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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Project # Project Title Principal Investigator Start and End Date Original Contract Total
002 Characterize the roles of callose and phloem proteins in citrus huanglongbing (HLB) symptom development Albrigo, Gene

University of Florida

4/1/2010 - 9/30/2012 $250,000.00
045 Examine the response of different genotypes of citrus to citrus greening (huanglongbing) under different conditions Dawson, Bill

University of Florida

7/1/2010 - 6/30/2011 $453,322.00
046 Identify and deliver antibacterial peptides and/or proteins for control of citrus greening (huanglongbing or HLB) Dawson, Bill

University of Florida

8/1/2010 - 7/31/2012 $618,388.00
048 Attempts to in vitro culture Candidatus Liberibacter asiaticus isolates in order to fulfil Koch’s postulates Dollet, Michel 4/1/2010 - 4/30/2012 $177,120.00
065 Genomic sequencing to closure of a curated Florida citrus greening strain of Candidatus Liberibacter asiaticus Gabriel, Dean

University of Florida

4/1/2010 - 3/31/2012 $186,285.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Objectives
  1. Characterize the blockage materials with immunoassay techniques with TEM and test for induction signals and virulence factors.
  2. Transgenic approaches to disrupt HLB-associated callose and phloem protein plugging by reducing their production.
  3. Reduce sieve element blockage in field and greenhouse HLB-infected plants through treatment with chemicals that are expected to alter callose and p-protein production. The information learned from the proposed research may lead to alternative control methods and will contribute to the understanding of the virulence mechanisms of Las and will be distributed through an extension program.
  1. Continue to examine the susceptibility of citrus genotypes to Las.
    1. Compare the response of commercial varieties and more citrus relatives to Las.
    2. Examine the sensitivity of elite lines and select hybrids for the Plant Improvement Group.
  2. Examine the reduction in Las titer in Poncirus trifoliate.
  3. Characterize the pathogen that induces symptoms in Citrus latipes.
  1. Continue to screen peptides for activity against Las.

    1a. Screening against Las in citrus.

    1b. Screening against Liberibacter in tobacco.

  2. Examine ability to export peptides from the rootstock.
  3. Improve Citrus tristeza virus (CTV)-based vector.

    3a. Develop a vector to express 2–5 peptides.

    3b. Develop a vector to overcome cross protection.

    3c. Remove aphid transmissibility from the vector.

    3d. Develop a vector that is restricted to transgenic tree parts.

  4. Examine survival of peptides in fruit and juice.
  5. Develop transgenic citrus trees with effective peptides—mature transformation.
To obtain a pure axenic culture of several strains of Ca. Liberibacter asiaticus maintained at this time in periwinkle and in infectious D. citri populations by the use of feeder cells: in this case insect cells cultures. Objectives of the project are numerous but the first of all is to obtain pure axenic cultures of Liberibacters in order to be able to fulfil Koch’s postulates. Furthermore, some indirect objectives are to enable and facilitate development of technologies for improved detection of Ca. Liberibacter spp. and epidemiological studies, to facilitate host/pathogen/insect vector interactions studies, and to facilitate studies on genomics and molecular biology of Liberibacters.
The principal objective of this funding request is to completely close the Las UF506 genome.
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Project # Project Title Principal Investigator Start and End Date Original Contract Total
090 An effective trap for Asian citrus psyllid that can be used to monitor groves and plants for sale Mizell, Russell

University of Florida

11/1/2010 - 10/31/2012 $98,282.00
091 Efficacy of seasonal insecticide programs for suppressing HLB in new citrus plantings Hall, David

USDA

7/1/2010 - 9/30/2012 $199,402.00
093 Pathogen–vector relations between asian citrus psyllid and liberibacter asiaticus Hall, David

USDA

7/1/2010 - 8/31/2012 $296,414.00
123 Bioinformatic characterization and development of a central genome resources website for Ca. Liberibacter asiaticus Lindeberg, Magdalen

Cornell University

11/1/2010 - 4/30/2013 $286,337.00
125 Development of SSR markers for detection, genotyping, phenotyping, and genetic diversity assessment of Candidatus Liberibacter strains in Florida Lin, Hong

USDA

11/1/2010 - 1/31/2013 $215,166.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Objectives
  1. Develop a better method for monitoring both psyllids and HLB pathogens in the environment.
  2. Identify attractants for the psyllids. Project will address the needs of regulatory agencies as well as citrus growers.
In our proposed project, we would evaluate ACP and HLB control under intensive and moderate insecticide programs.
  1. Development of HLB in young citrus subjected to intensive or moderate insecticide control programs or an oil program for psyllid control.
  2. Development of HLB in young citrus subjected to an intensive insecticide program for ACP control compared to development of HLB in young citrus subjected to a moderate insecticide program in combination with growing Murraya paniculata as a potential trap plant or source of ACP natural enemies.
  3. The effect of planting density on development of HLB in young citrus subjected to an intensive insecticide program for ACP control.
  4. Protecting citrus resets from HLB.
  1. To study pathogen–vector cellular interactions, transmission barriers, and other factors affecting vector specificity and vector competence.
  2. To clarify various acquisition and transmission parameters between the psyllid vector and the HLB pathogen.
To characterize the molecular interactions between the citrus-FLS2 and CLas/X. citri flagellins and increase disease resistance by regulating the expression of the functional FLS2 To understand the roles of FLS2 and a leucine-rich repeat receptor kinase (LRR-RK) gene by mutating them using the newly developed CRISPR/Cas9 system.
Identify molecular mechanisms associated with host response during heat treatment of Las-infected citrus plants. We hypothesized that novel mechanisms will be identified in heat-treated Las-infected citrus tissues that are absent in tissues exposed to only heat or Las infection.

Induced citrus plants to mimic the molecular conditions associated with heat-induced Las resistance. 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. We hypothesized that there will be a net gain in fitness and resistance to Las in genetically modified plants compared to non-genetically modified plants.

Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Project # Project Title Principal Investigator Start and End Date Original Contract Total
15-003 Metabolomic profiling to accelerate development of HLB-tolerant rootstocks Bowman, Kimberly

USDA

7/1/2015 - 6/30/2018 $539,104.00
15-009 Exploiting the Las phage for potential control of HLB Gabriel, Dean

University of Florida

8/1/2015 - 7/31/2017 $419,500.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Objectives
Susceptibility of existing citrus rootstock cultivars to HLB and other diseases poses a great risk for tree and crop loss. The availability of resistant rootstocks with other qualities essential for superior production of citrus would effectively eliminate disease as a threat and permit continuous higher production at much lower production cost. The proposed research will be complementing the USDA rootstock development program and is expected to identify metabolite profiles in citrus rootstock cultivars, and conferred to commercial citrus scions, which are associated with tolerance to HLB, tolerance to other stresses, and the excellent production of citrus fruit. Metabolite profiles of field-grown plants established during the first year of research will be correlated with data from previous and ongoing rootstock trials regarding HLB tolerance, growth performance, and citrus fruit production. Continuous experiments involving greenhouse and field studies during the following years will allow us to refine metabolic profiles and to integrate findings into our breeding efforts. The ultimate goal of this project is the early selection of the most promising candidate rootstocks prior to long-term field testing, therefore reducing the time and expense of testing, and accelerating the release of trees for commercial use. 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.

Objectives

Control of HLB using the putative Las LexA target

Control of HLB using the psyllid repressor as target

Control of HLB using Las peroxidase and Las lytic cycle activator(s) as targets

Field and greenhouse testing of lead compounds

Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Project # Project Title Principal Investigator Start and End Date Original Contract Total
15-016C High-throughput inoculation of transgenic citrus for HLB resistance Hall, David

USDA

8/1/2015 - 7/31/2018 $375,000.00
15-017 Disrupt LuxR solo quorum sensing that mediates plant virulence and insect transmission of Candidatus Liberibacter asiaticus to control the disease Killiny, Nabil

University of Florida

8/1/2015 - 7/31/2017 $157,144.00
15-020 Create citrus varieties resistant to huanglongbing (HLB) through transgenic and nontransgenic approaches Mou, Zhonglin

University of Florida

7/1/2015 - 6/30/2018 $358,922.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Objectives
Citrus plants transformed to express AMPs must be inoculated in order to evaluate HLB resistance. A startup inoculation program was established in 2011 using funds from the Citrus Research and Development Foundation (CRDF) (Hall-502, ends July 2015). Colonies of ACP are maintained in cages on potted citrus plants infected by CLas and showing HLB symptoms. A two-step inoculation program is used. First, individual plants are subjected to a 2-week no-choice infestation by ACP from these colonies; and second, the plants are held for 6 months in a greenhouse with an open infestation of ACP coming from CLas-infected source plants. Over 7,000 transformed scion or rootstock plants have passed through this program and are currently being evaluated for resistance. Meanwhile, the citrus breeders continue developing new transformed germplasm. This proposal asks CRDF for renewed/expanded support for inoculations. To maintain the inoculation program, funding is needed for labor (we request funds for two technicians 100% dedicated to the project), insect cages, materials, and supplies, notably for qPCR assays, which have proved critical in selecting which ACP colonies to use in the first inoculation step. Our funding request is based on an inoculation pace of at least 300 plants monthly. This pace will require 10 to 20 individual colonies of hot ACP.
To enhance citrus resistance to CLas by quenching the quorum-sensing signals; CLas quorum-sensing signals will be quenched by expressing acyl-homoserine lactonase (AHL-lactonase) in citrus plants.

To test the effect of AHL-producing citrus plants on the pathogencity of CLas; an AHL-producing citrus plant will be produced to study the effect of AHL on the pathogencity of CLas.

To prevent CLas from infecting citrus plants by jamming bacterial communication of CLas; quorum-sensing (QS) antagonists will be used to block the cell-to-cell signaling of CLas. 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.

Test commonly used bioassay strains to find the best reporter strain for the AHLs present in ACP or AHL mimic compounds present in the phloem sap of citrus. In addition, we aim to develop a more sensitive reporter (double sensors).

The overall goal of this proposal is to create citrus varieties resistant or tolerant to HLB using two different approaches. One approach is to generate HLB-resistant/tolerant transgenic citrus plants, and the other approach is to screen for HLB-resistant/tolerant citrus mutants. These two approaches complement each other.

Confirm HLB resistance or tolerance in citrus transgenic lines and putative mutants.

Stack the ELP3 and NPR1 genes in citrus.

Generate and test transgenic citrus plants overexpressing newly cloned disease resistance genes.

Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Project # Project Title Principal Investigator Start and End Date Original Contract Total
15-023 Citrus nutrition studies for improved survival of HLB-affected trees Schumann, Arnold

University of Florida

7/1/2015 - 6/30/2018 $281,804.00
15-024 Predicting when, why, and where asian citrus psyllids move to increase effectiveness of insecticide sprays Stelinski, Lukasz

University of Florida

8/1/2015 - 7/31/2017 $161,116.00
15-027 Developing a culture medium for Liberibacter asiaticus through comparative multi-omics analysis with its closest cultured relative, L. crescens Triplett, Eric

University of Florida

7/1/2015 - 6/30/2017 $325,912.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Objectives
Our main goal is to find the reasons for inconsistent responses of HLB-affected citrus to EN programs and to develop feasible and economical remedies that can consistently replicate successful HLB mitigation with ENs in all Florida groves. Specific objectives are
  1. Establish nutrient sufficiency guidelines for leaf tissues of HLB-affected trees that have successfully responded to nutritional programs in order to help growers replicate the successes and achieve higher nutrient efficiencies.
  2. Determine soil conditions that favor root hair and VAM proliferation of citrus roots, thus maximizing root uptake of calcium and other deficient nutrients in HLB-affected citrus trees.
  3. Establish testing protocols and remedial soil amendments that can be deployed to maximize root uptake of calcium and other deficient nutrients in nonresponsive HLB-affected groves.
  4. Deliver results of the research project to the Florida citrus industry through extension/outreach to all stakeholders (growers, contractors, supporting industries).
Overall Goal: Determine the effects of abiotic factors on Asian citrus psyllid dispersal and flight capability.

Determine the fight initiation thresholds of ACP depending on temperature and humidity. Determine the effect of wind speed on flight and the direction of psyllid flight with respect to wind.

Determine the effects of barometric pressure changes on psyllid dispersal.

Measure how psyllid dispersal is affected by abiotic factors in the field.

Establish a model to predict the risk of ACP dispersal/invasion based on prevailing abiotic conditions. Deliver this model as an online tool for growers.

Overall goal: The development of a robust, defined medium that provides sustained and reproducible growth of an L. asiaticus culture. Use comparative genomics approaches to identify means to culture L. asiaticus, including metabolic reconstruction, based on our new defined media and the identification of the essential gene set needed to culture L. crescens.

Continue to integrate multi-omics approaches to improve the medium for L. crescens, which, by extension, will get us closer to a robust medium for L. asiaticus.

Overarching—embedded with all other objectives. Test each new media design for its ability to sustain growth by L. asiaticus.

Characterization of the physiology and genome of the L. asiaticus culture.

Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Project # Project Title Principal Investigator Start and End Date Original Contract Total
15-028 Control citrus huanglongbing (HLB) by counteracting the SA hydroxylase of Candidatus Liberibacter asiaticus Wang, Nian

University of Florida

7/1/2015 - 6/30/2018 $430,697.00
15-033C Support role of the Citrus Core Transformation Facility remains crucial for research leading to production of citrus plants that may be tolerant or resistant to diseases Orbović, Vladimir

University of Florida

7/1/2015 - 6/30/2018 $270,000.00
15-036C Correlating pesticide residue analysis with psyllid feeding to improve protection of young trees Rogers, Michael

University of Florida

7/1/2015 - 6/30/2018 $451,603.00
15-038C Insecticide resistance monitoring and management in Florida citrus to maintain sustainable control of Asian citrus psyllid within Citrus Health Management Areas Stelinski, Lukasz

University of Florida

7/1/2015 - 6/30/2017 $129,491.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Objectives
The goal of this project is to develop management strategies that boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las).

Control HLB by optimization of application of SA and its analogs. Application of SA and its analogs have potential to neutralize the SA hydroxylase. Based on our previous study, foliar spray of SA and its analogs slowed down the increase of Las population in citrus and HLB disease severity, whereas trunk injection of SA and its analogs significantly reduced Las population. The previous study suggest that we can improve the HLB management by optimizing the application methods of SA and its analogs.

Control HLB using a combination of SA, SA analogs, or SA hydroxylase inhibitors. By combining SA or SA analogs with SA hydroxylase inhibitors, we could improve the efficacy of plant defense inducing to control HLB. SA hydroxylase inhibitors can directly counteract Las SA hydroxylase. We have identified six SA hydroxylase inhibitors in our previous studies. We will continue to optimize those SA hydroxylase inhibitors.

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. Practical applications of this project are Cost-effective completion of important phase of research contained in many projects.

Human resources as well as equipment in different research groups can be used for other activities while CCTF produces transgenic material for them.

Timely realization of ideas into life through production of transgenic plants that can be challenged by pathogens/vectors to test their susceptibility to diseases within a short period of time.

Residue analysis
We have already documented resistance in regional FL ACP populations, where prescribed MOAs are applied up to 12 times/yr to suppress new HLB infections, which began in earnest in 2007. A recent investigation from Mexico has shown that ACP had become 100-fold and 4,000-fold resistant to organophosphates and neonicotinoids. This is alarming and illustrates how lack of resistance management can allow ACP populations to become grossly resistant to our best tools leading to product failures. Our goal is to prevent this from happening proactively by monitoring resistance within CHMAs and prescribing appropriate management protocols.
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Project # Project Title Principal Investigator Start and End Date Original Contract Total
15-039C Secure site for testing transgenic and conventional citrus for HLB and psyllid resistance Stover, Ed

USDA

1/1/2016 - 12/31/2018 $260,980.00
15-042 Control citrus huanglongbing using endophytic microbes from survivor trees Wang, Nian

University of Florida

7/1/2015 - 6/30/2018 $467,000.00
16-001 Enhancing genetic transformation efficiency of mature citrus Li, Yi

University of Connecticut

7/1/2016 - 6/30/2019 $316,168.00
16-005 GFP labeling of Candidatus Liberibacter asiaticus in vivo and its applications Wang, Nian

University of Florida

10/1/2016 - 9/30/2019 $472,753.00
16-020C Dyed kaolin to repel Asian citrus psyllid in field conditions Vincent, Christopher

University of Florida

12/1/2016 - 11/30/2019 $273,908.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Objectives
Transgenic strategies for controlling HLB and its psyllid vector will be tested in a secure environment in which ideal care is provided for the invaluable research material in full compliance with regulatory requirements. Nontransgenic citrus with reasonable probability of resistance will be tested and may provide resistant planting materials with fewer regulatory constraints. This care will be provided with no compromise to the research teams’ ownership of tested intellectual property and no USDA/ARS expectation of inclusion in the related patents secured when USDA/ARS was not involved in development of the plant materials, expression vectors, or other material tested.
The goal of the proposed study is to characterize the effect of using endophytic microbes in controlling HLB. 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. GFP labeling of Candidatus Liberibacter asiaticus in vivo
  2. Elucidation of plant–Las interaction through real-time monitoring of Las movement and multiplication in planta using GFP labeled Las
  3. Investigate the effect of different control approaches on the dynamic population of Las in planta using GFP labeled Las
  1. Determine the optimum adjuvant-kaolin rate combinations to improve rainfastness of dyed kaolin particle film on citrus foliage.
  2. Determine the optimum concentration of red-dyed kaolin residue to deter ACP.
  3. Determine the effect of dyed kaolin particle films on ACP populations and CLas transmission under field conditions.
  4. Assess horticulturally relevant physiological impacts of optical properties of red-dyed kaolin particle films as compared to undyed films.
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Project # Project Title Principal Investigator Start and End Date Original Contract Total
162 Dissecting the disease complex of citrus huanglongbing in Florida Duan, Yongping

USDA

7/1/2010 - 6/30/2013 $670,183.00
164 Sampling plans to guide decision making for control asian citrus psyllid Qureshi, J. A.

University of Florida

8/2/2010 - 7/31/2012 $124,786.00
176 Effects of nutrition and host plant on biology and behavior of the Asian citrus psyllid and implications for managing psyllid populations Rogers, Michael

University of Florida

4/1/2010 - 3/31/2012 $87,607.04
212 Enhanced biological control of asian citrus psyllid in Florida through introduction and mass rearing of natural enemies Stansly, Phil

University of Florida

5/1/2010 - 4/30/2011 $148,775.00
214 Quantitative measurement of the movement patterns and dispersal behavior of Asian citrus psyllid in Florida for improved management Stelinski, Lukasz

University of Florida

8/1/2010 - 7/31/2011 $71,614.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Objectives
Genome sequencing and gap closure using computational approaches, construction of a large-insert library, combinatorial PCR, and chromosomal walking.

Culturing Las bacterium in vitro running metabolic pathway analyses.

Role of the microbial community and genetic diversity of Las bacteria in HLB development.

Seed transmission of Las bacterium.

Monitor Las population dynamics in citrus and periwinkle plants after chemical and heat treatment.

  1. Evaluate and refine the use of the tap sample to monitor adult psyllids and methods of assessing flush density, and infestation rates in blocks of growing and dormant citrus trees of different ages and varieties planted at experimental and commercial groves.
  2. Test the influence of these parameters on precision of estimated means and the distribution of the population within blocks using the appropriate aggregation and regression models.
  3. Evaluate and integrate methods for assessing these parameters into a user friendly system accessible to consultants and managers.
Determine the effects of varying rates of N, P, and K fertilization rates on the duration and survivorship of nymphal stages; adult fecundity and longevity and body mass.

Examine psyllid fitness on different commercial citrus varieties grown in Florida and the effects of rootstock selection on suitability of scion material for psyllid development.

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.

  1. Import, release, and evaluate new strains and species of parasitoid specific to D. citri.
  2. Identify genetic markers that can be used to track T. radiata in the environment.
  3. Develop efficient methods for mass rearing and release of T. radiata and possibly other species to increase biological control through augmentation of natural populations.
  4. Transfer technology to industry clientele.
Determine the seasonality and frequency of psyllid dispersal behavior.

Determine how flush availability impacts ACP movement and whether HLB-infected ACP disperse more than noninfected counterparts.

Quantify ACP dispersal distance within managed groves and distance of dispersal between managed groves.

Confirm the impact of abandoned groves on psyllid populations in neighboring managed groves.

Determine height at which psyllids disperse.

Develop management recommendations based on understanding of ACP dispersal behavior patterns.

Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Project # Project Title Principal Investigator Start and End Date Original Contract Total
232 Characterization of the virulence mechanism of the citrus huanglongbing pathogen Candidatus Liberibacter asiaticus Wang, Nian

University of Florida

4/1/2010 - 3/31/2012 $218,134.00
306 Culturing Liberibacter asiaticus Davis, Michael J.

University of Florida

5/1/2010 - 4/30/2011 $65,000.00
307 Infection traits and growth of “Candidatus Liberibacter asiaticus” inside microfluidic chambers De La Fluente, Leonardo

Auburn University

9/1/2010 - 5/30/2013 $100,000.00
312 Functional study of the putative effectors of “Candidatus Liberibacter asiaticus” using Citrus tristeza virus vector Gowda, Siddrame

University of Florida

5/1/2010 - 4/30/2013 $238,400.00
314 Insight into the causative agent of citrus greening disease (HLB) using computational structure/function analysis of genome-encoded proteins Grishin, Nick

Southwestern Medical

3/1/2010 - 8/30/2011 $99,902.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Objectives
  1. Transcriptional and microscopic analyses of different citrus varieties that are either susceptible or tolerant to Ca. L. asiaticus infection at different infection stages in greenhouse and citrus grove.

    1.1. Identify citrus genes with altered regulation caused by inoculation with Ca. Liberibacter asiaticus in susceptible and tolerant citrus cultivars.

    1.2. Characterize the relationship between the population of Ca. Liberibacter asiaticus in the phloem, anatomical changes of phloem, nutrient transport, and symptom development in susceptible and tolerant citrus cultivars.

  2. Transcriptional and microscopic analyses of model system tobacco to Las infection.
We are proposing to attempt the establishment of co-cultures of Las with these cell lines. Such cultures could then be used as a source of inoculum in attempts to grow Las axenically.
Optimize culture conditions of Las inside microfluidic chambers.

Collect inoculum and phloem fluid from HLB-affected and healthy plants.

Observe movement and aggregation of Las inside microfluidic chambers.

  1. Identify the putative effectors based on the genome sequence of Las and cloning into CTV vector.
  2. Expression of selected putative effectors of Las in citrus using CTV vector and transcriptional analysis of the host in response to expression of Las effectors.
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.

Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
315 Speedy evaluation of citrus germplasm for psyllid resistance Hall, David

USDA

5/1/2010 - 7/31/2013 $497,503.00
325 Development and evaluation of psyllid management programs for protection of resets and young tree plantings from HLB Rogers, Michael

University of Florida

5/1/2010 - 4/30/2012 $127,288.40
330 Targeting the asian citrus psyllid feeding mechanism as a means of blocking psyllid feeding on citrus Shatters, Bob

USDA

5/1/2010 - 6/30/2014 $483,000.00
334 How does Liberibacter infection of psyllids affect the behavioral response of this vector to healthy versus HLB-infected citrus trees? Stelinski, Lukasz

University of Florida

4/1/2010 - 3/31/2011 $30,867.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
Screen citrus germplasm for resistance to the Asian citrus psyllid (ACP, Diaphorina citri); explore host plant resistance as a management tactic for the Asian citrus psyllid. Two different types of plant resistance would be good candidates for psyllid control in commercial citrus: antixenosis and antibiosis (Painter 1951). A plant that adult psyllids avoid for either food or oviposition would constitute the antixenosis type of resistance. Plant species avoided by the adult psyllid for food and oviposition could lack specific plant traits (e.g., kairomones, color, or plant morphology) that attract the psyllid, or they could contain traits that repel the psyllid (e.g., kairomones).
  1. Determine the most effective rates and application methods for soil-applied systemic insecticides to control psyllids on nonbearing trees of different sizes.
  2. Evaluate the use of Kaolin clay particle films in conjuction with various adjuvants/stickers to increase the rainfastness of these applications.
  3. Determine the ability of soil-applied systemic insecticide applications in combination with Kaolin clay particle film or dimethyl disulfide (DMDS) to protect nonbearing trees from HLB in a commercial grove setting.
Conduct chemical and molecular analyses of the isolated ACP salivary sheaths to determine structure and mode of synthesis. (year 1)

Identify specific salivary proteins secreted in both the gelling and the watery saliva. (year 1 and 2)

Use information from 1 and 2 above to construct a model describing the ACP feeding process. (end of year 2)

Use artificial diet system to screen for salivary sheath/feeding inhibitory compounds. (initiate in year 1 and continue through year 2)

Conduct plant spray tests to determine if inhibitory compounds currently identified (i.e., EDTA and related chelators) or identified in the future, will work to prevent ACP from feeding on plants. (initiate in year 1 and continue to field trials in years 2 and 3)

Use information to identify a transgenic plant approach to create citrus that block the ACP feeding process and thus are not at risk of becoming HLB infected. (year 3)

  1. To determine whether healthy ACP distinguish between healthy versus HLB-infected citrus plants
  2. To determine whether behavior of Ca. Las-infected ACP to healthy versus infected citrus plants differs from the behavior of ACP that are not carrying the pathogen
  3. 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. To identify the active synthetic plant compounds that mediate ACP response to infected plants by GC-MS
  5. To determine whether behavior of ACP to healthy versus HLB-infected trees differs in the field
  6. To determine whether synthetic chemicals identified from HLB-infected plants attract ACP
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
335 Evaluation of methyl salicylate as a simultaneous repellent of Asian citrus psyllid and attractant for psyllid natural enemies Stelinski, Lukasz

University of Florida

4/1/2010 - 9/30/2011 $42,054.00
336 Genome-enabled metabolic reconstruction of Ca. Liberibacter asiaticus and its use in culturing and controlling the pathogen Triplett, Eric

University of Florida

7/1/2010 - 3/31/2014 $834,163.00
407 Culturing Liberibacter asiaticus Davis, Michael J.

University of Florida

5/1/2011 - 4/30/2013 $130,000.00
418 Analysis of the colonization of citrus seed coats by “Candidatus Liberibacter asiaticus” the causal agent of citrus huanglongbing and their use as a concentrated, pure source of bacteria for research Hilf, Mark

USDA

6/1/2011 - 5/31/2013 $175,000.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
  1. Determine if field release of methyl salicylate (MeSA) will enhance biological control organisms.
  2. Determine if MeSA is repellent to ACP.
Build a genome-scale metabolic model of Ca. Liberibacter asiaticus and use this model to identify the likely growth conditions necessary to culture Ca. L. asiaticus.

Determine which fully sequenced, culturable bacterium is most closely related metabolically to Ca. L. asiaticus and then apply those growth conditions to culture Ca. L. asiaticus.

Perform the experiments necessary on citrus with the cultured pathogen to satisfy Koch’s postulates. Predict the antibiotic sensitivity of Ca. L. asiaticus based on its sequenced genome and verify this sensitivity in the cultured organism as well.

Identify genes in Ca. L. asiaticus that can be targeted for inhibition based on comparative genomic analyses with all other sequenced bacteria.

We propose to continue modifying our media formulations until continuous culture of Las is obtained. This study is to provide culturing tools for Las that will support efforts to manage HLB. The capacity to grow Las in axenic culture is an advantage to many areas of study of HLB and its control.
Develop an effective protocol for isolation of viable “Ca. L. asiaticus” cells from citrus seed coats.

Evaluate the viability of extracted “Ca. L. asiaticus” cells.

Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
424 Functional disruption of the NodT outer membrane protein of Candidatus Liberibacter asiaticus for rootstock-mediated resistance to citrus greening using a phloem-directed, single-chain antibody McNellis, Timothy

Pennsylvania State University

7/1/2011 - 6/30/2017 $55,000.00
425 Effect of application rate, tree size, and irrigation scheduling on leaf imidacloprid concentration, psyllid populations, and soil leaching Morgan, Kelly

University of Florida

8/1/2011 - 10/31/2013 $199,225.00
434 Mass rearing and release of parasitic wasps to augment biological control of the Asian citrus psyllid (ACP) Stansly, Phil

University of Florida

6/1/2011 - 5/31/2014 $370,975.36
447 Role of nutritional and insecticidal treatments in mitigation of HLB in new citrus plantings Stansly, Phil

University of Florida

2/15/2012 - 2/14/2016 $324,430.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
The ultimate objective of this proposal is to develop citrus rootstocks that can be used to control HLB in fruiting variety scions. A transgenic approach will be used. Transgenic citrus plants producing a phloem-targeted, single-chain antibody recognizing an outer membrane protein of Ca. L. asiaticus will be developed and tested for resistance to HLB. The antibody will be translationally fused with the FLT protein, which is a phloem-mobile protein that gets efficiently translocated from lower parts of the plant to upper parts of the plant, even through graft unions. The FLT-antibody fusion protein is expected to be functional and to move systemically through the phloem, including from a transgenic rootstock into a nontransgenic scion. However, the current proposal is limited to producing and testing the transgenic plants for resistance to HLB and does not include testing of graft transmissibility of any HLB resistance that may be observed.
  1. Documentation of citrus greening management by application of the imidacloprid over time after selected irrigation applications.
  2. Determine sorption and transformation of imidacloprid in soils.
  3. Assess the soil transport and plant uptake of the applied imidacloprid and its metabolites over time after selected irrigation applications.
  4. Characterize root zone water movement using bromide tracer.
  5. Monitor psyllid populations and incidence of HLB over time.
  1. Augment biological control of D. citri through mass production and release of previously established and imported colonies of T. radiata and D. aligarhensis.
  2. Assess parasitism rates at release and control sites using feral populations of D. citri and infested sentinel plants.
  3. Develop a system of genetic identification of the parasitoids recovered from the field to prove the establishment and effectiveness of released biotypes.
  4. Transfer technology to industry clientele.
The overall objective of this research is to evaluate the individual and combined contributions of vector control and foliar nutrients in order to bring a new solid-block planting of juice oranges into profitable production.
  1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, and eventually yield in newly planted citrus blocks.
  2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters.
  3. Evaluate the effectiveness of reflective mulch to control ACP.
  4. Provide economic analysis of costs and projected benefits.
  5. Extend results to clientele.
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
502 High-throughput screening of transgenic citrus for HLB resistance Hall, David

USDA

6/1/2012 - 7/31/2015 $190,200.00
516 Develop citrus resistant or tolerant to HLB using the CTV vector and transgenic approaches Dawson, Bill

University of Florida

8/1/2012 - 1/31/2016 $1,239,174.00
523 Screening and cloning of resistance related genes by RNA-seq in huanglongbing (HLB) resistant and susceptible citrus breeding lines Duan, Yongping

USDA

5/1/2012 - 4/30/2015 $458,000.00
535 Exploiting the Las and Lam phage for potential control of HLB Gabriel, Dean

University of Florida

5/1/2012 - 4/30/2013 $117,028.00
536 Identification and mapping of the genes controlling resistance to huanglongbing (HLB) Gmitter, Fred

University of Florida

12/1/2012 - 11/30/2014 $190,000.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
The purpose of this proposal is to support a high-throughput facility to evaluate transgenic citrus for HLB resistance. Briefly, individual plants are caged with infected psyllids for 1 week, and housed for 6 months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB symptoms, and Las titer. To expedite this process, additional labor, space, and supplies are needed.
The first goal is to find anti-HLB or antipsyllid genes that are effective in controlling the disease and that will also be acceptable for consumers and regulatory processes. I see this as two different stages. We now are in the first stage—finding genes that are efficacious in controlling the disease. Screening for effective genes is the most difficult and time-consuming challenge of my career. It would be ideal to quickly screen the activity of the gene products against the bacterium in culture.

The second phase will be to choose from the effective genes ones that can be effectively marketed or to modify selected ones such that they are suitable.

  1. Generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology.
  2. Identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications, such as RT-PCR.
  3. Create transgenic citrus cultivars with new constructs containing the resistant gene(s).
A principal objective of this funding request is to establish a model in E. coli that can be used to assay if chemicals can be used to activate phage lytic (late) genes.

Cloning of previously identified early/late gene promoter regions fused with lacZ as a reporter.

Cloning and expression of both Las and the Lam repressors and determining responsiveness of the lacZ reporter.

Cloning and expression of all four Las and the one possible Lam antirepressors, and determining responsiveness of the reporter and clones from Milestone 2.

Development of a chemical assay for Las-responsive SOS.

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. Specifically, this project is intended to
  1. Phenotype 92 replicated progeny and determine the genetic basis and mode of inheritance of the HLB resistance in Poncirus.
  2. Construct a linkage map of the Poncirus genome and locate and map the Poncirus genes conferring resistance to HLB.
  3. Increase the marker density and mapping resolution to refine the map location of identified resistance genes.
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
538 Host genetic control of interference in Asian citrus psyllid life cycles Gmitter, Fred

University of Florida

12/1/2012 - 11/30/2014 $100,000.00
567 Acoustic trap for Asian citrus psyllids Mankin, Richard

USDA

7/1/2012 - 12/31/2013 $112,200.00
579 Citrus Core Transformation Facility (CCTF) as a platform for testing of different genes and/or sequences that have potential to render citrus plants tolerant or resistant to diseases Orbović, Vladimir

University of Florida

5/1/2012 - 6/30/2015 $255,000.00
581 Key unknowns about Asian citrus psyllid biology in Florida: Overwintering sites and alternative hosts Pelz-Stelinski, Kirsten

University of Florida

5/1/2012 - 9/30/2014 $200,029.00
581-1 Enhancement—Key unknowns about Asian citrus psyllid biology in Florida: Overwintering sites and alternative hosts Pelz-Stelinski, Kirsten

University of Florida

7/1/2013 - 6/30/2014 $11,868.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
This project will address another approach that can contribute to the management of HLB disease spread, by targeting the vector, specifically to study the potential for host interference in ACP life cycles. If life cycles are significantly disrupted or impeded, population levels of ACP in the field may be reduced, which in turn should likewise reduce the potential spread of Clas and HLB disease. We will test whether the previously observed characteristic, of decreased fitness of ACP when reared on Cleopatra mandarin (Citrus reshni) seedlings, can be transmitted to hybrid offspring of Cleopatra.
In the proposed study:

Characteristics of the female reply that most effectively induce attraction of calling males will be identified in a laboratory setting.

Optimal replies will be tested in a field or greenhouse environment.

The best reply will be incorporated into a sticky (or alternative) trap with a piezoelectric buzzer that plays back the reply whenever a male call is detected.

The trap will be tested and modified as needed in laboratory and field tests with male ACP to determine whether infestations can be detected and targeted more precisely than with current methods.

If the trap is demonstrated to have enhanced efficacy, additional devices will be constructed for general field use.

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.
Identify overwintering habitats and alternative hosts of ACP.

Determine the capacity of alternative hosts as food sources and oviposition sites.

Directly sample ACP in citrus tree canopies and surrounding habitats all season long.

Determine effects of cold and heat acclimation on susceptibility of ACP to insecticides.

Enhancement
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
590 Enhancing psyllid control through a better understanding of the effects of pesticide applications on psyllid feeding and mortality Rogers, Michael

University of Florida

5/1/2012 - 4/30/2015 $889,701.00
600 Management tactics based on psyllid movement and distribution in Florida citrus Stansly, Phil

University of Florida

6/1/2012 - 5/31/2015 $296,000.00
603 Non-neurotoxic chemicals as alternatives to conventional insecticides for Asian citrus psyllid management and prevention of insecticide resistance Stelinski, Lukasz

University of Florida

4/1/2012 - 12/31/2014 $191,322.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
The goal of this proposed work is to improve psyllid control and minimize the spread of the HLB pathogen through more effective use of pesticide applications.

Objective 1, an electrical penetration graph (EPG) 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.

Objective 2 will examine the duration of psyllid control provided by foliar-applied insecticides in a typical grove setting. This objective will address whether certain insecticides perform better at certain times (seasons) of the year and examine the use of adjuvants to increase the longevity of psyllid control provided.

Objective 3 will examine the use of different application methods of neonicotinoid insecticides for young and intermediate-sized trees to maximize the level of psyllid control provided by soil-applied systemic products.

Objective 4 is a multiyear evaluation of 4 different season-long approaches to young tree care to determine which approach is most likely to prevent HLB in young trees in order to bring them into production.

Objective 5 will examine whether, once a young tree has become infected with the HLB pathogen, there is a benefit to continuing to control psyllids on those plants already infected.

  1. Assess effects of abiotic factors (light quality, photoperiod, air flow, and temperature fluctuations) on psyllid movement.
  2. Evaluate physiological limits and biotic factors effecting of movement including feeding, egg load, infection status, and population density.
  3. Evaluate techniques for tracking psyllid movement in the field for mark recapture studies.
  4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field.
  5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide-treated trap crops (such as Bergera koenigii) to attract and kill.
The goal of this project is to evaluate the efficacy of non-neurotoxic chemicals, known for their insecticidal properties, against ACP. The mortality, feeding behavior, host selection, development, and fecundity will be determined in controlled laboratory experiments.

Subsequently, thorough field investigations will be conducted with the most promising chemicals under Florida conditions.

Objective 1. Determine the effect of non-neurotoxic compounds on mortality of ACP.

Objective 2. Investigate sublethal effects of non-neurotoxic compounds on development and fecundity of ACP.

Objective 3. Quantify the effect of non-neurotoxic compounds on ACP feeding behavior.

Objective 4. Determine the effectiveness of proven non-neurotoxic chemicals against ACP under field conditions in Florida citrus.

Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
609 Control of citrus huanglongbing by exploiting the virulence mechanisms of Candidatus Liberibacter asiaticus and inducing plant defense Wang, Nian

University of Florida

5/1/2012 - 4/30/2015 $413,783.00
701 Exploitation of visual stimuli for better monitoring and management of ACP in young citrus plantings Allan, Sandra

USDA

5/1/2013 - 4/30/2015 $119,096.00
701-1 Enhancement—exploitation of visual stimuli for better monitoring and management of ACP in young citrus plantings Allan, Sandra

USDA

7/1/2013 - 6/30/2014 $115,000.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
This project is a combination of projects No. 609 and 613 as requested by CRDF. 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. The virulence mechanisms of Las are largely unknown. Understanding the virulence mechanisms is important for HLB management. Importantly, we identified an enzyme salicylate hydroxylase encoded by sahA of Las, which breaks down salicylic acid (SA) and its derivatives. SA plays a central role in plant defenses. Degradation of SA is likely one important strategy of Las to suppress plant defense. In addition, phloem blockage caused by Las due to phloem proteins and callose has been suggested as a plant defense response. However, the deposition of phloem proteins and callose caused by Las seems to be very slow and could not be observed in the asymptomatic leaves, even though Las is present. Phloem-feeding insects cause rapid and significant host gene expression to insect feeding. It is probable that psyllid feeding could significantly affect citrus gene expression. Thus, modifying the promoters of phloem proteins and callose synthase genes with psyllid-induced promoters has the potential to block Las from moving by accelerating local phloem blockage around psyllid feeding sites. In order to achieve the goal of this study, the following objectives will be conducted: to characterize how Las causes HLB disease symptoms and how Las manipulates plant defense response by investigating the roles of putative virulence factors, to test different compounds in controlling HLB and characterize their mechanisms in controlling HLB, and to control HLB by blocking the translocation of Las inside the phloem from psyllid feeding sites.
The long-term goal of this project is to develop a push-pull system for protection of young citrus plantings from infestation of Asian citrus psyllids to complement insecticidal control and to protect young trees from citrus greening. This push-pull system will be developed through developing a better understanding of the visual cues that influence ACP attraction and repellency.

Objective 1. Evaluate factors that enhance visually induced flight behavior, including takeoff tendency, flight duration, and light choice of psyllids.

Objective 2. Optimize attraction of psyllids to visual targets to provide a pull system.

Objective 3. Develop and evaluate push-pull systems in small orchards and commercial groves.

Enhancement
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
711 Identification of Bacillus thuringiensis endotoxins active against adult Asian citrus psyllid Bonning, Bryony

Iowa State University

5/1/2013 - 4/30/2017 $500,000.00
726L A bacterial virus based method for biocontrol of Liberibacter Gonzalez, Carlos

Texas AgriLife Research

5/1/2013 - 10/31/2016 $386,902.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
The overall goal of this application is to identify a Bt crystal toxin with toxicity to ACP and to further enhance toxicity by genetic modification. The long-term goal of the proposed research is an effective management strategy for the psyllid and associated huanglongbing disease that is more sustainable, less costly, and more environmentally benign than the repeated application of broad spectrum insecticides. 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. We will screen toxins from up to 200 Bt strains with diverse insect toxicities and toxin profiles for ACP toxicity.

Modify a Bt toxin for psyllid toxicity. We will isolate peptides that bind to ACP gut membrane by screening a phage display library, and confirm gut binding of selected peptides.

Isolate and characterize Xac phages. As previously stated a large pool of virulent phages having a diversity of surface receptors is necessary for the development and implementation of a successful phage-based biocontrol system for control of Xac. However, due to limit of 1-year funding we will work to isolate and characterize a limited pool of phages. It must be reiterated that only virulent and nontransducing phages should be used for the development of an effective, sustainable, and ethical phage-based control system for citrus canker.

Therefore, full characterization is necessary before phages are released as biocontrol agents. All phages will be initially characterized as previously outlined for the ability to form clear plaques, not form lysogens and morphology. The genomes of candidate virulent phages will then be sequenced and annotated to ensure virulent status. Receptor diversity will be based on limited host range studies due to time limit.

Initiate phage persistence studies on plant tissue. Studies will be conducted as previously outlined in Objective IIIa using virulent phage Xfas303, a fully characterized KMV-like phage shown to have activity against Xac field strains (North 40, Block 22, Fort Besinger; see preliminary results) obtained from Florida. Phage will be sprayed with and without a protective carrier. The protective carriers developed by Balogh et al. (2003 and 2008) and along with others will be evaluated.

Determine therapeutic efficacy of Xac phages. Studies will be conducted as previously outlined in Objective IIIb in cooperation with Dr. Nian Wang.

Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
733 Molecular basis of citrus greening and related diseases gleaned from genome analyses of hosts and pathogens Grishin, Nick

Southwestern Medical

4/1/2013 - 3/30/2015 $200,000.00
750 Identification of key components in HLB using effectors as probes Ma, Wenbo

University of California, Riverside

4/1/2013 - 3/31/2016 $299,781.00
766 Biotic and abiotic factors that cause Asian citrus psyllids to accept hosts: potential implications for young plantings and pathogen transmission Stelinski, Lukasz

University of Florida

4/1/2013 - 9/30/2015 $145,039.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
The overall goal of the project is to generate hypotheses about molecular mechanisms of CLas pathogenicity from the comparative genomic analysis of the host, pathogen, and vector, and to present the results as a website that shows predictions of spatial structures and functions for all proteins in analyzed genomes for the citrus research community to use. The following specific objectives will be the steps toward the goal:

Obj. 1. Using the computational pipeline developed for the analysis of CLas proteome, predict spatial structures and functions for all proteins in two available citrus genomes and drafts of the psyllid genome.

Obj. 2. Improve the prediction pipeline initially developed for prokaryotic genome and adapt it to the analysis of eukaryotic genomes.

Obj. 3. Present results of analysis as a comprehensive website with a dedicated webpage for each protein in these organisms, showing details of predictions.

Obj. 4. Compare analyzed genomes with each other. Find predicted physical and functional interactions and associations between proteins from different genomes.

Obj. 5. Foster collaborations with experimentalists, helping them with the analysis of proteins they have chosen as research targets.

The main goal of this proposed research is to identify the targets of CLas effectors in citrus. These effector targets are likely to be key components in HLB pathogenesis. We will then explore strategies to modify these targeted processes or disrupt the interaction of effectors with their targets, which may lead to improved resistance/tolerance of citrus to HLB. Three objectives will be pursued to accomplish this overall goal:
  1. Identify citrus proteins associating with three selected CLas effectors using yeast two hybrid screens.
  2. Confirm the effector-host target interactions using a series of in vitro and in vivo assays.
  3. Design control strategies aiming to enhance the resistance/tolerance of citrus to HLB based on effector activities and the functions of their targets.
Overall Goal: Define the factors governing ACP host acceptance in citrus groves.

Objective 1. Determine how biotic and abiotic parameters differ between resets, mature trees, and “party trees.”

Objective 2. Define the biotic factors that affect ACP host acceptance and fitness.

Objective 3. Investigate whether ACP population abundance differs among resets, mature trees, and party trees, and if party trees remain consistently attractive to ACP throughout seasons and years.

Objective 4. Quantify how time since tree inoculation with the HLB pathogen and abundance of HLB-infected trees affects ACP host acceptance behavior within groves.

Objective 5. Determine if and where ACP disperse from resets and party trees and how this might contribute to HLB transmission within groves.

Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
769 A team approach to culturing Ca. Liberibacter asiaticus Triplett, Eric

University of Florida

4/16/2013 - 10/15/2015 $448,257.00
805 Functional genomics of Liberibacter in a model system Long, Sharon

Stanford University

4/1/2014 - 12/30/2016 $540,197.00
834 Optimizing heat treatment in the fields and understanding the molecular mechanism behind the success of thermotherapy for the control of citrus HLB Duan, Yongping

USDA

4/1/2014 - 3/31/2016 $385,900.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
The overall goal of this project is to culture Ca. Liberibacter asiaticus.

Determine the nutrient content of citrus and periwinkle phloem, which are well colonized by CLas, and murraya and tomato phloem, which are not good habitats for CLas. Team members involved: Nabil Killiny-Mansour (primary), Mark Hilf, and Eric Triplett.

Determine the nutrient content of the psyllid hemolymph. Team members involved: Kirsten Pelz-Stelinski (primary), Nabil Killiny-Mansour, David Hall, and Eric Triplett.

Prepare and test media based on the findings from Objectives 1 and 2 to attempt to culture CLas. Team members involved: Mike Davis (primary), Mark Hilf, and Eric Triplett.

Digitize the media recipes attempted to date by Prof. Davis into a searchable relational database. Team members involved: Eric Triplett (primary) and Mike Davis.

Once cultured, CLas will be characterized physiologically and made available to the world through two bacterial strain repositories. Team members involved: Eric Triplett (primary), Mike Davis, and Mark Hilf.

Determine the role of CLas prophage in the culture deterioration over time. If prophage activation is correlated with CLas culture deterioration, efforts will be made to inhibit prophage activation as well as search for CLas strains that lack the SC1 prophage in the genome. Team members involved: Eric Triplett (primary) and Dean Gabriel.

Clone a preliminary set of CLas regulatory genes, and synthesize/clone target promoters, and provide proof of principle for use of this synthetic model system approach.

Use activator/promoter pairs to control expression of fluorescence in S. meliloti. Use these engineered strains to carry out high-throughput screen of compounds that inhibit the CLas regulatory protein.

  1. To investigate the effect of heat stress on Las, associated prophages, and those genes involved in the phage lytic cycle.
  2. To monitor healthy and HLB-affected citrus genome-wide response to heat stress.
  3. To optimize field thermotherapy to produce a standard operating procedure.
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
850 Scheduling ACP spring spray selection based on the Citrus Flowering Model Albrigo, Gene

University of Florida

4/1/2014 - 3/31/2017 $90,000.00
853 Why is Poncirus trifoliata resistant to colonization by Asian citrus psyllid? LaPointe, Stephen

USDA

5/1/2014 - 8/30/2016 $187,681.00
858 New nonphytotoxic composite polymer film barrier as ACP repellent for controlling HLB infection Santra, Swadeshmukul

University of Central Florida

4/1/2014 - 3/31/2017 $350,000.00
910 An integrated approach for establishment of new citrus plantings faced with the HLB threat Powell, Chuck

University of Florida

4/1/2014 - 9/1/2016 $369,714.00
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
The overall objectives of these studies are to provide advanced knowledge of spring leaf flush and flowering for spring flush ACP control and predict the best 4- to 5-week window for bee foraging in citrus.
  1. Collect weather, flowering, and leaf flush data for recent 10 years and use to improve the Citrus Flowering Monitor System. Particular goals are to improve near-bloom temperature responses of the model and add leaf flushing time to the flowering time in the monitor system. Additionally, it would be desirable to add the 5% open flower point as the point of likely bee activity.
  2. Establish a 4- to 5-week peak flowering period as part of the monitor and test it for use of chemicals with ACP effectiveness but minimum bee toxicity, after which all effective ACP control chemicals would again be used. Work with growers and beekeepers to see if this approach is feasible for both concern of the grower for adequate ACP control and beekeepers for adequate access to citrus flowers for their bees.
Identify host plant–produced volatile chemicals and leaf/plant metabolites that are attractive or repellant to adult male and/or female ACP.

Test preference (antixenosis) and development (antibiosis) of ACP adults on susceptible and resistant host plants.

Identify attractive or repellent volatiles, metabolites, or their blends, and study the behavior of ACPs to these odorants or taste blends using Y-tube olfactometer assay, caged vial assay, flight tunnel assay, and SPLAT probing assay.

  1. OSCF formulation development and optimization (Santra) In this objective we will perform the following tasks. (1) Prepare OSCF materials from readily available chemicals (such as silica precursors, acid/base catalyst, ionic cross-linker, and PAM), (2) Systematically characterize OSCF materials, (3) Optimize the synthesis protocol to produce OSCF formulations that will exhibit strong rainfastness, (4) Develop a scalable synthesis protocol and prepare formulations for field trials, and (5) Study OSCF liquid formulation stability and evaluate its shelf-life.
  2. EPG testing to evaluate change of ACP feeding behavior (Rogers).
  3. Field trial for evaluation of ACP population control (Graham and Rogers).
  4. Evaluation of HLB infection using PCR method (Graham and Irey).
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. This will be broken into three stages: treatment of field trees, analysis of the effect of the treatment on trees, making conclusions about optimum HLB control strategies.
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Project # Project Title Principal Investigator Start and End Date Original Contract Total
916 Screening and application of antibacterial producing microbes to control citrus huanglongbing Wang, Nian

University of Florida

7/1/2014 - 6/30/2017 $431,180.00
922 Control citrus canker by manipulating the EBE (effector binding element) of CsLOB1, which is the citrus susceptibility gene for citrus canker disease Wang, Nian

University of Florida

4/1/2014 - 3/31/2017 $436,045.00
928.1C Field trial of naturally occurring microbes Sutherland, Dudley

Glades Crop Care, Inc

4/11/2014 - 3/31/2017 $100,788.00
934C Soil drenches of products to combat initial HLB infection in young citrus trees Curtis, John

Better Crops, LLC

6/1/2014 - 9/30/2016 $15,000.00
941C Influence of thermal therapy on transmission of Candidatus Liberibacter asiaticus Pelz-Stelinski, Kirsten

University of Florida

2/1/2015 - 7/31/2016 $105,782.00
944C RSA—small plant assay for testing the efficacy of antimicrobial materials against HLB Pelz-Stelinski, Kirsten

University of Florida

3/1/2015 - 7/31/2016 $125,797.33
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×
Objectives
Test antibacterial-producing bacteria against Liberibacter crescens and other Rhizobiaceae bacteria that are closely related to Las.

Control HLB using antibacterial-producing bacteria.

Generate nontransgenic EBECsLOB1-modified citrus.

Test how modification of the EBE of CsLOB1 gene affects citrus resistance against citrus canker and other important traits.

Test how modification of the EBE of CsLOB1 gene affects citrus resistance against citrus canker.

2.2 Experimental evolution analysis of the Xcc strain on the EBECsLOB1-modified grapefruit to investigate how stable is the resistance.

2.3 Test how modification of the EBE of CsLOB1 gene affects other important citrus traits.

Objectives of the Project: Test five soil-applied products, with mulch subplots, plus an untreated control (six treatment plots) on health and HLB status of orange trees over 3 years.
Test four soil-applied treatments plus a water-treated control (five treatment plots) on health and HLB status of orange trees over 3 to 5 years. The five treatment plots of 20 trees each will be replicated four times.
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. Moreover, the magnitude of heat treatments on Las transmission will be evaluated over time for 1 year following application. A second objective is to evaluate the acquisition and transmission efficiency of nymphs subsequent to development on infected plants subjected to heat treatments.
RSA-The objectives of this assay are to test the efficacy of antimicrobial materials at various rates against CLas; mobility of the material in the plant, particularly in the phloem; and phytotoxicity.
Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
×

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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Suggested Citation:"Appendix D: Selected Citrus Research Development Foundation Projects." National Academies of Sciences, Engineering, and Medicine. 2018. A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. Washington, DC: The National Academies Press. doi: 10.17226/25026.
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Huanglongbing (HLB) or citrus greening, first observed more than a hundred years ago in Asia, is the most serious disease threat to the citrus-growing industry worldwide due to its complexity, destructiveness, and incalcitrance to management. First detected in Florida in 2005, HLB is now widespread in the state and threatens the survival of the Florida citrus industry despite substantial allocation of research funds by Florida citrus growers and federal and state agencies.

As the HLB epidemic raged in 2008, Florida citrus growers began allocating funds for HLB research in hopes of finding short-, medium-, and long-term solutions. This effort created the Citrus Research and Development Foundation (CRDF), an organization with oversight responsibility for HLB research and development efforts in Florida. This report provides an independent review of the portfolio of research projects that have been or continue to be supported by the CRDF. It seeks to identify ways to retool HLB research—which, despite significantly increasing understanding of the factors involved in HLB, has produced no major breakthroughs in controlling the disease—and accelerate the development of durable tools and strategies that could help abate the damage caused by HLB and prevent the possible collapse of the Florida citrus industry.

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