A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation Fighting a Ravaging Disease (2018) / Chapter Skim
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Appendix D: Selected Citrus Research Development Foundation Projects
Appendix D: Selected Citrus Research Development Foundation Projects
Pages 227-270
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Appendix D Selected Citrus Research Development Foundation Projects 227
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under different conditions 046 Identify and deliver Dawson, Bill 8/1/2010 $618,388.00 antibacterial - 7/31/2012 peptides and/or University of proteins for control Florida of citrus greening (huanglongbing or HLB) 048 Attempts to in vitro Dollet, Michel 4/1/2010 $177,120.00 culture Candidatus - 4/30/2012 Liberibacter asiaticus isolates in order to fulfil Koch's postulates 065 Genomic sequencing Gabriel, Dean 4/1/2010 $186,285.00 to closure of a - 3/31/2012 curated Florida University of citrus greening Florida strain of Candidatus Liberibacter asiaticus
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5. Develop transgenic citrus trees with effective peptides -- mature transformation.
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230 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 090 An effective trap for Mizell, Russell 11/1/2010 $98,282.00 Asian citrus psyllid - 10/31/2012 that can be used to University of monitor groves and Florida plants for sale 091 Efficacy of seasonal Hall, David 7/1/2010 $199,402.00 insecticide programs - 9/30/2012 for suppressing USDA HLB in new citrus plantings 093 Pathogen–vector Hall, David 7/1/2010 $296,414.00 relations between - 8/31/2012 asian citrus psyllid USDA and liberibacter asiaticus 123 Bioinformatic Lindeberg, 11/1/2010 $286,337.00 characterization Magdalen - 4/30/2013 and development of a central genome Cornell University resources website for Ca. Liberibacter asiaticus 125 Development Lin, Hong 11/1/2010 $215,166.00 of SSR markers - 1/31/2013 for detection, USDA genotyping, phenotyping, and genetic diversity assessment of Candidatus Liberibacter strains in Florida
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We hypothesized that citrus plants can be genetically engineered to express the molecular mechanisms associated with heat-induced Las resistance. Develop genetically modified citrus plants that will enhance defense response to HLB infection.
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232 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 15-003 Metabolomic Bowman, Kimberly 7/1/2015 $539,104.00 profiling to - 6/30/2018 accelerate USDA development of HLB-tolerant rootstocks 15-009 Exploiting the Las Gabriel, Dean 8/1/2015 $419,500.00 phage for potential - 7/31/2017 control of HLB University of Florida
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In addition, we expect to identify metabolites that are associated with resistance to HLB and that may be employed for development and improvement of early detection methods of HLB and therapeutic strategies to reduce Las titer levels in infected citrus trees. The overall goal of the project is to enable the practical exploitation of the presence of lytic prophage in all known Florida Las strains by identification of chemicals or a biological control method that could be used to artificially trigger the phage lytic cycle in psyllids and thereby cure the psyllids by provoking an innate immune response and cure citrus by provoking a stronger innate immune response; suppress the expression of phage-encoded lysogenic conversion genes, particularly peroxidase in citrus; disable phage peroxidase enzymatic function in citrus; or any combination of the above in existing Las infected trees in the field and thereby cure the trees.
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234 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 15-016C High-throughput Hall, David 8/1/2015 $375,000.00 inoculation of - 7/31/2018 transgenic citrus for USDA HLB resistance 15-017 Disrupt LuxR solo Killiny, Nabil 8/1/2015 $157,144.00 quorum sensing - 7/31/2017 that mediates plant University of virulence and Florida insect transmission of Candidatus Liberibacter asiaticus to control the disease 15-020 Create citrus Mou, Zhonglin 7/1/2015 $358,922.00 varieties resistant - 6/30/2018 to huanglongbing University of (HLB) through Florida transgenic and nontransgenic approaches
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Identification of AHLs in Asian citrus psyllids hemolymph using GC-MS. Because different types of symbionts are confined in a small environment "arena" within their insect host, cooperative interactions and communication among theses.
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236 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 15-023 Citrus nutrition Schumann, Arnold 7/1/2015 $281,804.00 studies for improved - 6/30/2018 survival of HLB- University of affected trees Florida 15-024 Predicting when, Stelinski, Lukasz 8/1/2015 $161,116.00 why, and where - 7/31/2017 asian citrus psyllids University of move to increase Florida effectiveness of insecticide sprays 15-027 Developing a Triplett, Eric 7/1/2015 $325,912.00 culture medium for - 6/30/2017 Liberibacter asiaticus University of through comparative Florida multi-omics analysis with its closest cultured relative, L crescens
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. Overall Goal: Determine the effects of abiotic factors on Asian citrus psyllid dispersal and flight capability.
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238 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 15-028 Control citrus Wang, Nian 7/1/2015 $430,697.00 huanglongbing - 6/30/2018 (HLB) by University of counteracting the Florida SA hydroxylase of Candidatus Liberibacter asiaticus 15-033C Support role of Orbović, Vladimir 7/1/2015 $270,000.00 the Citrus Core - 6/30/2018 Transformation University of Facility remains Florida crucial for research leading to production of citrus plants that may be tolerant or resistant to diseases 15-036C Correlating pesticide Rogers, Michael 7/1/2015 $451,603.00 residue analysis with - 6/30/2018 psyllid feeding to University of improve protection Florida of young trees 15-038C Insecticide resistance Stelinski, Lukasz 7/1/2015 $129,491.00 monitoring and - 6/30/2017 management in University of Florida citrus to Florida maintain sustainable control of Asian citrus psyllid within Citrus Health Management Areas
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The most important result of this project will be availability of an efficient service for production of transgenic plants according to a variety of ideas representing different approaches to fight citrus diseases. Considering the magnitude of crisis the citrus industry is in, CCTF as a place that routinely produces transgenic citrus plants will encourage researchers not to abandon any scientifically sound ideas but to test them through production of transgenic plants.
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240 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 15-039C Secure site for Stover, Ed 1/1/2016 $260,980.00 testing transgenic - 12/31/2018 and conventional USDA citrus for HLB and psyllid resistance 15-042 Control citrus Wang, Nian 7/1/2015 $467,000.00 huanglongbing using - 6/30/2018 endophytic microbes University of from survivor trees Florida 16-001 Enhancing genetic Li, Yi 7/1/2016 $316,168.00 transformation - 6/30/2019 efficiency of mature University of citrus Connecticut 16-005 GFP labeling Wang, Nian 10/1/2016 $472,753.00 of Candidatus - 9/30/2019 Liberibacter University of asiaticus in vivo and Florida its applications 16-020C Dyed kaolin to repel Vincent, 12/1/2016 $273,908.00 Asian citrus psyllid Christopher - 11/30/2019 in field conditions University of Florida
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Our hypothesis is the outcome of the interaction among Las, psyllid, and citrus is affected by the citrus phytobiome. In order to achieve the goal of this study, the following objectives will be conducted: To characterize the phytobiomes and endophytic microbes from HLB survivor trees and HLB diseased trees To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB Using P19 co-expression to improve Agrobacterium infection/transient expression efficiency of mature citrus tissues Using transient down regulation of AGO2 and NRPD1a expression to improve Agrobacterium stable transformation efficiency of mature citrus tissues Using H2A co-expression to improve Agrobacterium stable transformation efficiency of mature citrus tissues Using a combination of genes to develop a super-transformation-vector that is genotype independent, highly effective for transformation and regeneration of mature citrus Using root specific auxin biosynthetic gene to engineer rootstock of citrus to improve success rates of micrografting 1.
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A 8/2/2010 $124,786.00 to guide decision - 7/31/2012 making for control University of asian citrus psyllid Florida 176 Effects of nutrition Rogers, Michael 4/1/2010 $87,607.04 and host plant - 3/31/2012 on biology and University of behavior of the Florida Asian citrus psyllid and implications for managing psyllid populations 212 Enhanced biological Stansly, Phil 5/1/2010 $148,775.00 control of asian - 4/30/2011 citrus psyllid in University of Florida through Florida introduction and mass rearing of natural enemies 214 Quantitative Stelinski, Lukasz 8/1/2010 $71,614.00 measurement of the - 7/31/2011 movement patterns University of and dispersal Florida behavior of Asian citrus psyllid in Florida for improved management
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Evaluate psyllid fitness when reared on non-commercial citrus species and other rutaceous noncitrus host plants. Examine the effects of host plant quality on feeding behaviors of adult psyllids.
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244 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 232 Characterization Wang, Nian 4/1/2010 $218,134.00 of the virulence - 3/31/2012 mechanism of the University of citrus huanglongbing Florida pathogen Candidatus Liberibacter asiaticus 306 Culturing Davis, Michael J 5/1/2010 $65,000.00 Liberibacter asiaticus - 4/30/2011 University of Florida 307 Infection traits De La Fluente, 9/1/2010 $100,000.00 and growth of Leonardo - 5/30/2013 "Candidatus Liberibacter Auburn University asiaticus" inside microfluidic chambers 312 Functional study of Gowda, Siddrame 5/1/2010 $238,400.00 the putative effectors - 4/30/2013 of "Candidatus University of Liberibacter Florida asiaticus" using Citrus tristeza virus vector 314 Insight into the Grishin, Nick 3/1/2010 $99,902.00 causative agent - 8/30/2011 of citrus greening Southwestern disease (HLB)
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To classify and predict spatial structures and functions of all Candidatus Liberibacter asiaticus proteins and suggest their relevance in the citrus disease. To compile a database from all the data obtained in the course of the project and make it available at our group website and at the central depository www.citrusgreening.org.
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246 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 315 Speedy evaluation of Hall, David 5/1/2010 $497,503.00 citrus germplasm for - 7/31/2013 psyllid resistance USDA 325 Development Rogers, Michael 5/1/2010 $127,288.40 and evaluation of - 4/30/2012 psyllid management University of programs for Florida protection of resets and young tree plantings from HLB 330 Targeting the asian Shatters, Bob 5/1/2010 $483,000.00 citrus psyllid feeding - 6/30/2014 mechanism as a USDA means of blocking psyllid feeding on citrus 334 How does Stelinski, Lukasz 4/1/2010 $30,867.00 Liberibacter - 3/31/2011 infection of psyllids University of affect the behavioral Florida response of this vector to healthy versus HLB-infected citrus trees?
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To determine whether the settling preference of healthy and Ca. Las-infected ACP on healthy versus HLB-infected citrus plants differs from the behavior of ACP that are not carrying the pathogen 4.
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5/1/2011 $130,000.00 Liberibacter asiaticus - 4/30/2013 University of Florida 418 Analysis of the Hilf, Mark 6/1/2011 $175,000.00 colonization of - 5/31/2013 citrus seed coats USDA by "Candidatus Liberibacter asiaticus" the causal agent of citrus huanglongbing and their use as a concentrated, pure source of bacteria for research
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Build a genome-scale metabolic model of Ca. Liberibacter asiaticus and use this model to identify the likely growth conditions necessary to culture Ca.
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250 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 424 Functional McNellis, Timothy 7/1/2011 $55,000.00 disruption of - 6/30/2017 the NodT outer Pennsylvania State membrane protein University of Candidatus Liberibacter asiaticus for rootstock mediated resistance to citrus greening using a phloem directed, single-chain antibody 425 Effect of application Morgan, Kelly 8/1/2011 $199,225.00 rate, tree size, and - 10/31/2013 irrigation scheduling University of on leaf imidacloprid Florida concentration, psyllid populations, and soil leaching 434 Mass rearing and Stansly, Phil 6/1/2011 $370,975.36 release of parasitic - 5/31/2014 wasps to augment University of biological control Florida of the Asian citrus psyllid (ACP) 447 Role of nutritional Stansly, Phil 2/15/2012 $324,430.00 and insecticidal - 2/14/2016 treatments in University of mitigation of HLB in Florida new citrus plantings
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Transgenic citrus plants producing a phloem-targeted, single-chain antibody recognizing an outer membrane protein of Ca.
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252 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 502 High-throughput Hall, David 6/1/2012 $190,200.00 screening of - 7/31/2015 transgenic citrus for USDA HLB resistance 516 Develop citrus Dawson, Bill 8/1/2012 $1,239,174.00 resistant or tolerant - 1/31/2016 to HLB using University of the CTV vector Florida and transgenic approaches 523 Screening and Duan, Yongping 5/1/2012 $458,000.00 cloning of resistance - 4/30/2015 related genes USDA by RNA-seq in huanglongbing (HLB) resistant and susceptible citrus breeding lines 535 Exploiting the Las Gabriel, Dean 5/1/2012 $117,028.00 and Lam phage for - 4/30/2013 potential control of University of HLB Florida 536 Identification and Gmitter, Fred 12/1/2012 $190,000.00 mapping of the - 11/30/2014 genes controlling University of resistance to Florida huanglongbing (HLB)
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The overall goal of this project is to accelerate the utilization of the HLB resistance in Poncirus for production of HLB-resistant citrus cultivars. This project aims to develop the knowledge and tools needed for this goal.
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254 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 538 Host genetic control Gmitter, Fred 12/1/2012 $100,000.00 of interference in - 11/30/2014 Asian citrus psyllid University of life cycles Florida 567 Acoustic trap for Mankin, Richard 7/1/2012 $112,200.00 Asian citrus psyllids - 12/31/2013 USDA 579 Citrus Core Orbović, Vladimir 5/1/2012 $255,000.00 Transformation - 6/30/2015 Facility (CCTF) as a University of platform for testing Florida of different genes and/or sequences that have potential to render citrus plants tolerant or resistant to diseases 581 Key unknowns Pelz-Stelinski, 5/1/2012 $200,029.00 about Asian Kirsten - 9/30/2014 citrus psyllid biology in Florida: University of Overwintering sites Florida and alternative hosts 581-1 Enhancement -- Key Pelz-Stelinski, 7/1/2013 $11,868.00 unknowns about Kirsten - 6/30/2014 Asian citrus psyllid biology in Florida: University of Overwintering sites Florida and alternative hosts
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The objective of this project is for CCTF to continue to produce transgenic plants, maintain its capacity from the aspect of output and turnaround time, and provide a service to scientists involved in the Citrus field of research, especially those who have a goal to create Citrus lines that will be tolerant/resistant to bacterial and other emerging diseases. By doing so, CCTF will reconfirm its prominence as the central place for production of transgenic Citrus plants and present itself as platform for testing of genes and sequences that are candidates to render Citrus resistant to diseases.
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256 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 590 Enhancing psyllid Rogers, Michael 5/1/2012 $889,701.00 control through a - 4/30/2015 better understanding University of of the effects Florida of pesticide applications on psyllid feeding and mortality 600 Management Stansly, Phil 6/1/2012 $296,000.00 tactics based on - 5/31/2015 psyllid movement University of and distribution in Florida Florida citrus 603 Non-neurotoxic Stelinski, Lukasz 4/1/2012 $191,322.00 chemicals as - 12/31/2014 alternatives to University of conventional Florida insecticides for Asian citrus psyllid management and prevention of insecticide resistance
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monitor will be used to determine whether insecticides can disrupt the psyllid feeding behaviors responsible for pathogen transmission. For insecticides that disrupt psyllid feeding behavior, the longevity of feeding disruption provided will be determined.
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258 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 609 Control of citrus Wang, Nian 5/1/2012 $413,783.00 huanglongbing - 4/30/2015 by exploiting University of the virulence Florida mechanisms of Candidatus Liberibacter asiaticus and inducing plant defense 701 Exploitation of Allan, Sandra 5/1/2013 $119,096.00 visual stimuli for - 4/30/2015 better monitoring USDA and management of ACP in young citrus plantings 701-1 Enhancement -- Allan, Sandra 7/1/2013 $115,000.00 exploitation of - 6/30/2014 visual stimuli for USDA better monitoring and management of ACP in young citrus plantings
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The goal is to develop management strategies that boost plant defense to protect citrus from HLB by exploiting the virulence mechanisms of Candidatus Liberibacter asiaticus (Las) , how Las manipulates plant defense, and control HLB by blocking the translocation of Las inside the phloem from psyllid feeding sites.
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260 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 711 Identification of Bonning, Bryony 5/1/2013 $500,000.00 Bacillus thuringiensis - 4/30/2017 endotoxins active Iowa State against adult Asian University citrus psyllid 726L A bacterial virus Gonzalez, Carlos 5/1/2013 $386,902.00 based method - 10/31/2016 for biocontrol of Texas AgriLife Liberibacter Research
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This work will provide the foundation for sustainable management of the ACP through the use of transgenic citrus, or through delivery of the ACP-active Bt toxin using a nonpathogenic phloem-limited virus, such as the Citrus tristeza virus vector. Screen Bt strains for activity against the ACP and identify ACP-active Cry toxins.
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262 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 733 Molecular basis Grishin, Nick 4/1/2013 $200,000.00 of citrus greening - 3/30/2015 and related diseases Southwestern gleaned from Medical genome analyses of hosts and pathogens 750 Identification of key Ma, Wenbo 4/1/2013 $299,781.00 components in HLB - 3/31/2016 using effectors as University of probes California, Riverside 766 Biotic and abiotic Stelinski, Lukasz 4/1/2013 $145,039.00 factors that cause - 9/30/2015 Asian citrus University of psyllids to accept Florida hosts: potential implications for young plantings and pathogen transmission
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Overall Goal: Define the factors governing ACP host acceptance in citrus groves. Objective 1.
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264 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 769 A team approach Triplett, Eric 4/16/2013 $448,257.00 to culturing Ca. - 10/15/2015 Liberibacter asiaticus University of Florida 805 Functional genomics Long, Sharon 4/1/2014 $540,197.00 of Liberibacter in a - 12/30/2016 model system Stanford University 834 Optimizing heat Duan, Yongping 4/1/2014 $385,900.00 treatment in - 3/31/2016 the fields and USDA understanding the molecular mechanism behind the success of thermotherapy for the control of citrus HLB
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Team members involved: Eric Triplett (primary) , Mike Davis, and Mark Hilf.
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266 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 850 Scheduling ACP Albrigo, Gene 4/1/2014 $90,000.00 spring spray - 3/31/2017 selection based on University of the Citrus Flowering Florida Model 853 Why is Poncirus LaPointe, Stephen 5/1/2014 $187,681.00 trifoliata resistant - 8/30/2016 to colonization by USDA Asian citrus psyllid? 858 New nonphytotoxic Santra, 4/1/2014 $350,000.00 composite polymer Swadeshmukul - 3/31/2017 film barrier as ACP repellent for University of controlling HLB Central Florida infection 910 An integrated Powell, Chuck 4/1/2014 $369,714.00 approach for - 9/1/2016 establishment of University of new citrus plantings Florida faced with the HLB threat
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. The objective of this project is to determine the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB.
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268 APPENDIX D Principal Start and Original Project # Project Title Investigator End Date Contract Total 916 Screening and Wang, Nian 7/1/2014 $431,180.00 application of - 6/30/2017 antibacterial University of producing microbes Florida to control citrus huanglongbing 922 Control citrus Wang, Nian 4/1/2014 $436,045.00 canker by - 3/31/2017 manipulating the University of EBE (effector Florida binding element) of CsLOB1, which is the citrus susceptibility gene for citrus canker disease 928.1C Field trial of Sutherland, Dudley 4/11/2014 $100,788.00 naturally occurring - 3/31/2017 microbes Glades Crop Care, Inc 934C Soil drenches of Curtis, John 6/1/2014 $15,000.00 products to combat - 9/30/2016 initial HLB infection Better Crops, LLC in young citrus trees 941C Influence of Pelz-Stelinski, 2/1/2015 $105,782.00 thermal therapy Kirsten - 7/31/2016 on transmission of Candidatus University of Liberibacter asiaticus Florida 944C RSA -- small plant Pelz-Stelinski, 3/1/2015 $125,797.33 assay for testing Kirsten - 7/31/2016 the efficacy of antimicrobial University of materials against Florida HLB
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The objective of this study will be to determine how each of these heat treatment methods may influence acquisition of a systemic, circulative infection of Candidatus Liberibacter asiaticus by the Asian citrus psyllid, and subsequent inoculation (transmission) to susceptible hosts.
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