Progress 09/15/23 to 09/14/24
Outputs
Target Audience:Citrus growers, public, consumers, high school students, graduate students, scientific communities, juice industry, ag industry, regulatory agencies Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?3 graduate students, 2 undergraduates and 4 postdocs have attended scientific meetings to present research progress related to this project. How have the results been disseminated to communities of interest?Extension articles, extension talks, website, field days. What do you plan to do during the next reporting period to accomplish the goals?Wang lab will continue to generate transgene-free genome edited citrus lines and test them for HLB resistance/tolerance and will continue to conduct field trials to test horticultural approaches to control HLB. Tripti: will continue field trials and collect tree health and harvest data. In addition, Vashisth lab will initiate an in-depth greenhouse study to understand the mechanism by which GA and micronutrients are benefiting the HLB-affected trees. El-Kereamy will finish the second season of the field trial and collect data. Seymour: will regenerate plants from callus that were edited for the target genes. Madhu: Will continue the field trials and collect data, will conduct genome editing for select genes for Rio Red grapefruit. Dandekar will continue to develop embryogenic tissue cultures for Eureka lemon and Lisbon lemon and develop protocols to conduct non-transgenic genome editing for Eureka and Lisbon lemon. Davie will continue with data collection under greenhouse and field conditions. El-Mohtar: Graft stable CTV overexpression vectors into sweet orange citrus seedlings to test resistance against HLB. Orozco-Cárdenas lab will continue with genome editing of Tango mandarin and Sour orange for the target genes. Jude will initiate the callus lines annually to have robust new totipotent embryogenic callus lines for protoplast experiments and callus transformation (citrus embryogenic callus lines lost their totipotency over time, and a high regeneration capacity is required for recovering plants from Crispr and transformation experiments). We will continue to provide the project team with the embryogenic callus and cell suspension cultures of cultivars important for the success of the project. Coltrane lab: Regulatory approval, registration, and commercialization of non-transgenic genome-edited lines, CTV constructs and uric acid Y. Wang lab will conduct sensory and consumer preference and flavor analyses of fruit by genome-edited lines when ready. Guan lab will conduct economic analyses of different management approaches. The team will work together on outreach and extension.
Impacts
What was accomplished under these goals?
Objective 1. Field trials. Multiple field trials are ongoing in Florida, California, and Texas to evaluate the effect of antioxidants, micronutrients or plant hormones on HLB management. In Florida, Tripti led two field trials to evaluate the right timing of GA application for sweet oranges for improving health and productivity of HLB-affected trees. Tripti and team have finished the harvest of the Valencia and Hamlin harvest in 2023-2024. We have also collected harvest data from GA and other PGRs study. In this study Tripti and team are using GA in combination with Methyl salicylate to induce vegetative growth as well as Systemic acquired resistance response. GA + MeSA improved yield by 20%. In addition, GA alone and combination treatment slowed the canopy decline as compared to control. Figures for fruit drop and canopy density are shown below. Wang led one field trial to evaluate antioxidants and immunoregulators for mitigating citrus HLB symptoms under field conditions. The treatments GA at 7.5 mg/l and SA at 1.0 mM significantly reduced H2O2 contents in leaf tissues at 24 hours post application and the other treatments reduced ROS (H2O2) levels compared the untreated control, although the differences were not statistically significantly. Kadyampakeni conducted two greenhouse studies on Zinc and Molybdenum fertilization comparing the response of HLB and non-HLB trees was completed. A follow-up field study is underway for both micronutrients. In California, El-Kereamy and team are working on the data collected from the first season and have completed the field trials for the second year to test the effect of the GA and Uric acid on the growth, yield and fruit quality of Tango Mandarin and Navel oranges under California condition. In Texas, Kunta and team have successfully implemented foliar spray treatments of micronutrients (Manganese, Zinc, Iron, and Boron) at two rates (2 quarts/acre and 3 quarts/acre) on HLB-affected citrus trees and tested antioxidants, including Gibberellic acid, Uric acid, and GABA (applied in high and low rates) as a secondary treatment under each micronutrient rate. These applications aimed to assess their effectiveness in mitigating oxidative stress caused by HLB. Objective 2. El-Mohtar lab in collaboration with the Wang lab engineered and inoculated citrus with 14 citrus vectors to increase the expression of antioxidant enzymes and silence key genes involved in CLas-triggered ROS production. Plants that were silenced for one gene showed significant HLB tolerant, which was confirmed using CRISPR genome editing. Other genes are under investigation. Objective 3. Optimization of transgene-free citrus genome editing technology. We have successfully developed two different transgene-free citrus genome editing technologies. The first one is based on transformation of embryogenic protoplasts with Cas12a/crRNA ribonucleoprotein. The second one is based on a co-editing strategy. It generates transgene-free, gene-edited plants via Agrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFP in planta. Using this approach, transgene-free genome-edited plants were efficiently generated for various genes (either individual or multiplex) in citrus in the T0 generation. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 8% to 50%. Development of embryogenic tissue cultures. Grosser lab is responsible for initiating and maintaining different calluses of sweet orange, grapefruit, and lemon. During this period, Grosser lab has initiated callus of the following cultivars: Sweet oranges: (Valencia, Hamlin, EV1 & EV2 (early-maturing Valencia selections), Vernia (mid-season), N7-3 (seedless Valencia), and Valencia somaclones T1-56 and B9-65 (selected for high yield and soluble solids). Red grapefruits: (N11-7, Rio Red, N11-11, N11-29, and cybrid Flame C4-3-32) Mandarin (W. Murcott, Tango, and Sun Chu Sha Kat Mandarin). Lemon (Lisbon lemon and Eureka Lemon). Dandekar lab has been focusing on developing embryogenic tissue cultures for two lemon varieties Lisbon and Eureka. Seymour lab: Cell cultures and callus have been successfully developed for Washington navel orange and five additional sweet orange cultivars, including Cara Cara, Moro, Powell, Shahani, and Olinda. Viable protoplasts were generated from each cultivar and transformations were conducted using Cas12 RNP with gRNA for six target genes. Orozco-Cárdenas lab has developed embryogenic tissue cultures for Tango mandarin and Sour orange. Genome editing of the two cultivars is ongoing. Kunta lab has been developing embryogenic tissue culture of Rio Red grapefruit. Transgene-free citrus genome editing of target genes. So far non-transgenic genome-edited plants were generated for 4 target genes. We are in the process of propagating them. In addition, other genes have been edited and in the regeneration process. Objective 4. In Florida. 20 extension presentations were given to citrus growers at Citrus Expo, Citrus Show, Citrus Institute, and other grower meetings (2 by Vashisth, 2 by Kadyampakeni, 6 by Zekri, 6 by Chris, 2 by Wang); 2 extension articles have been published. In Texas, Kunta made multiple presentations to the citrus growers, and students.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Wang N, Sundin GW, Fuente L, Cubero J, Tatineni S, Brewer MT, Zeng Q, Bock CH, Cunniffe NJ, Wang C, Candresse T, Chappell T, Coleman JJ, Munkvold G. Key Challenges in Plant Pathology in the Next Decade. Phytopathology. 2024 May;114(5):837-842. doi: 10.1094/PHYTO-04-24-0137-KC.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Khan A, `vara A, Wang N. Comparing Apples and Oranges: Advances in Disease Resistance Breeding of Woody Perennial Fruit Crops. Annu Rev Phytopathol. 2024 Sep;62(1):263-287. doi: 10.1146/annurev-phyto-021622-120124.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Pandey SS, Li J, Oswalt C, Wang N. Dynamics of 'Candidatus Liberibacter asiaticus' Growth, Concentrations of Reactive Oxygen Species, and Ion Leakage in Huanglongbing-Positive Sweet Orange. Phytopathology. 2024 May;114(5):961-970. doi: 10.1094/PHYTO-08-23-0294-KC.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Kamsikiri, K. and D. Kadyampakeni. 2024. Therapeutic impacts of molybdenum on young huanglongbing-affected citrus trees. Proc. Florida State Hort. Soc. (in press)
- Type:
Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Peddapuli, M., A. Atta, and D. Kadyampakeni. 2024. Does variable macronutrient application impact the performance of Huanglongbing (HLB)-affected sweet oranges? Proc. Florida State Hort. Soc. (in press)
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Singh K; M. Huff, J. Liu, J.-W. Park, T. Rickman, M. Keremane, R. R. Krueger, M. Kunta, M. L Roose, C. Dardick, M. Staton, and C. Ramadugu. 2024. Chromosome-scale, de novo, phased genome assemblies of three Australian limes, Citrus australasica, C. inodora, and C. glauca, towards finding insights into disease resistance to citrus huanglongbing. Plants 13:1460. https://doi.org/10.3390/plants13111460
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Su H, Wang Y, Xu J, Omar AA, Grosser JW, Wang N. Cas12a RNP-mediated co-transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome. Front Plant Sci. 2024 Aug 1;15:1448807. doi: 10.3389/fpls.2024.1448807.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
de Leon, V. S., J. Chen, G. McCollum, J.-W. Park, E. S. Louzada, M. S�tamou and M. Kunta. 2024. Diversity of Candidatus Liberibacter asiaticus strains in Texas revealed by prophage sequence analyses. Plant Disease 108: 1455-1460.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Carter EW, Peraza OG, Wang N. The protein interactome of the citrus Huanglongbing pathogen Candidatus Liberibacter asiaticus. Nat Commun. 2023 Nov 29;14(1):7838.
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Progress 09/15/22 to 09/14/23
Outputs
Target Audience:Citrus growers, public, consumers, high school students, graduate students, scientific communities, juice industry, ag industry, regulatory agencies Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?13 students or postdocs have attended scientific meetings to present research progress related to this project. How have the results been disseminated to communities of interest?Via publications, extension presentations to citrus growers at Citrus Expo, Citrus Show, Citrus Institute, and other grower meetings, workshops, and extension articles What do you plan to do during the next reporting period to accomplish the goals?Wang lab will continue to generate transgene-free genome edited citrus lines and test them for HLB resistance/tolerance and will continue to conduct field trials to test horticultural approaches to control HLB. Tripti: will continue field trials and collect tree health and harvest data. In addition, Vashisth lab will initiate an in-depth greenhouse study to understand the mechanism by which GA and micronutrients are benefiting the HLB-affected trees. El-Kereamy will continue the field trials. Seymour: will focus on protoplast-based editing of the focal navel varieties and their subsequent regeneration. Madhu: Will investigate micronutrients, GA, and uric acid on HLB management, will conduct genome editing for select genes for Rio Red grapefruit. Dandekar will continue to develop embryogenic tissue cultures for Eureka lemon and Lisbon lemon and develop protocols to conduct non-transgenic genome editing for Eureka and Lisbon lemon. Davie will continue greenhouse study on micronutrients to accomplish goals of Objective 1, conduct a field study to leverage preliminary work of Objective 1, and conduct more outreach efforts. El-Mohtar: Will check the stability of different CTV constructs in Citrus macrophylla by RT-PCR and will start grafting into sweet orange seedlings and screening for the HLB tolerance/resistance. Orozco-Cárdenas lab will conduct PEG-mediated transformation to improve transfection efficiency of Tango protoplasts, editing specific genes of interest (e.g., RBOHD) for Tango mandarin. Martha lab will also continue work to establish embryogenic tissue cultures for sour orange and conduct genome editing when ready. Jude will initiate the callus lines annually to have robust new totipotent embryogenic callus lines for protoplast experiments and callus transformation (citrus embryogenic callus lines lost their totipotency over time, and a high regeneration capacity is required for recovering plants from Crispr and transformation experiments). We will continue to provide the project team with the embryogenic callus and cell suspension cultures of cultivars important for the success of the project. Coltrane lab: Regulatory approval, registration, and commercialization of non-transgenic genome-edited lines, CTV constructs and uric acid Y. Wang lab will conduct sensory and consumer preference and flavor analyses of fruit by genome-edited lines when ready. Guan lab will conduct economic analyses of different management approaches. The team will work together on outreach and extension.
Impacts
What was accomplished under these goals?
Objective 1. Dynamics of Candidatus Liberibacter asiaticus titers, concentrations of reactive oxygen species, and ion leakage in HLB-positive sweet orange. To help determine the best timing to mitigate ROS, we have conducted monthly dynamics of CLas titers, ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. The CLas titers varied significantly with the time of the year. There are two peaks for CLas titers in Florida citrus groves, with one peak in the late spring and early summer and another peak in the late fall. In both Hamlin and Valencia asymptomatic tissues, CLas titers are strongly negatively correlated with the difference between the monthly average mean temperature and the optimum temperature for CLas colonization in plants (25.7oC). ROS levels are significantly higher in symptomatic or asymptomatic branches than that in healthy branches in most months. ROS concentrations are higher in symptomatic branches than in asymptomatic branches in most months. CLas triggers significant increases in ion leakage in most months for asymptomatic and symptomatic branches than healthy controls. A positive correlation exists between CLas titers and ROS concentrations, CLas titers and ion leakage levels, and ROS and ion leakage in asymptomatic branches of Hamlin. This study sheds lights on the pathogenicity of CLas and provides guidance regarding the application of antioxidants and antimicrobial agents to control HLB. Field trials. Multiple field trials are ongoing in Florida, California, and Texas to evaluate the effect of antioxidants, micronutrients or plant hormones on HLB management. In Florida, Tripti led three field trials. Field Trial 1-To evaluate the right timing of GA application for sweet oranges for improving health and productivity of HLB-affected trees Field Trial 2- To evaluate the effectiveness of GA and foliar nutrients in improving health and productivity and reducing fruit drop in severely symptomatic HLB-affected sweet orange trees Field Trial 3-To evaluate the effectiveness of GA and other gibberellin derivatives in improving health and productivity of HLB-affected sweet orange Wang led two field trials to evaluate different combinations of GA, micronutrients, and antioxidants on HLB management. The field trials were started in 2022. One trial includes 12 different treatments and the 2nd trial includes 13 different treatments. Kadyampakeni conducted a greenhouse study to evaluate the impact of B and Zn on HLB-affected trees. In California, El-Kereamy started the field trials to test the effect of the GA and uric acid on the growth, yield and fruit quality of Tango Mandarin and Navel oranges under California condition. In Texas, Kunta have selected the suitable groves for the testing micronutrients, GA, and uric acid on HLB management. Objective 2. El-Mohtar lab in collaboration with the Wang lab engineered and inoculated citrus with 14 citrus vectors to increase the expression of antioxidant enzymes and silence key genes involved in CLas-triggered ROS production. F of the CTV vectors are positive in Citrus macrophylla mother plants. 3 CTV vector infected plants were checked for stability by RT-PCR and will be working to graft into sweet orange plants. Objective 3. Optimization of transgene-free citrus genome editing technology. We have successfully developed two different transgene-free citrus genome editing technologies. The first one is based on transformation of embryogenic protoplasts with Cas12a/crRNA ribonucleoprotein. It was used develop transgene-free genome editedCitrus sinensislines in the T0 generation within 10 months. Among the 39 regenerated lines, 38 are biallelic/homozygous mutants, demonstrating a 97.4% biallelic/homozygous mutation rate. No off-target mutations are detected in the edited lines. The second one is based on a co-editing strategy. It generates transgene-free, gene-edited plants via Agrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFP in planta. Using this approach, transgene-free genome-edited plants were efficiently generated for various genes (either individual or multiplex) in citrus in the T0 generation. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 8% to 50%. Whole genome sequencing further confirmed transgene-free and absence of off-target mutations in the edited plants. Development of embryogenic tissue cultures. Grosser lab is responsible for initiating and maintaining different calluses of sweet orange, grapefruit, and lemon. During this period, Grosser lab has initiated callus of the following cultivars: Sweet oranges: (Valencia, Hamlin, EV1 & EV2 (early-maturing Valencia selections), Vernia (mid-season), N7-3 (seedless Valencia), and Valencia somaclones T1-56 and B9-65 (selected for high yield and soluble solids). Red grapefruits: (N11-7, Rio Red, N11-11, N11-29, and cybrid Flame C4-3-32) Mandarin (W. Murcott, Tango, and Sun Chu Sha Kat Mandarin). Lemon (Lisbon lemon and Eureka Lemon). Dandekar lab has been focusing on developing embryogenic tissue cultures for two lemon varieties Lisbon and Eureka. Seymour lab has produced callus for parent navel and 5 other sweet orange varieties (Olinda Valencia, Shahani, Moro, Powell, and Cara cara). Orozco-Cárdenas lab has developed embryogenic tissue cultures for Tango mandarin, in the development for Sour orange. Kunta lab has initiated embryogenic tissue culture of Rio Red grapefruit. Identification of target genes. To identify putative genetic determinants of HLB pathogenicity, we have conducted genome-wide association mapping and analysis of allele-specific expression between susceptible, tolerant, and resistant accessions further refined candidates underlying the response to HLB. We first developed a phased diploid assembly of Citrus sinensis 'Newhall' genome and produced resequencing data for 91 citrus accessions that differ in their response to HLB. These data were combined with previous resequencing data from 356 accessions for genome-wide association mapping of the HLB response. Multiple genes determinants for HLB pathogenicity were identified. In addition, we conducted RNA-seq analyses on HLB-susceptible Valencia sweet orange and HLB-tolerant mandarin 'LB8-9' in winter, spring, summer, and fall. Significant variations in differentially expressed genes (DEGs) related to HLB were observed among the four seasons. For both cultivars, the highest number of DEGs were found in the spring. CLas infection stimulates the expression of immune-related genes such as NBS-LRR, RLK, RLCK, CDPK, MAPK pathway, reactive oxygen species (ROS), and PR genes in both cultivars, consistent with the model that HLB is a pathogen-triggered immune disease. This study also further defined the target genes for editing. Transgene-free citrus genome editing of target genes. For the select genes, we have conducted transgene-free citrus genome editing for C. sinensis cv. Hamlin and the plants are being regenerated. Other varieties will be initiated with the embryogenic protoplasts being developed. Objective 4. In Florida. 19 extension presentations were given to citrus growers at Citrus Expo, Citrus Show, Citrus Institute, and other grower meetings (3 by Vashisth, 1 by Kadyampakeni, 12 by Zekri, 3 by Wang); 10 workshops were held; 21 extension articles have been published. Guan led two on-going studies, one on the potential impact of a technology breakthrough (effective HLB management) on players along the supply chain (growers, processors, consumers), the other on the impact of the HLB outbreak on the market power and pricing behaviors of growers and processors. In Texas, Kunta made one presentation to the citrus growers at Citrus Center Advisory Board meeting.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
1. Su H, Wang Y, Xu J, Omar AA, Grosser JW, Calovic M, Zhang L, Feng Y, Vakulskas CA, Wang N. Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation. Nat Commun. 2023 Jul 5;14(1):3957.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Gao Y, Xu J, Li Z, Zhang Y, Riera N, Xiong Z, Ouyang Z, Liu X, Lu Z, Seymour D, Zhong B, Wang N. Citrus genomic resources unravel putative genetic determinants of Huanglongbing pathogenicity. iScience. 2023 Jan 23;26(2):106024.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
3. Pandey SS, Xu J, Achor DS, Li J, Wang N. Microscopic and Transcriptomic Analyses of Early Events Triggered by 'Candidatus Liberibacter asiaticus' in Young Flushes of Huanglongbing-Positive Citrus Trees. Phytopathology. 2023 Jun;113(6):985-997.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Ribeiro C, Xu J, Hendrich C, Pandey SS, Yu Q, Gmitter FG Jr, Wang N. Seasonal Transcriptome Profiling of Susceptible and Tolerant Citrus Cultivars to Citrus Huanglongbing. Phytopathology. 2023 Feb;113(2):286-298.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
6. Danda, T., J.-W. Park, K. L. Timmons, M. S�tamou, E. S. Louzada, and M. Kunta. 2023. A field deployable real-time loop-mediated isothermal amplification targeting five Copy nrdB gene for the detection of Candidatus Liberibacter asiaticus in citrus. Plant Pathology. J. 39(4): 309-318. https://doi.org/10.5423/PPJ.OA.02.2023.0030
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
7. Basu, S., E. Sineva, L. Nguyen, N. Sikdar, J. W. Park, M. Sinev, M. Kunta & G. Gupta. 2022. Host-derived chimeric peptides clear the causative bacteria and augment host innate immunity during infection: A case study of HLB in citrus and fire blight in apple. Front. Plant Sci. 13:929478. doi: 10.3389/fpls.2022.929478
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