Plant Defense Inducers. The Seventh International Research Conference on Huanglongbing (IRCHLB) Conference, March 26-29, 2024, Riverside, CA, Riverside CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Demard E, Ebert T, Roldan E, Dutt M, Pelz-Stelinski KS, and Diepenbrock L. 2024 Characterization of the feeding behavior of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae) on finger limes and finger lime hybrids using electropenetrography. International Electropenetrography (EPG) Conference, Lake Alfred, FL, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Mou Z, Dawson W, Grosser J, Orbovic V, Dutt M, Levy A, El Mohtar C, Batuman O and Irey M. 2024. Multiple approaches towards Huanglongbing tolerance. Journal of Plant Pathology 11:1 p. 31
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Grosser J, Gmitter F, Dutt M, and Chater J. 2024. Finding HLB Tolerance for Commercial Citrus From Expected and Unexpected Sources. Journal of Plant Pathology 11:1 p. 42
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Dutt M, Mahmoud LM, Deol JG, Weber K, and Grosser JW. 2024. Exploring the Feasibility of Australian Limes to Create Hybrid Rootstocks with HLB tolerance. Journal of Plant Pathology 11:1 p. 44
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Dutt M, Killiny N, Mahmoud LM, Gmitter F and Grosser JW. 2024. Harnessing the genetic potential of citrus species to develop new hybrids with enhanced tolerance to Huanglongbing. Journal of Plant Pathology 11:1 p. 70
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Mahmoud LM, Weber K, Deol JG, Grosser JW and Dutt M. 2024. Mechanisms Underlying Huanglongbing (HLB) Tolerance in the Finger Lime (Citrus australasica) and Its Hybrids. Journal of Plant Pathology 11:1 p. 72
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Mahmoud LM, Deol J G, Grosser JW, Killiny N and Dutt M. 2024. Molecular Analysis of Pummelo-Finger Lime Hybrids following Huanglongbing (HLB) infection. Abstracts book of 37th Annual Meeting of the Florida State Horticultural Society p. 32
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Progress 09/15/22 to 09/14/23
Outputs
Target Audience: The project's outreach efforts disseminate information about strategies to control HLB, as well as updates on the project's advancements, to various stakeholders such as farmers, industry representatives, government regulators at state and federal levels, as well as students and the general public. Batuman et al. Can finger limes help Valencia and Hamlin trees be more tolerant of HLB? Citrus Expo. August 16-17, Tampa, FL Batuman, O. HLB and its management in citrus groves. Presentation to Citrus Advisory Board. June 9, 2023 (Virtual) Batuman O. Citrus Pathology Program for Huanglongbing (HLB) management. Presentation to USDA/Cornell Research Group. June 1, 2023 (Virtual) Batuman, O. IPCs' beneficial effects on citrus diseases and challenges they create. UF-IFAS In-service training to extension Agents. May 3, 2023; Immokalee, FL Batuman, O. Emerging viral diseases of citrus and management of their insect vectors. UF-IFAS In-service training to extension Agents. Mar 28, 2023; Immokalee, FL Batuman, O. Citrus Pathology Program, and Projects. Presentation to specialty crop county extension agents. Apr 27, 2023; Clewiston, FL Batuman, O. Plant pathogens that may exacerbate the HLB effects on citrus trees. UF-IFAS Citrus Seminar Series for citrus stakeholders Jan 10, 2023; Immokalee, FL Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A student intern from Zamorano university in Honduras was invited for a short term 3 month internship. He participated in the detailed evaluation of the Australian lime hybrids. This student also learned the basics of DNA and RNA extraction. He also conducted evaluation of the trees for CaLas titre using real time PCR. One postdoc was trained in experimental design, advising graduate students, presentation,and progress report deveopment. How have the results been disseminated to communities of interest?We routinely disseminate the outcome of our research to interested stakeholders through in-person meetings and presentations. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We will continue to screen our rootstock trees for HLB by routinely monitoring for CaLas presence using qPCR. Differential gene expression will also be studied in these trees and EPG technology will be used to understand the probing behaviors of adult ACP. Tree health will be monitored through the evaluation of several physiological parameters (starch content, Total phenolic content (TPC), Proline content, Malondialdehyde (MDA) content in both leaves and roots as well as chlorophyll a, chlorophyll b and total chlorophyll content in the leaves.We will complete processing the EPG data and interpreting the trend to understand the biological mechanisms involved in this feeding interaction. Objective 2: HLB infected interstock trees will be continuously monitored for bacterial titer accumulation using qPCR and defense gene expression in the HLB susceptible scions will be recorded. Objective 3: The phloem sap metabolite study will be completed. Additionally, leaf and root metabolites will also be characterized to produce a detailed understanding of the hybrid germplasm.
Impacts
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).
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Dutt M, Mahmoud LM P, Grosser JW. 2023. Field Performance of Valencia Sweet Orange Trees Grafted onto Pummelo Interstocks and Swingle Citrumelo Rootstocks under Huanglongbing (HLB) Endemic Conditions. Horticulturae 9 (6):719
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