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Suggested Citation:"3 HLB Research and Development Efforts." 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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3

HLB Research and Development Efforts

In 2009, at the request of Florida Department of Citrus, the National Academies appointed the National Research Council (NRC) Committee on Strategic Planning for the Florida Citrus Industry: Addressing the Citrus Greening Disease (huanglongbing). This committee released its report, Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease, in early 2010. Included in this report were organizational, informational, and research and technology recommendations. Among the organizational recommendations was to identify one organization and empower it to have oversight responsibility over huanglongbing (HLB) research and development efforts (Recommendation O-2). The Citrus Research and Development Foundation (CRDF), a nonprofit corporation that was created through the initiative of the Florida citrus industry in 2009, currently serves as the organization in Florida with oversight responsibility for HLB research and commercial product development and delivery.

This chapter examines the HLB research areas that have received or continue to receive support from CRDF1 and discusses whether these research efforts are in line with the recommendations in the NRC 2010 report (notable outcomes from HLB research and pitfalls are discussed in Chapter 4 of this report). This chapter also examines the HLB research efforts that are being funded by other agencies2 (state and federal) and overlaps or

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1 Information on CRDF-funded projects (project number, principal investigator, funding duration, funding amount, and project objectives), can be found in Appendix D of this report.

2 Information on projects funded by other agencies is available online at http://citrusrdf.org/wp-content/uploads/2012/10/US-Citrus-Research-Inventory.pdf. Accessed June 8, 2018.

Suggested Citation:"3 HLB Research and Development Efforts." 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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duplications with CRDF-funded research efforts. The chapter ends with a section addressing sociological and economic factors that influence grower decision making with respect to citrus cultivation and disease management, as well as market and consumer acceptance of fruit and juice produced in various ways. These factors were not mentioned specifically in the 2010 report, but the committee deems them to be critical to the success of any future HLB management efforts and, indeed, to the health of the citrus industry in Florida.

HLB PATHOGEN, VECTOR, AND HOSTS

HLB Causal or Associated Bacteria

CRDF-Funded Research

CRDF has funded 26 projects, totaling $8.6 million, with at least one major objective related to understanding the biology of Candidatus Liberibacter asiaticus (CLas). CLas-focused projects address several major themes, including efforts to grow the bacterium in culture, characterize its associated bacteriophages and other biocontrol microbiota, understand its genetic diversity and distribution, and identify essential proteins as control targets. Several projects address more than one of these themes. The largest number support efforts to isolate and culture CLas in vitro (Projects #048, 162, 306, 407, 307, 336, 418, 769, and 15-027, totaling about $2.9 million). Investigators of five past (#535, 726L, 711, 803, and 15-009) and one current (#15-042) CRDF project (totaling about $2.5 million) have studied the properties of bacteriophages or bacteria that coexist with CLas in order to identify ways that these may be used for bacterial control. Relevant projects include studies of bacteriophage viruses encoded in the CLas genome and of antagonistic microbes found in citrus phloem. Two past (#045 and 15-008) and one current project (#16-001), totaling $979,000, have aimed to understand the patterns of multiplication and movement of CLas within the plant host, or to develop improved tools for pathogen tracking in the plant. Four completed projects (#125, 065, 123, and 162), totaling $1.4 million, focused on the genetic or genomic characterization of CLas to understand its diversity and distribution, identify virulence factors or targets, and generate public resources for genome sharing and analysis. Five projects (#424, 15-028, 805, 15-017, and 711), totaling $1.7 million, have aimed to use bacterial proteins to develop or optimize CLas inhibitory strategies.

Research Toward Culturing CLas. In addition to evaluating a wide array of culture media formulations, researchers have worked to mimic aspects of the insect and plant environment through the addition of insect

Suggested Citation:"3 HLB Research and Development Efforts." 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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cells to the culture or the use of phloem-mimicking flow chambers, and through profiling the molecular components of host, vector, and pathogen to identify potential metabolic needs of CLas. Researchers have also optimized methods for obtaining CLas starter culture from plant or insect tissues having the largest titers of bacteria. These efforts are in line with the NRC 2010 recommendation NI-10: Develop in vitro culture techniques for CLas to facilitate experimental manipulation of the bacterium for insights into gene function.

One project funded by CRDF focused on culturing the nonpathogenic, plant-associated bacterium Liberibacter crescens to provide the closest-available proxy system to CLas for use in experimentation (Fagen et al., 2014). This effort is in line with the 2010 recommendation NI-5: Support development of HLB model systems.

Genome Sequencing and Bioinformatics Analysis. The completion of the CLas genome (Duan et al., 2009) in 2009 (with funding from the Florida Citrus Production Research Advisory Council, the CRDF’s precursor agency) was later followed by the completion of the genome of other HLB-causing species Candidatus L. americanum (CLam) and Ca. L. africanum (CLaf) (Wulff et al., 2014), and other Liberibacter species that cause diseases in other plant species. Genomic comparison among these and with genomes of related free-living species, including a culturable nonpathogenic Liberibacter strain, have been performed toward identification of essential pathogenesis genes (Hartung et al., 2011; Kuykendall et al., 2012; Leonard et al., 2012). These efforts are in line with the 2010 recommendations NI-5: Support development of HLB model systems and NI-6: Exploit the CLas genome sequence for new strategies of HLB mitigation.

Functional Comparative Genomics and the Identification of Infection-Associated Genes and Proteins. CRDF funded projects (#123, 733, and 314) aimed to curate the genomic sequences of HLB pathogen, vector, and host; perform comparative bioinformatics analysis of proteins among the genomes of each species; predict interactions among proteins from different genomes; and provide a user-friendly website to the community3 (Cong et al., 2012). Another CRDF project (#123) focuses on bioinformatics efforts in Liberibacter genome analysis and extending to bioinformatics characterization of vector and hosts.4 For many of the virulence-associated proteins identified through genomics, translational work has begun to identify targeting strategies. Small-molecule screens aim to find inhibitors of specific virulence factors, such as the master regulator LdtR and the CLas salicylic acid–degrading enzyme (Pagliai et al., 2014). Other research projects (#805, 314, and 424) have sought to identify and characterize other secreted CLas

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3 See http://prodata.swmed.edu/citrusgreening. Accessed June 8, 2018.

4 See https://www.citrusgreening.org. Accessed June 8, 2018.

Suggested Citation:"3 HLB Research and Development Efforts." 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.
×

proteins in order to find or engineer proteins that may bind or degrade them. These efforts are in line with the 2010 NRC recommendation NI-6: Exploit the CLas genome sequence for new strategies of HLB mitigation.

Understanding Antagonistic Phage and Bacteria. More than 6% of the sequenced CLas genome is composed of two bacteriophages (antibacterial viruses) that may exist as lytic (bacteria-killing state) or lysogenic phages (repressed state) (Zhang et al., 2011). Phage-associated sequences are highly variable among CLas isolates (Zhang et al., 2011; Wang et al., 2012); research has been aimed at using this variability to understand the diversity, evolution, and geographic origin of CLas populations (Zhou et al., 2011; Zheng et al., 2017). Phage could also play a critical functional role in limiting CLas growth in different host contexts. These efforts are in line with the 2010 NRC recommendation NI-6: Exploit the CLas genome sequence for new strategies of HLB mitigation.

Impacts of the Citrus Microbiome. CLas is among thousands of bacterial species present in citrus phloem and in psyllid guts (Fagen et al., 2012). Bacterial communities could affect HLB development and transmission through nutrient competition or cooperation, chemical antibiosis or growth promotion, triggering plant defense, or activating regulatory signals in CLas; alternatively, CLas effects on other bacteria might increase stress on the plant. CRDF-funded projects (#916 and 15042) have sought to characterize or manipulate the citrus microbiome to understand the role of cooperative or antagonistic microbes in HLB. CRDF-funded researchers helped form a Citrus Microbiome Consortium in 2015, aimed at coordinating microbial community exploration in citrus tissues, including the effects of variety, diseases, and management on the microbiome (Wang et al., 2015). Other research has focused on the role of individual microbial species for disease-controlling properties. Microbial species that have been tested include 327 citrus-inhabiting bacteria (Riera et al., 2017) and Burkholderia spp. (Zhang et al., 2017). A current project is focused on optimizing the effectiveness of a form of Bacillus thuringiensis (Bt) toxin against the Asian citrus psyllid (ACP) (#711) (see also the sections Pathogen–Vector Interactions, page 82, and Insect Control, page 90). A phage-suppressing protein derived from the endosymbiont Wolbachia is also being investigated to determine its role in CLas behavior (Jain et al., 2017). These efforts are in line with the 2010 NRC recommendation NI-7: Support research aimed at developing alternative ACP management strategies.

Research Funded by Other Agencies

In 2016, the National Institute of Food and Agriculture (NIFA) Specialty Crop Research Initiative (SCRI) awarded funding to three different projects related to the biology of CLas. Two projects (#2016-70016-24824

Suggested Citation:"3 HLB Research and Development Efforts." 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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and 2016-70016-248441) with $2.1 million and $4 million funding, respectively, aim to identify possible new culturing strategies that will increase the span of viability through comprehensive analyses of phloem sap and insect gut composition, the effect of the physical environment, the role of CLas chemical signals, and several other factors. The third NIFA project (#2016-70016-24781), a $3.3 million effort, aims to identify CLas-secreted proteins as candidates for targeting by plant-expressed proteases.

The NIFA Plant-Biotic Interactions program awarded $863,000 to Project #2017-03060,5 which aims to characterize the role of the master regulator LdtR in the persistence of CLas in citrus. A current project (#5300-160) funded by the California Citrus Research Board (CRB) aims to identify the core secreted proteins of CLas in order to develop bacterial inhibition and diagnostics strategies. Finally, K. Mandadi at Texas A&M was awarded the Foundation for Food and Agriculture New Innovator Award in 2017, receiving $600,000 from the Foundation for Food and Agriculture Research and Southern Gardens Citrus to develop a root-based bioassay for culturing studies and antimicrobial screening.6

HLB Vector: Asian Citrus Psyllid

CRDF-Funded Research

CRDF has previously funded 15 projects, totaling $5.9 million, that have at least one major objective related to understanding the biology and ecology of ACP. These are in line with the 2010 recommendation L-3: Support analysis of ACP behavior, ACP–plant interactions, and ecology to enhance the knowledge base available for new ACP management strategies.

Dispersal Dynamics of ACP. One previously funded project (#214) evaluated the dispersal dynamics of ACP to determine the distances traveled by adults, the seasonal phenology of these movements, and cues for movement of psyllids into and out of citrus groves. In this study, a protein-marking technique was employed to investigate dispersal dynamics using marked individuals from abandoned citrus groves to adjacent managed citrus (Lewis-Rosenblum et al., 2015). Additional studies, based on the specific color-morphs of Diaphorina citri, were carried out to determine patterns of flight initiation and duration using flight mills (Martini et al., 2014a). Paris et al. (2017) similarly investigated how calling behavior, mate seeking, and phototaxis were affected by changes in atmospheric pres-

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5 Project information available at https://www.nsf.gov/bio/pubs/awards/IOS_awards_list.htm. Accessed January 8, 2018.

6 Announcement of award available at https://foundationfar.org/new-innovator/kranthi-mandadi/. Accessed January 8, 2018.

Suggested Citation:"3 HLB Research and Development Efforts." 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.
×

sure. Another project (#15-024) aimed to improve the understanding of ACP flight and dispersal behavior, and more specifically how temperature, humidity, barometric pressure, and wind speed affect the probability that ACP initiate and perform long-distance flights.

Infection Status and Psyllid Dispersal. One past study investigated the influence of CLas infection status and how it influences ACP dispersal behavior, flight capacity, and sexual attraction (Martini et al., 2015a).

Effects of Host Variety and Nutrition on Vector Fitness or Behavior. The influence of citrus variety and nutrition on ACP biology, fitness, and behavior was also investigated (Tsagkarakis and Rogers, 2012; Project #176). Moreover, the effect of different plant growth regulators on citrus tree vegetative growth and subsequent impact on ACP fitness were also evaluated in greenhouse and growth chamber experiments.

Increasing the Efficiency of ACP Monitoring/Trapping. To learn more about the visual cues that influence ACP attraction and repellency, light-emitting diodes, emitting light of wavelengths within the insect visible spectrum (Paris et al., 2015), were designed to study the responses of ACP to variations in light and light quality. Mankin et al. (2013; Project #567) investigated the possibility of increasing trapping efficiencies by attracting male ACP using vibrational communications produced by females. A micro-controller platform was evaluated for its capacity to regulate vibrational sensing and output devices and has subsequently been programmed to send mimics of different D. citri signals to a buzzer or calling device.

Disrupting ACP Feeding Mechanisms. Specific compounds that inhibit the formation of salivary sheaths and/or degrade hemipteran style sheaths (Project #330; Appl. No. US20160316762 A17) were investigated for direct topical delivery to plants by spraying or root applications. These compounds and delivery methods were designed to prevent and/or reduce the transmission of vascular tissue–associated hemipteran vector-borne pathogens to plants. Specific investigations focused on the characterization of the ACP feeding process and testing of compounds that block or inhibit psyllid feeding as a mechanism for disease control.

Research Funded by Other Agencies

U.S. Department of Agriculture (USDA) NIFA funding supported research on ACP behavioral responses to the presence of CLas in trees. CLas infection leads to changes in the host’s odor, which influences attraction of noninoculative vectors and repellency of inoculative individuals. Shatters et al.8 (2015–2017) compared the proteome and transcriptome of

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7 See https://www.google.com/patents/WO2016176061A1. Accessed December 20, 2017.

8 See https://www.ars.usda.gov/research/project/?accnNo=427147. Accessed January 9, 2018.

Suggested Citation:"3 HLB Research and Development Efforts." 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.
×

ACP alimentary canal and midgut regions, as well as salivary glands with and without the CLas infection. Another USDA NIFA project (Handler et al.9; 2015–2017) focused on the development of efficient transformation protocols to create vector-incompetent, transgenic ACP to replace vector-competent strains in the field.

Hunter et al.10 (2015–2017) conducted deep sequencing runs to assist in the completion of the ACP genome, releasing this assembly on two websites for free access by the research community. The transcriptome dataset was used to promote the Innocentive Challenge, whose aim is to produce and screen genetic sequences for RNA-interference (RNAi) products that work against psyllids. These efforts are in line with the 2010 recommendation NI-11: Sequence, assemble, and annotate the ACP genome to provide a basis for new approaches to ACP management.

Transcriptome assemblies and other sequence data are available at websites of the International Asian Citrus Psyllid Genome Consortium11 and the National Center for Biotechnology Information.12 Bioinformatic and annotation resources for genomes of ACP and other HLB components have been assembled in a NIFA-funded comprehensive, publicly available resource, CitrusGreening Solutions.13 Investigations of insect gustation (act or sensation of tasting) have examined the function of ACP sugar receptors and identified compounds that enhance or inhibit their activity, testing whether the presence of these compounds alters feeding behavior. USDA Animal and Plant Health Inspection Service (APHIS) Multi-Agency Coordination (MAC) Group funding (2016–2017) was focused primarily on pathogen detection in ACP in discrete, hot-spot locations, along with the development of attract- and-kill procedures based upon vector host location cues.

HLB Hosts: Citrus

CRDF-Funded Research

During the early rounds of funding CRDF provided approximately $11.5 million in support of research and development (R&D) in citrus transformation (genetic engineering) and has continued to support efforts in gene discovery and genetic mapping14 (for example, Projects #071 and 356), in line with the 2010 recommendation L-1: Support the development of transgenic HLB-resistant and ACP-resistant citrus and NI-4: Accelerate

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9 See https://www.ars.usda.gov/research/project/?accnNo=430081. Accessed January 9, 2018.

10 See https://www.ars.usda.gov/research/project/?accnNo=429787. Accessed January 9, 2018.

11 See http://psyllid.org/download. Accessed December 20, 2017.

12 See http://www.ncbi.nlm.nih.gov/bioproject/29447. Accessed December 20, 2017.

13 See www.citrusgreening.org. Accessed January 24, 2018.

14 Identifying the respective loci of genes on a genome, and the distances between them.

Suggested Citation:"3 HLB Research and Development Efforts." 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.
×

the sequencing, assembly, annotation, and exploitation of a sweet orange genome to provide a powerful tool for all future citrus improvement research.

Citrus Transformation. While varying levels of tolerance to HLB have been noted in a variety of rootstock and fruiting cultivars, there appear to be no sources of high-level resistance that are viable in the sense that commercially acceptable fruiting citrus cultivars could be produced in time to sustain the Florida citrus industry. Currently, the most promising approach is the development of high-level resistance through genetic engineering using genes from citrus-related (sexually compatible) or unrelated species or organisms (NRC, 2010). Transgenic rootstocks, sweet oranges (Citrus sinensis (L.) Osb.), grapefruits (C. paradisi Macf.), mandarins (C. reticulata Blanco), limes (Citrus × aurantiifolia), and lemons (C. limon (L.) Osb.) have been produced using juvenile tissues with funding from CRDF (Dutt and Grosser, 2010; Orbović and Grosser, 2015). CRDF also funded efforts to transform mature tissues (Marutani-Hert et al., 2012; Orbović et al., 2015; Wu et al., 2015).

Transgene promoters have been characterized and used to target gene expression to particular tissues, most importantly the phloem, where CLas is located (Dutt et al., 2012; Benyon et al., 2013; Belknap et al., 2015), and methods are being developed for the production of transgenic plants without the use of antibiotic-resistance-selectable markers, utilizing a visual anthocyanin marker (Stover et al., 2013a; Dutt et al., 2016). Such a marker could be used alone or it could be used to select plants already containing another marker in order to retransform, or stack, transformation events.

Transgenic Plants. Several projects, together aimed at producing hundreds of independent lines of transgenic plants via genetic transformation, have received funding from CRDF. These projects focused on creating transgenic citrus plants that express a variety of antimicrobial proteins (AMPs) (Stover et al., 2013b; Dutt et al., 2015b), disease resistance genes or R genes isolated from citrus and other plant species (Lu et al., 2013; Dutt et al., 2015a,b), antibodies against CLas proteins (Project #606), genes to improve regeneration and transformation (Hu et al., 2016), and promoters targeting specific tissues (Dutt et al., 2012; Benyon et al., 2013; Belknap et al., 2015). RNA interference (RNAi) strategies have also been explored (Hajeri et al., 2014), in line with the 2010 recommendation NI-9: Support demonstration of RNA interference (RNAi) effects for possible suppression of ACP. Efforts to edit clustered regularly interspersed short palindromic repeats (or CRISPR) have also received CRDF funding (Jia et al., 2016, 2017; Project #922). There is potential for rootstock transmission of resistance to the scion, which would allow the scion itself to be nontransgenic.

Resources Required for Genetic Engineering. Genetic engineering involves gene identification, gene isolation, promoter selection, vector con-

Suggested Citation:"3 HLB Research and Development Efforts." 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.
×

struction, and vector quality testing, sometimes in model plants such as Arabidopsis or tobacco. The test of the effectiveness of a gene or promoter relies on the production and testing of transgenic plants. Transformation is tedious and time consuming as all work must be done using sterile techniques, explants must be transferred onto fresh media multiple times, all putative transformants must be checked for the presence of transgenes and individual transgenic clones multiplied for inoculation tests, and shoots must be rooted and hardened off for moving from the culture room to the greenhouse for further testing. Yields of transformants are generally quite low. Not all laboratories have the trained personnel or facilities to undertake transformation but may have potentially useful genes to evaluate in planta. The Core Citrus Transformation Facility at the Citrus Research and Education Center in Lake Alfred, Florida, which receives funding from CRDF (Projects #579 and 15-033C), serves this purpose. Some laboratories that receive CRDF funding have their own transformation facilities, while others rely on an outside facility such as the one supported by CRDF.

In addition to the difficulty of transforming most citrus genotypes, rapid and reliable screening of transgenic plants is a significant obstacle to the development of HLB-resistant genetically engineered citrus. Projects #15-016C and 502, which aim to improve and speed the inoculation and HLB resistance screening process, have been supported by CRDF. These projects employed a high-throughput screening using ACP, which better simulates the natural route of infection than graft inoculation and thus would better identify trees having field resistance. CRDF has supported field testing at the Picos Farm in Fort Pierce, Florida (Project #15-039C). Such a site is critical for the long-term testing of HLB resistance, tree performance, and fruit quality and for developing additional data necessary for regulatory agencies on genotypes that will be submitted for approval (see also the section Vector–Host Interactions, page 76).

Genetic Engineering Traits. CRDF funding has enabled researchers to begin to investigate a large number of potentially useful disease resistance genes from citrus and other species. Promoters have been utilized to target transgene expression to phloem.

HLB Resistance and Immunity. A project aimed at testing transgenic trees for resistance or tolerance (Project #15-020) and those focused on HLB tolerance/resistance candidate gene discovery through differential expression of genes in infected susceptible and tolerant genotypes (for example, Project #724) have been supported by CRDF.

Citrus Tristeza Virus Gene Delivery. While the development of new highly HLB-resistant citrus cultivars through conventional breeding or genetic engineering would revitalize and stabilize the citrus industry, both approaches will take time, which a suffering industry can ill afford. CRDF is supporting (about $1.25 million) an innovative approach to provide a

Suggested Citation:"3 HLB Research and Development Efforts." 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.
×

more rapid, if only temporary, route to resistance that could be applied quickly and provide the industry with HLB-resistant material at least until long-term solutions are available. This technology involves using a mild strain Citrus tristeza virus (CTV) vector to deliver and express resistance transgenes in existing citrus cultivars, even in mature trees that are already established in groves (El-Mohtar and Dawson, 2014). The CTV vector is phloem limited and therefore expresses the transgenes (AMPs) where CLas is located. CTV persists for at least 4 to 6 years in citrus trees (Folimonov et al., 2007; NRC, 2010). This technology, which is applied simply through grafting, may not only provide protection from infection but could cure infected trees or reduce symptoms. One previously funded project aimed to assess AMPs for activity against CLas and to develop transgenic trees with effective AMPs (Project #046). Delivery of RNAi by CTV to target ACP is another approach that was investigated (Hajeri et al., 2014). Maximizing expression of transgenes in the CTV vector is an issue and was also a focus of investigation (Project #516).

Research Funded by Other Agencies

A multifaceted project ($3.3 million; funded from 2015 to 2020) that aims to identify candidate HLB resistance genes in resistant or tolerant citrus relatives and utilize these for gene editing producing non-genetically modified HLB-resistant citrus cultivars is funded by NIFA (#2015-70016-23027). CRB also funded efforts to produce non-genetically engineered (non-genetically modified organism but engineered to contain resistance genes only from other edible plants) “consumer-friendly” HLB-resistant citrus (Project #5200-144).

Overlaps and Duplications

While there are no obvious overlaps or duplications of efforts and a number of CRDF-funded projects are multi-institutional, there appears to be a need for improved communication and coordination of citrus genetic improvement projects.

VECTOR–HOST INTERACTIONS

CRDF-Funded Research

By the end of 2017 CRDF had funded more than a dozen projects, 13 completed and 1 still active (#15-039C), that contain a substantial component addressing vector–host associations, generally in the context of developing new approaches or enhancing existing strategies for ACP

Suggested Citation:"3 HLB Research and Development Efforts." 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.
×

management. These efforts are in line with the 2010 recommendation L-3: Support analysis of ACP behavior, ACP–plant interactions, and ecology to enhance the knowledge base available for new ACP management strategies. In total, these projects represent $2.6 million of support from CRDF.

Host Range of ACP. Identifying the host range of ACP is vital to managing HLB. Finding resistant plant species or varieties can facilitate the characterization of potential genetic resistance mechanisms or phytochemicals that can function as repellents or deterrents. Moreover, in view of the presence of other native and introduced rutaceous species in Florida, characterizing the host range can pinpoint unmanaged host reservoirs where populations can build up and, via immigration, thwart efforts to control ACP in citrus groves.

Major efforts to find resistance to HLB have been focused on Poncirus trifoliata (hardy or trifoliate orange). P. trifoliata is closely related to Citrus and in fact is sometimes placed within the genus Citrus; it is genetically compatible with sweet orange and can hybridize to produce “citranges.” CRDF projects have been aimed at characterizing the extent of trifoliate orange resistance and its underlying basis. In terms of current projects, Project #15-039C has as its objectives to establish the Picos Farm of the U.S. Horticultural Research Laboratory in Fort Pierce, Florida, as a secure experimental site for field testing citrus germplasm for resistance to HLB and ACP, and to make it available to the greater research community. Part of this project included testing of more than 1,000 Poncirus hybrid trees for resistance to facilitate gene mapping in a collaborative project with the USDA Agricultural Research Service (ARS), University of California, Riverside, and University of Florida.

With respect to completed projects, Project #315 set out to screen citrus germplasm for resistance to ACP with the ultimate goal of exploiting plant resistance as a management tactic. Plant trait targets for screening included kairomones,15 color, phenology, and morphology. Although Citrus species are generally all capable of supporting ACP growth and development, host species can differ in their impact on ACP morphology and related life history traits; investigators quantified such differences. In a publication resulting from this project, Hall and Hentz (2016) compared flushing characteristics of nine rutaceous species and/or genotypes vulnerable to colonization by ACP: Afraegle paniculata, Bergera koenigi, Citrus aurantiifolia, C. macrophylla, C. maxima, C. medica, C. reticulata, C. taiwanica, and Murraya paniculata. Project #581/581-1 was focused on identifying overwintering habitats and alternative host plants of ACP and to assess the suitability of alternative hosts for oviposition and develop-

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15 Chemical substances emitted by an insect or plant that have an adaptive benefit, such as a stimulus for oviposition, to another species.

Suggested Citation:"3 HLB Research and Development Efforts." 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.
×

ment. This project yielded at least one publication, reporting on trapping of ACP in habitats without known rutaceous host plants (Martini et al., 2013). Project #538 was aimed at determining whether previously reported resistance of Cleopatra mandarin (C. reshni) seedlings to ACP (Tsagkarakis and Rogers, 2010) is genetically based and thus potentially useful in breeding for resistance in hybrid offspring of Cleopatra.

Semiochemical and Other Host-Associated Cues for Host Orientation, Assessment, and Acceptance. Both previous and ongoing studies have examined a multitude of sensory modalities used by ACP to find host plants; collectively, these studies indicate that multimodal cues interact and vary with context, including age, sex, and previous experience of the psyllid. Olfactory cues deriving from the host plant itself play a major role in ACP host orientation; as well, host orientation is influenced by chemical cues used to facilitate mate finding. With respect to phytochemicals, in most herbivore–plant interactions, volatile cues are primarily responsible for host attraction, whereas gustatory cues are important for host acceptance. CRDF-funded investigators set out to identify semiochemicals involved in psyllid host finding, host assessment, and host acceptance, with the ultimate goal of developing and optimizing multicomponent blends for use in monitoring and control.

One past project (#853) was designed to take advantage of ACP resistance exhibited by P. trifoliata in order to characterize phytochemicals that influence ACP behavior and physiology. The project’s three major objectives were to identify host plant-produced volatile chemicals and leaf/plant metabolites that are attractive or repellent to adult ACP; to test preference and performance of ACP adults on susceptible and resistant host plants; and to evaluate ACP behavior in response to odorant and tastant blends.

For many herbivorous insects, particularly hemipterans, visual cues play a significant role in host orientation and attraction. One past project (#701/701-1), which was part of an effort to develop a push-pull system to increase the efficacy of ACP control through enhanced attraction, had among its objectives to characterize attractants including visual cues for inducing take-off propensity, flight duration, and color preferences. In this project, investigators compared the size and shape of ACP reared on four species of Citrus (C. aurantiifolia, C. macrophylla, C. maxima, and C. taiwanica) and two species in other rutaceous genera (B. koenigii, M. paniculata) to determine whether host plant identity influences dispersal potential through changes in wing aspect ratio (Paris et al., 2016).

Although the plant is an important source of semiochemical signals, other signals that influence vector behavior arise from the insect itself. This dimension of vector–host interactions was the focus of two successively funded projects: #15-024 and 766. The first of these (#15-024), which is ongoing, has the overarching goal of evaluating effects of abiotic factors on

Suggested Citation:"3 HLB Research and Development Efforts." 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.
×

dispersal and flight capability of ACP. Specific objectives are to determine temperature and humidity effects on flight initiation thresholds, effects of wind speed on psyllid flight and directionality, and effects of barometric pressure changes on dispersal. The researchers also plan to develop a model to predict invasion risk based on abiotic factors. This project was a continuation of earlier funding to examine biotic factors affecting dispersal and flight capacity. Among biotic factors examined were contributions of female ACP-produced odors relative to mating state, the chemical composition of female-produced chemical cues, and impacts of male reproductive experience on responses. The second project (#766) focused on vector-influencing signals that arise from the insect itself and had the general objective of ascertaining the mechanistic basis for ACP acceptance of citrus hosts. Initial objectives of this project included characterizing differences in abiotic and biotic factors in resets, mature trees and trees having unusually high populations of CLas-infected ACP, or CLas-infected trees having high populations of ACP that are likely to be sources of inoculum. The goals were to define the biotic factors influencing ACP acceptance and performance, to measure ACP population size differences in resets and mature trees, to establish the impacts of time since HLB inoculation and proportion of infected trees on ACP host acceptance within groves, and to assess differences in ACP dispersal from resets and mature trees in the context of HLB transmission (Stelinski, 2016).

With respect to insect-derived attractants, there were efforts to examine the chemical composition of and behavioral responses to volatiles produced by female adult ACP in the presence and absence of volatiles of intact or ACP-damaged foliage (Mann et al., 2012). The behavioral responses of male and female D. citri to cuticular extracts in the laboratory and in the field and sex differences in the chemical composition of their cuticular extracts as well as effects of volatiles from insects themselves and from intact or insect-damaged plants on attractiveness were also been investigated (Mann et al., 2013; Martini et al., 2014b). There was also a study on the impacts of developmental and adult experience on learning to recognize olfactory and visual host plant stimuli (Stockton et al., 2016).

Microclimate Effects on Host–Vector Interactions. Inasmuch as the host plant is, for most insect herbivores, also its habitat for much of its life cycle, abiotic factors have a profound influence on population development. Abiotic environmental factors affect dispersal initiation and distances, while host plant dispersion patterns are important in determining dispersal success rates. CRDF funded projects aimed at characterizing psyllid responses to abiotic environmental factors with the ultimate goal of acquiring data useful in optimizing trapping and predicting risks of psyllid attack. A completed project examining abiotic factors was Project #214, which aimed to evaluate seasonality and frequency of psyllid dispersal. Among the host

Suggested Citation:"3 HLB Research and Development Efforts." 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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plant attributes evaluated was flush availability; abiotic factors evaluated included movement between managed and abandoned groves, geographic barriers, wind direction, and movement in the absence of severe weather. Another completed project was #600; objectives relevant to determining impacts of the abiotic environment were to assess effects of light quality, photoperiod, air flow, and temperature on psyllid movement; to evaluate physiological limits and life history traits including feeding behavior, infection status, fecundity, and population density; and to characterize seasonal patterns of ACP distribution and movement at different scales in the field.

As mentioned earlier, a component of Project #15-024 was to evaluate effects of abiotic factors on dispersal and flight capability of the ACP. Among the publications produced from this project, Martini et al. (2015b) documented the effects of windbreaks on dispersion patterns of mature and reset-replacement trees on ACP populations. Also mentioned earlier, Project #581 had objectives of identifying overwintering sites and alternative hosts, and describing effects of latitude and row orientation on psyllid density during winter (Pelz-Stelinski et al., 2017).

Influence of CLas on Insect–Plant Interactions. In many systems in which bacterial phytopathogens depend on a vector insect for transmission, pathogen-induced changes in the plant host affect the attractiveness and suitability of the host for the insect (Rid et al., 2016). This phenomenon was investigated in completed CRDF-funded projects, particularly with respect to the volatiles released by the host plant. Three completed projects examined the tritrophic interaction between CLas, ACP, and citrus, as mediated by host plant chemistry. One goal of Project #093 was to examine impacts of pathogen–vector interactions that influence vector specificity and vector competence. Project #334 was aimed at determining whether healthy ACP distinguish between healthy versus HLB-infected citrus plants, whether ACP infected with CLas behave differently relative to healthy and infected plants with respect to settling behavior in the laboratory and the field, and whether chemically mediated interactions vary in the presence of bacterial infection. Finally, Project #335 had as its goal to determine if methyl salicylate is repellent to ACP; researchers examined how CLas infection alters the volatile chemistry of citrus (including methyl salicylate) and the attractiveness to and settling behavior by ACP, and whether pathogen-induced volatiles attract parasitoids of ACP (Mann et al., 2012; Martini et al., 2014b).

Research Funded by Other Agencies

Of 16 NIFA SCRI grants supporting HLB research, evidently no funding was directed specifically toward elucidating the interaction between ACP and its host plants. CRDF partnered with USDA NIFA Coordinated

Suggested Citation:"3 HLB Research and Development Efforts." 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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Agricultural Project Award No. 2012-51181-20086 and the Florida Citrus Advanced Technology Program, Contracts 21 and 510, on a project to construct complementary DNA (cDNA) libraries from CLas-infected and CLas-free ACP adults and nymphs to characterize gene expression profile with the goal to identify psyllid bacterial effectors that interact with factors required for circulative, propagative transmission of CLas (Vyas et al., 2015).

The National Institutes of Health (NIH) National Center for Research Resources (5P20RR016469) and the National Institute for General Medical Science (8P20GM103427), USDA ARS U.S. Horticultural Research Laboratory, Subtropical Insects Research Unit, Fort Pierce, Florida, and the CRB together supported a project to characterize the transcriptomes of egg, nymph, and adult ACP (Reese et al., 2014), although the focus was on identifying insecticide-resistance genes (some of which may actually contribute to phytochemical detoxification).

USDA NIFA grant 2015-70016-23028 supported annotation to complement ACP genome sequencing supported by NIH National Center for Research Resources (5P20RR016469), USDA ARS, and CRB (Saha et al., 2017). This sequencing effort resulted in annotation of 60 cytochrome P450 genes, some of which are likely to be involved in host–vector associations (e.g., detoxification of phytochemical resistance factors, CYP3 clan, degradation of volatile attractants, and CYP4 clan).

CRB has supported several projects on vector–host interactions, including studies of ACP responses to volatiles produced by flushing shoots (Patt and Sétamou, 2010), identification of volatiles for surveillance and management of ACP (Coutinho-Abreu et al., 2014a; Project #5500-186), odor coding in ACP underlying odor detection in ACP antennae (Coutinho-Abreu et al., 2014b; Project #5500-186), characterization of the ACP transcriptome (Reese et al., 2014), and collaborative work on mitogenome analysis to identify region of origin for ACP in California (Wu et al., 2016).

Overlaps and Duplications

With respect to characterizing interactions between ACP and its host plants, there was little overlap in objectives between projects funded by CRDF and other funding agencies. Although both agencies funded projects aimed at characterizing volatile attractants and repellents, there were differences in scope and experimental design that generated findings that were complementary rather than duplicative.

Suggested Citation:"3 HLB Research and Development Efforts." 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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PATHOGEN–VECTOR INTERACTIONS

CRDF-Funded Research

The 2010 NRC report listed several gaps in understanding the ACP–CLas interaction affecting transmission and HLB epidemiology. These included a need to study the role of ACP nymphs in CLas transmission and HLB epidemiology, the effects of CLas acquisition and infection on ACP biology and fitness, and the potential for vertical transmission of CLas through ACP (NRC, 2010). Numerous investigators developed projects based on these recommendations, and CRDF funded several projects to investigate the various aspects of CLas transmission by ACP. As is often the case for plant pathogen transmission by insects, all of the measured parameters can vary widely depending on pathogen and insect populations, pathogen source materials, experimental design and methods, and environment. Nonetheless, important information has been generated that is being used to develop and modify HLB management recommendations.

Five projects funded by CRDF (totaling about $1.5 million) are directly and indirectly researching CLas–ACP interactions related to transmission. A majority of the transmission-related projects are aimed at identifying or developing molecules that could have direct or indirect effects on the ability of ACP to transmit CLas. Projects include studies on the effects of thermotherapy (see also the section HLB Management, Bacterial Control, page 86) on ACP transmission efficiency (#941C); the ability of other bacteria to initiate an immune response in ACP that would affect their ability to acquire and transmit CLas (#15-021); the identification of ACP gut receptors targeting the binding and uptake of Bt toxins (#711); the identification and expression of quorum sensing–related compounds that can disrupt bacterial cell-to-cell communication and biofilm formation in CLas populations (#15-017), thus resulting in less efficient survival and transmission of bacteria from ACP; and the manipulation of the CLas phage to induce a lytic phase in CLas harbored by the ACP to essentially cure the insect of the bacteria (#15-009). One CRDF-supported project relates to CLas having a single-component LuxR quorum-sensing mechanism and lacking the acyl-homoserine lactone (AHL) second component. AHL mimics produced in citrus in response to CLas infection may bind to LuxR and trigger cell aggregation and limit CLas movement in plants. ACP endosymbionts may also produce AHL mimics and allow CLas to form biofilms on the ACP gut. Feeding ACP on LuxR-expressing citrus results in a decrease in gut biofilm formation and a decrease in CLas titer in the insect, presumably because the LuxR from citrus binds the AHL mimic in ACP and disrupts the LuxR (bacteria)–AHL interaction. This continuing work (Project #15-017) is focused on developing resistance in citrus, although investigation of aspects related to acquisition

Suggested Citation:"3 HLB Research and Development Efforts." 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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and transmission of CLas from the AHL-expressing citrus plants is planned (Killiny et al., 2014, 2017). Several additional ongoing projects described in the Vector–Host Interactions section (page 76) are looking at ACP survival or deterrence of host finding or feeding. The outcomes from these projects may also provide ways to reduce transmission, although many of these projects are focused only on insect survival and do not incorporate CLas transmission.

Toward achieving the goals set out in the 2010 NRC report, CRDF and other agencies (mentioned below) have funded projects aimed at understanding the biological parameters affecting transmission efficiency (Pelz-Stelinski et al., 2010; Mann et al., 2011; Hall et al., 2012; Wang and Trivedi, 2013; Albrecht et al., 2014; Pelz-Stelinski, 2014; Coy and Stelinski, 2015; Martini et al., 2015a; Hall et al., 2016; Pelz-Stelinski and Killiny, 2016) and how the bacteria move within and interact with the ACP (Ammar et al., 2011a,b, 2016). In addition, they have funded studies to determine how different compounds may be used to prevent these interactions (Tiwari et al., 2011; Yan et al., 2013; Hoffmann et al., 2014; Pelz-Stelinski, 2014; Ramsey et al., 2015, 2017; Chu et al., 2016; Arp et al., 2017; Gill et al., 2017). A key component of the latter was the development of transgenic citrus expressing various compounds that may interfere with ACP feeding or ACP–CLas interactions. It also included the use of CTV vectors for transient expression (El-Shesheny et al., 2013; El-Mohtar and Dawson, 2014; Hajeri et al., 2014; Dawson et al., 2015) (see also the section Citrus Tristeza Virus Gene Delivery, page 75). Efforts in this research area are in line with the 2010 recommendation NI-9: Support demonstration of RNA interference [RNAi] effects for possible suppression of ACP. Several laboratories are currently identifying genes, proteins, and metabolites that are being tested for activity. Intellectual property concerns have kept many of the specifics out of this review. The expression and quantification of compounds either in transgenic plants or transiently using the CTV vector are not straightforward tasks, but progress is being made with promising, albeit inconclusive, results. It is appropriate to mention the recently ended 5-year, $9 million NuPsyllid project (#780nu, 781nu, 782nu, 783nu, 784nu, 785nu, 786nu, 787nu, 788nu, 789nu, 790nu, 791nu, 792nu, 793nu, 794nu, 795nu, 796nu, 797nu, 798nu, 799nu, 800nu, and 801nu) that was designed to generate a genetically modified ACP that would not be able to transmit CLas and could displace wild ACP populations.

Research Funded by Other Agencies

The USDA APHIS MAC program has many projects focused on the management of ACP, but because few of them integrate CLas transmission with vector control it is difficult to assess what ACP management strategies will have any meaningful effect on reducing HLB incidence. One MAC--

Suggested Citation:"3 HLB Research and Development Efforts." 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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funded project (#15-8130-0485-CA) investigated the use of thermotherapy to reduce the ability of ACP to acquire and transmit CLas; this type of work was also funded by CRDF (Project #941C).

Past CRDF funding has been instrumental in generating preliminary data and methods that have allowed larger, multi-institution and multidisciplinary projects to be developed and funded by other agencies. Most of these are fundamental studies aimed at medium- to long-term solutions to HLB. Relevant vector-pathogen projects funded by USDA NIFA include the use of cell-penetrating peptides to inhibit CLas and endosymbiont gene expression in ACP as a way of reducing CLas in the insects and reducing or disrupting transmission and/or insect fitness (Project #2016-70016-24781). This research appears to overlap with CRDF funding (Project #15-026).

NIFA (Project #2016-70016-24779) and CRB are funding research to characterize ACP populations differing in CLas transmission efficiency (Project #5300-163) that would enable a system to study the genetics of transmission competency in ACP and characterize ACP proteins regulating the movement and survival of CLas in the insect. Another NIFA-funded project (#2015-70016-23028) is focused on the much needed integration and curation of omics data becoming available for ACP, citrus, and CLas and to further use these data along with de novo generated genomic, proteomic, and metabolomics data to discover molecules that can block pathways related to pathogenesis and transmission.

CRB funded projects that aimed to discover an infectious CTV vector from a California CTV isolate (#5300-158), to discover ACP and CLas proteins that are involved in pathogen–vector interactions (#5300-155), and to characterize factors influencing transmission efficiency (#5500-203).

HOST–PATHOGEN INTERACTIONS

CRDF-Funded Research

CRDF has funded 16 projects, including 12 past projects (totaling $3.1 million) and four currently active projects (totaling about $1.7 million), with at least one objective that is directly related to host–pathogen interactions. The past projects were focused on determining the physiological response of host plants to HLB infection (#002 and 045), identifying host resistance or tolerance genes to HLB (#523 and 536), analyzing the genomes of the HLB pathogen and host to better understand their interactions (#733, 314, and 123), and studying pathogen effectors or host gene effectors to understand HLB and develop control strategies (#312, 750, 232, and 609).

The currently funded projects are focused on the application of knowledge acquired on the citrus microbiome (#15-042), host metabolomic re-

Suggested Citation:"3 HLB Research and Development Efforts." 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.
×

sponse (#15-003), and effector and tool development for resistance and control of HLB (#15-028 and 16-005).

Host Physiological Responses to CLas Infection. Effort was made to understand the roles of plant callose and phloem proteins in symptom development and disease control by manipulating callose and phloem proteins in HLB (Koh et al., 2012; Aritua et al., 2013).

Identification of HLB Resistance Candidate Genes. Two projects aimed to identify host genes that are highly upregulated in resistant citrus compared to susceptible varieties through transcriptomic and informatics analyses (Folimonova et al., 2009; Nwugo et al., 2013; Pitino et al., 2016), and a study by Gmitter and colleagues (Rawat et al., 2015, 2017) was focused on identifying and mapping HLB resistance gene(s) in Poncirus.

Target Pathogen Effectors or Host Gene of Effectors for Understanding of HLB and Development of Control Strategies. Four past CRDF projects aimed to investigate the molecular interaction of host genes and pathogen effectors. One project (#312) involved development of a virus-based vector for gene expression in citrus plants. Using this system, putative effector genes of interest from Liberibacters were delivered into host cells using CTV for functional analysis (Zou et al., 2012). A second project (#750) focused on four CLas effectors and their host target proteins. It was hypothesized that the interaction of effectors and their targets would play important roles in pathogenesis of HLB. Tentative host target proteins were identified from yeast two-hybrid, or Y2H, screening, and exploration of their biological relevance is in progress. Two projects (#232 and 609) addressed the role of Liberibacter salicylate hydroxylase in HLB pathogenesis. The pathogen uses the enzyme to break down the host salicylic acid (SA) and its derivatives, messengers of the host defense system, leading to weakened resistance in host plants (Li et al., 2017).

Research Funded by Other Agencies

In 2016, NIFA SCRI funded two currently active projects (totaling about $7.3 million) with objectives directly related to host–pathogen interactions. One focuses on the identification of effectors in Liberibacter spp. and elucidation of their role in HLB pathogenesis (#2016-70016-24833), while another aims to identify host candidate genes for HLB resistance or tolerance (#2015-70016-23027).

CRB is currently funding two projects (totaling about $1.2 million) with objectives directly related to host–pathogen interactions. One project aims to identify and characterize small RNAs and mRNAs (#5300-131), and the other aims to identify and characterize citrus targets from CLas (#5300-160).

Suggested Citation:"3 HLB Research and Development Efforts." 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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HLB MANAGEMENT

Bacterial Control

Despite aggressive research and development efforts, there are no fully effective strategies for field management of HLB in Florida citrus. Keeping citrus free of CLas is a goal not only in fruit production groves but also in seedling nurseries and germplasm-development greenhouses. Development of approaches directed at reducing or eliminating the pathogen are hampered by the phloem-restricted microbial habitat and the uncultivable nature of CLas, but researchers are exploring a variety of avenues to target the pathogen as a means of disease management.

CRDF-Funded Research

CRDF has supported a number of past research projects studying HLB management by targeting the bacterial pathogen; many, but not all, of these have resulted in publications in peer-reviewed journals and have served as a foundation for current research efforts, as noted below. Funding these projects is in line with the NRC recommendation L-2: Support development and testing of bactericides, therapeutics, or systemic acquired resistance (SAR) activators. More recent projects aim to evaluate a variety of approaches, including the application of bactericidal or bacteriostatic chemical compounds or antibiotics (nine current projects), the use of high temperature (thermotherapy) to eliminate the pathogen from living tissues (two projects), biocontrol through triggering of the lytic cycle of bacteriophages native to CLas (two projects), nutritional strategies and microbiome enhancements (three projects), and understanding and inducing natural defense responses in the citrus host (four projects). Considering all of the funded research, past and present, related to bacteria-targeted disease management, CRDF has awarded just over $16,864,000 in grants.

Induction of Host Defense Gene Expression. Multiple factors and triggers for induction of genes encoding compounds related to host defense to bacterial invasion have been identified in citrus. These factors and inducers can be mediated not only by pathogens but also by beneficial microorganisms and a variety of chemical compounds. Their application can result in suppression of disease progress and reduction of disease severity and bacterial multiplication. One past project (#129) used the model plant Arabidopsis to investigate the effect of overexpressing the citrus gene CsNDR1 (an ortholog of an Arabidopsis gene that positively regulates SA accumulation) on the production of SA, which is a signaling molecule important for both basal and induced host defense responses in many plants, and the expression of the defense marker gene PR1 (Lu et al., 2013).

Suggested Citation:"3 HLB Research and Development Efforts." 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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Another project (#609) evaluated several combinations of β-aminobutyric acid, 2,1,3-benzothiadiazole, 2,6-dichloroisonicotinic acid, ascorbic acid, and the glucose analog 1-deoxy-D-glucose for their effects on CLas replication in the host and HLB disease severity (Li et al., 2016). A chemical genomics approach was used in a proxy system involving the CLas-related bacterium Ca. Liberibacter psyllaurous, which causes zebra chip disease of potatoes and vein-greening disease of tomato (Prager and Trumble, 2018), in Arabidopsis (Patne et al., 2011; Project #326). In addition to screening many small, drug-like molecules to identify chemicals that induce plant tolerance or resistance against the pathogen, this project (#326) aimed to also create Arabidopsis mutants deficient in specific defense mechanisms to determine the basis for observed tolerance in this host (Roose et al., 2014). Project #15-018 investigated the role of CLas salicylate hydroxylase in suppressing host defense, while another (#572) explored the infiltration into citrus of flagellin protein (shown in other systems to be involved in pathogen-associated molecular pattern host defense induction) from both Xanthomonas citri pv. citri (Xcc, causal agent of citrus canker)16 and CLas (Moore and Febres, 2014; Project #572).

Antibiotics and Chemotherapy. Although copper (Cu) bactericides have been used widely for controlling plant diseases, there are concerns about Cu accumulation in soil and leaching, as well as evidence of increasing microbial resistance (Young et al., 2017). Supplementation of bactericides with zinc (Zn) may help reduce the amount of Cu being released in the environment. One past project funded by CRDF (#15-037C) tested the effect of applying formulations designated “TSOL,” containing chelated Zn and inert ingredients to help in-plant translocation, on the incidence of HLB. CRDF also funded the development of the zinc-based nanoparticle treatment, Zinkicide, which has undergone initial testing in the field (Project #907). A patent application17 filed in 2013 is now listed as abandoned due to the absence of a statement of use; however, a current project18 related to this formulation is now being supported through NIFA SCRI.

A number of nonmetal antibiotics, including sulfadimethoxine and sulfathiazole, cell-wall inhibitors in the β-lactam group (ampicillin, carbenicillin, penicillin, and cephalexin), and the transcriptional inhibitor rifampicin have also been tested for their efficacy in eliminating or suppressing CLas (Zhang et al., 2014; Projects #617C and 617-1). In another CRDF-funded project (#775C), several tetracycline derivatives that lack activity against human bacterial pathogens were tested against strains of Liberibacter.

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16 Currently the citrus canker pathogen is referred to as Xanthomonas axonopodis pv. citri.

17 See https://www.trademarkia.com/zinkicide-86125365.html. Accessed December 20, 2017.

18 Grant #2015-70016-23010; Project #FLAW-2014-10120 (2015-2020).

Suggested Citation:"3 HLB Research and Development Efforts." 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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There has been a continual push to develop novel anti-CLas treatments, using both high-throughput and directed methods. Triplett et al. (2016; Project #782) tested 888 different chemical formulations for activity against L. crescens (a culturable proxy for CLas), including bacterial fermentation products obtained from both major and smaller startup companies. In an unusual approach to the identification of new antimicrobials having potential for HLB management, Pagliai et al. (2014, 2015; Project # 414) characterized a regulatory element containing a CLas transcriptional regulator (ldtR) and a transpeptidase (ldtP).

CRDF has also supported research in optimizing antibiotics and other antimicrobials for maximum efficacy. For all bactericidal treatments, the method of plant delivery is as critical as its chemistry; cost and damage issues associated with bactericidal injection and thermotherapy have obstructed their commercialization. One past project aimed to characterize the optimum parameters for trunk injection of oxytetracycline to achieve uniform antibiotic distribution in planta, and improve tree health, yield, and juice quality (Hu and Wang, 2016; Project #773). Two past projects focused on enhancing the efficacy of antimicrobials by combining them with adjuvants or delivering them as a nanoemulsion (Yang et al., 2015; Powell et al., 2016). “Soft” or nonclumping nanoparticles (SNPs) are being explored as vehicles for foliar application of natural biocides to infected trees (Moudgil et al., 2015; Projects #771 and 909). One past project (#15-031C) investigated the use of laser light in enhancing the uptake of foliar sprays (Etxeberria et al., 2016; Projects #818 and 15-031C). Other current CRDF projects are focused on the delivery mechanisms for agriculturally registered bactericides, essential oils, and other antimicrobials (Minter, 2017; Project #15-048C; Richardson and Shatters, 2017; Project #938C), their uptake and movement in planta (Tetard and Pelz-Stelinski, 2017; Project #16-017C), or their efficacy in the field (Richardson and Shatters, 2017; Project #938C).

A key point in the evaluation of antimicrobials is that their use may be enhanced when used in combination with other HLB management strategies in an integrated pest management (IPM) approach. For example, in one CRDF project (#910), Powell et al. (2016) aimed to determine the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for field citrus and control HLB.

A current CRDF project (Gabriel et al., 2017; #15-009) is investigating potential control of HLB using the putative CLas LexA-like (LC1) repressor protein, found in psyllids, as potentially a key phage lytic cycle regulator. Three protein repressors and target promoters are being screened for chemicals that might be used to activate the phage lytic cycle in CLas, in infected psyllids and citrus. Gabriel et al. (2017) also examined control of HLB using a CLas phage peroxidase, which degrades reactive oxygen species in

Suggested Citation:"3 HLB Research and Development Efforts." 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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the host defense, and a CLas lytic cycle activator, to enhance native citrus resistance to CLas. In an alternate approach, Gonzales (2016; Project #726) investigated phages from Xanthomonas axonopodis pv. citri for their HLB therapeutic potential.

Thermotherapy. CRDF has funded a number of research projects to explore the potential of thermotherapy for managing bacterial diseases such as HLB (Projects #910, 834, 586C, 586-1, and 941C). The impact of thermotherapy arises from two possible effects of elevated temperature: the direct effect of killing bacteria with heat and the indirect effect of triggering induction of a lytic phase in bacteriophages within the pathogen. In both cases it is necessary to identify an effective temperature that does not adversely affect the host plant. Both approaches have been effective in treating other bacterial diseases.

Host Plant Nutrition and Microbiome Approaches. The importance of both the entire plant-associated biotic community and the abiotic environment in shaping plant health has recently been reemphasized in the newly described phytobiome concept (Beattie et al., 2016), and how those principles apply to citrus and HLB was thoughtfully reviewed by Wang et al. (2017). All phytobiome species residing in plant-associated niches, external or internal, interact with one another and with the plant in beneficial, detrimental, and neutral ways that are now beginning to be appreciated, but these concepts may provide important clues not only to understanding complex diseases such as HLB but also to managing them. These principles have been explored in Projects #15-042, 916, and 608. This nontraditional approach to nutrient enhancement for citrus in the presence of HLB was reflected in the recent work of Zhang et al. (2017; Project #780C), who characterized the microbiomes of HLB-infected and healthy citrus trees and identified rhizosphere-to-rhizoplane-enriched taxonomic and functional properties associated with them. Another recent project (#15-042) addressed the effects of a number of beneficial bacteria on HLB disease index and CLas titers in the citrus host.

Research Funded by Other Agencies

Induction of Host Defense Gene Expression. The U.S. Department of Energy provided funding for a project that evaluated the application of epibrassinolide, a member of a group of essential plant-produced brassinosteroids, as a means of controlling HLB (Canales et al., 2016).

Nutritional Approaches. Most nutritional treatments for HLB management have focused on enhancement of tree health with the goals of extended productive lifespan and maintenance of fruit production and quality in HLB-infected trees (see also the section Cultural Control, page 91).

Suggested Citation:"3 HLB Research and Development Efforts." 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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Insect Control (Chemical and Biological)

CRDF-Funded Research

Since the release of the NRC report in 2010, CRDF has funded at least 26 projects dealing specifically with chemical control and IPM for ACP, in line with the 2010 NRC recommendation NI-1: Improve insecticide-based management of Asian citrus psyllid. Essentially, every insecticide registered for use on citrus has been examined by researchers, and many insecticides in the development stage have been tested against various psyllid stages (Boina and Blumquist, 2015). Collectively, approximately $5,560,000 was spent on these projects since 2010. The major themes of the research have been insecticide testing against all stages of the psyllid (examples include Projects #447, 590, 603, and 091), integration of insecticides into IPM programs to minimize resistance development (such as Projects #15-024, 850, and 15-038C), documentation of approaches that could be used in organic orchards (Projects #711, 858, and 217), and examination of attractant and repellent compounds with potential for use as control materials (Projects #853 and 16-020C). As part of these goals, projects were funded to examine pesticide application strategies on young and mature trees and at different times of the year based on tree phenology (Projects #15-036C, 325, and 425).

Six additional projects funded by CRDF were designed to determine how to sample for ACP in order to determine if pesticide use is justified (#15-024, 214, 090, 701-1, 164, and 567). These were funded for just over $1,150,000, and a detailed description of sampling techniques can be found in Monzo et al. (2015). These projects examined many different sampling approaches and proposed psyllid population thresholds designed to indicate a need for pesticide applications on both young and mature trees.

Considerable funding has also been expended for psyllid suppression using biocontrol strategies, including eight projects funded by CRDF for approximately $1.9 million (including #711, 760-1, 212, and 434). These projects had three major foci. The first was to evaluate the potential of indigenous predators and parasites. Because of intensive pesticide applications in active commercial groves, these beneficial insects appeared to have the greatest potential for effective use in abandoned orchards and in urban/suburban citrus. A second approach was to rear and release imported parasites, particularly Tamarixia radiata. This was by far the biggest recipient of support, with well over 95% of the funding. The third focus was to investigate the use of entomopathogens in psyllid suppression.

Suggested Citation:"3 HLB Research and Development Efforts." 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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Research Funded by Other Agencies

Additional funds of nearly $1.9 million for chemical control of ACP were provided by the USDA APHIS HLB MAC Group. CRB also funded projects on the use of pesticides for over $1 million, but this was not considered a duplication of funding because pesticides can have variable effects on a target insect depending on application strategy, crop, and environmental conditions.

The funding of the biological control programs supported by CRDF was supplemented by 10 projects and substantial funding from the MAC program (just over $4 million). This strategy is also being funded by CRB. Almost all of the additional funding from other agencies was designated for the parasite release program.

Overlaps and Duplications

Although funding for insecticide testing was provided by several agencies, this is a necessary duplication of effort. Insecticide efficacy can vary among cultivars, geographic locations, and environmental conditions. Thus, tests at multiple locations and on different cultivars are necessary and justified.

The biocontrol programs were well coordinated. Most of the predator and entomopathogen studies were conducted in Florida (with a small portion of the research occurring in Texas). The overlap between agencies for funding of the Tamarixia release projects was fairly minimal. Most of the CRDF funds were used to evaluate the effectiveness of the releases in Florida, while the CRB funding was used to support parasite releases in California. The much larger amounts of funding provided by the MAC program supported the Florida and California efforts by producing the large numbers of parasites needed for the releases in both states. Thus, these programs appeared to have good coordination with relatively little overlap.

Cultural Control

CRDF-Funded Research

CRDF has funded projects that aimed to investigate various aspects of cultural and on-farm management of HLB, CLas, and/or ACP. Studies were done to evaluate the effects of nutritional supplements on plant growth and development; these included the impacts of root and soil health (Projects #903, 15-013, 838, and 15-023), the use of endophytes to control CLas (Project #15-042), and the use of metallic mulches (Project #16-011C) and polymer films (Project #858) to repel ACP and improve tree growth.

Numerous CRDF-funded projects have investigated several potential

Suggested Citation:"3 HLB Research and Development Efforts." 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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cultural management strategies that could reduce the immigration of ACP, the inoculation efficiency of immigrating ACP, disease development in inoculated trees, and improvements in tree health and fruit production and quality (Projects #910, 903, 776C, 731,732, 943C, and 447). Advanced production systems for citrus have been partially supported by CRDF funding (Project #593) and have looked at integrating several production strategies, including high-density plantings, alternative irrigation and nutrient management, as well as using mulches and growing citrus in psyllid-proof enclosures (Campos-Herrera et al., 2013, 2014; Kadyampakeni et al., 2014, 2016; Ferrarezi et al., 2017a,b). High-density plantings are becoming more popular as growers replant in Florida, and the data generated by these studies are contributing to the enhanced citrus management systems being put in place by some growers. These efforts are in line with the 2010 recommendation NI-8: Support small-scale studies on the feasibility of alternative horticultural systems suited to endemic HLB.

Research Funded by Other Agencies

The USDA APHIS MAC program has contributed in excess of $8 million to research on numerous management topics, including removal of abandoned groves, reducing pH of irrigation water, thermotherapy, integrated management, mulch, and intensive grove management.

Diagnostics

CRDF-Funded Research

There are no projects currently funded by CRDF that are explicitly researching new CLas diagnostics; however, there are several projects that could contribute valuable information to the development of next-generation diagnostics. A study (Project #15-003) comparing the metabolomic profiles of germplasm from citrus that is susceptible to CLas and citrus that may have some level of resistance or tolerance would be valuable for laboratories looking for metabolomic biomarkers for disease detection. Another project (#15-042) is focused on characterizing the phytobiomes and endophytic microbes from HLB survivor trees and HLB-infected trees to determine if the endophytic microbes from survivor trees could be used to manage HLB; results of this study can be used by researchers who are investigating microbial population changes as biomarkers for disease detection. There is also a project (#15-008) that aimed to improve the understanding of the movement of the pathogen in or between trees, and to elucidate the nature of signals from the pathogen. The focus of this work is not diagnostics; however, understanding pathogen movement and systemic signals from

Suggested Citation:"3 HLB Research and Development Efforts." 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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the pathogen could be helpful in developing next-generation diagnostics. These projects can contribute to ongoing efforts that are in line with the 2010 recommendation NI-2: Support searches for biomarkers that may be exploited to detect CLas-infected citrus.

Past CRDF-funded projects with objectives related to CLas diagnostics include high-throughput screening of seedlings for resistance to HLB based on optical sensing (Project #880).

Research Funded by Other Agencies

Current CRB projects include research on metabolomics and proteomic biomarkers in trees related to CLas infection (Project #5300-150) and on improving detection using conventional methods, i.e., polymerase chain reaction-based amplification (for example, Project #5300-176). Canine detection is funded by the USDA, ARS, and USDA APHIS MAC program (Project #14-8130-0474-CA). MAC is also funding an antibody-based early detection project with apparent overlaps with a project funded by NIFA (Project #2016-2021) to identify CLas-secreted effector molecules that diffuse through the tree and can be detected by antibody-based diagnostics. Two additional NIFA-funded projects looked at CLas detection in ACP; one project was focused on the development of a yeast biosensor that can be used in the field to identify CLas-infected insects (#2016-70016-24779), and the other focused on the development of field assays using loop-mediated isothermal amplification (or LAMP) technology (#2015-70016-22992). These are subobjectives in larger projects aimed at understanding CLas transmission, ACP genetic variability, and ACP surveillance.

SOCIOLOGICAL AND ECONOMIC ASPECTS OF HLB AND ITS MANAGEMENT

The state of Florida has more acreage devoted to citrus production than any other U.S. state. In 2012 Salifu et al. (2012) estimated commercial production at just short of 600,000 acres (242,811 hectares) and annual proceeds at $9 billion, with about 76,000 jobs devoted to the Florida industry. However, since its detection in Florida in 2005, HLB has caused increasing economic losses each year to growers, processors, and the state as a whole (Roka et al., 2009; Muraro, 2012; Salifu et al., 2012; Court et al., 2017). These losses are incurred in a variety of ways and are felt by a variety of stakeholder groups.

The reactions and responses to the arrival and spread of HLB in Florida were influenced in a number of significant ways by the fact that in 2005, when HLB was first detected in Florida, growers and regulators were still occupied with containing a serious outbreak of citrus canker, a different

Suggested Citation:"3 HLB Research and Development Efforts." 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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disease caused by the bacterium X. axonopodis pv. citri. Canker, which had been found several times previously in Florida but successfully eradicated each time, was discovered again in 1996 near Miami (Gottwald et al., 2012). X. axonopodis pv. citri, disseminated primarily by wind, water, and the movement of infected plant parts, spread through substantial portions of the citrus-producing counties during the next 8 years. Because an eradication approach had been successful in past canker outbreaks it was implemented again, with targeted delimiting disease surveys and quarantines on the movement of citrus seedlings, buds, and fruit (USDA APHIS, 1999). A key feature of the canker eradication strategy was to rogue infected trees, including destruction (generally by burning) of trees that were symptomatic as well as all trees within a 1,900-ft. radius of a symptomatic tree (which may have already become infected but was not yet symptomatic). Although growers were compensated for approximately 60% of their evaluated losses, the impacts on the industry were heavy; losses to many growers were high and a number of farms were lost. Eradication costs rose above $200 million, and more than 10 million trees were destroyed, but the disease continued to spread (Gottwald et al., 2012). A series of hurricanes in 2004 further exacerbated the dissemination of X. axonopodis pv. citri within the state, and regulatory officials eventually amended their canker strategy; in 2006 USDA APHIS declared canker endemic and transitioned from eradication to disease management (USDA APHIS, 2017).

The arrival of HLB in Florida in 2005 was different in several important ways from that of canker in 1999. The ACP vector had been present in Florida since 1998 and by September 2000 it had been found in 31 counties (Halbert and Manjunath, 2004), making it extremely likely that if CLas ever made its way to Florida it would be impossible to eradicate. This expectation was indeed met after CLas arrived.

A traditional and generally successful approach to disease management for an insect-transmitted pathogen in a new location is to remove infected plants that could serve as reservoirs for the pathogen and practice strong insect control via chemical sprays and methods to discourage or prevent insect feeding. However, when HLB arrived in Florida the severe impacts of citrus canker—both the disease and the management strategies—were still fresh in the minds of growers and other industry personnel. Many growers had suffered financially from canker and had neither the funds nor the will to invest heavily in managing HLB. The disappointment and frustration that many growers felt over the efforts to manage canker influenced their willingness to accept new constraints, restrictions, regulations, and government involvement in the response to HLB. This reluctance continues today among some members of the citrus industry (Singerman, 2017) and must be factored into assessments of the potential success of any management strategies for HLB.

Suggested Citation:"3 HLB Research and Development Efforts." 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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Thus, a comprehensive assessment of future research directions in support of HLB management and of the potential of new research findings to be accepted and implemented according to guidances developed for them would be incomplete without consideration of factors such as economics and sociology, which influence decision making and behaviors of growers, processors, and the public. Although the research findings described below emerged from projects funded by sources other than CRDF, their implications for future HLB research are critical.

Economic Impacts of HLB

Eradication of HLB-infected and nearby trees in Florida eliminated about 10% of the citrus production capacity by 2008 (Irey et al., 2008), and the disease is estimated to have led to the loss of 6,600 jobs and as much as $1.6 billion in grower revenue ($418 million/year) (Hodges and Spreen, 2012; Court et al., 2017). Between 2005 and 2012 the increased number of recommended sprays for psyllid control and other disease-related actions resulted in significantly higher production costs: cultural practices rose 107% (reaching, for example, $1,187/acre for Valencia), fertilizer costs were up 160%, and chemical sprays over 170% (Muraro, 2012). As a result, growers must realize 33 to 39% greater income just to break even financially. Today, most Florida citrus growers no longer remove infected trees (Muraro, 2012) and even abandon (or stop managing) entire orchards that have become unproductive due to HLB. These findings could serve to inform decision making by state and federal officials with respect to targeting disease reduction and prevention.

Economic Aspects of ACP Management and CHMAs

Pests are essentially “common property,” ignoring farm boundaries such that a pest problem for one grower is likely to be a problem for the neighboring farmers (Singerman et al., 2017). The establishment of 35 Citrus Health Management Areas (CHMAs) was an attempt to encourage neighboring citrus growers to share and coordinate HLB management actions, especially those targeting the psyllid vector, to more effectively manage pathogen dissemination and limit disease spread (Muraro, 2012; UF IFAS, 2018). The effectiveness of the CHMAs was evaluated by comparing the yields from blocks of trees managed within CHMAs having high levels of grower participation with those from blocks having lower participation (Singerman et al., 2017). Yields from the high-participation blocks were significantly higher than those from low-participation blocks in each year of the 3-year study, and the difference between the two grew each year from 28% (year 1) to 98% (year 3). However, the evidence indicating that higher

Suggested Citation:"3 HLB Research and Development Efforts." 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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levels of CHMA participation results in greater economic benefits has not led to proportional levels of grower participation in them (Singerman et al., 2017); rather, grower participation levels have actually fallen off over the years since CHMA establishment. In surveys of grower perspectives, the most important reason given for nonparticipation by CHMA members was the belief that neighboring farmers were not complying with the agreed-upon spray strategy, and the resulting doubt about whether an individual’s own expense and effort would be effective in the absence of neighbor participation (Singerman et al., 2017). The second-most important reason given was that farmers preferred to spray on their own schedule, rather than following an area-wide schedule. Thus, “strategic uncertainty” has hindered the success of the CHMA approach. Singerman et al. (2017) made four recommendations for improving the success of CHMAs: (1) regulatory mandates for scheduled spraying, possibly with subsidies to farmers to defray a portion of chemical costs; (2) monitoring of sprays and consequences for noncompliance; (3) a process for in-kind transfers among growers and possible tax breaks; and (4) enhancement of communication and education among growers.

If CHMA programs continue, the use of newer ultra-low-volume sprayers, especially from aerial dissemination systems, should lower costs and enhance effectiveness (Muraro, 2012). However, the CHMA focus on pest control represents the more traditional formula for managing a vector-transmitted disease, a focus that many Florida citrus growers now question. Indeed, many growers are moving to new strategies having very different final objectives.

New Directions of HLB Management

Recent trends in HLB management in Florida have shifted from concerted efforts to stop the disease to actions intended to support and prolong the health and fruit production of HLB-infected trees (Roka et al., 2009; Muraro, 2012; Zansler, 2017). The idea is to live with the disease rather than eliminating it. One new approach is the use of advanced production systems, which have the potential to reduce the time to fruit production and to maximize and stabilize yields for up to 15 years using high-intensity cultural/agronomic approaches (Roka et al., 2009). Advanced production systems can include high-density planting (up to 360 trees/acre from the traditional 150) and a multifaceted approach to water and nutrient supplementation, including time-released fertilizers, computerized fertigation systems, hydroponic systems, and automated irrigation scheduling. Another new strategy, the Enhanced Foliar Nutritional Program (EFNP), involves the application of mixtures of foliar nutrients, generally as a replacement for traditionally applied fertilizers, in the attempt to compensate for nutri-

Suggested Citation:"3 HLB Research and Development Efforts." 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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ent losses caused by the blockage of conducting tissues in infected trees. When three different EFNP nutrient mixtures were applied to affected groves (Muraro, 2012), costs increased by $119 to 273/acre over that for traditional HLB management (tree removal). Irey et al. (2008) estimated that such new approaches to HLB management have increased production costs by about 40%.

Although some studies (not funded by CRDF) are under way to measure the impacts of the new nutritional/high-density approaches, it is still too early for the results of these analyses to be available. However, many Florida citrus growers have adopted some form of this strategy, and many believe that these approaches are working (Muraro, 2012), anecdotally reporting higher yield and more robust tree growth. The hope is that nurturing infected trees will allow them to remain productive much longer. Further, higher per-acre yields resulting from high-density planting (and proportionately higher grower revenues) should allow more frequent grove turnover as HLB symptoms become more severe. On the negative side, some researchers are concerned that the new emphasis on tree health in the face of HLB is leading many growers to reduce or eliminate their efforts to control the ACP, placing the two approaches in conflict with one another (Gottwald et al., 2012).

Any proposed new management approach will be practical only if the resulting yield and quality are such that grower application costs are more than compensated. For example, Roka et al. (2009) observed that advanced production systems will likely increase grower input costs by several thousand dollars per acre, so fruit yields must also increase to result in a favorable cost–benefit ratio; furthermore, market prices for citrus must be adequate to support that ratio.

Modeling

Because strategic uncertainty clearly influences grower behaviors in disease management (Singerman et al., 2017), it is critical to reduce their uncertainty by providing as much research-based evidence as possible for the benefits of various management strategies. Evidence should be based on realistic assessments of cost–benefit ratios of each approach as well as combinatorial approaches. Generally, a mixed set of approaches (i.e., insect control, removal of infected plants, and use of bactericides) will be more effective than a single one, but estimating cost–benefit ratios becomes more difficult and uncertain with each additional variable.

HLB is among the most complex of plant diseases, so it is among the most difficult to effectively manage. CLas, the associated pathogen, remains uncultivable, hampering many types of research into pathogen–host and vector–host interactions. Within the tree, it is restricted to the phloem sieve

Suggested Citation:"3 HLB Research and Development Efforts." 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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tubes and companion cells, through which it translocates erratically and unevenly throughout the tree. That fact and the fact that in-plant bacterial titers can remain very low for many months before symptom expression make sampling for disease detection frustrating and often misleading. On the host side, citrus is a perennial crop that is generally transplanted into the field as grafted rootstocks, so trees typically have two different genotypes. Young trees remain relatively unproductive until they are about 5 years old and then, if healthy, remain productive for about 20 to 25 years before production levels decline below profitability. The requirement of an insect vector for pathogen dissemination adds another layer of biological complexity, and elements of the psyllid’s epidemiology (flight behavior, feeding and mating behaviors, and attraction to plant colors and volatiles) are strong influencers of disease epidemiology. Thus, there is a multitude of interactions among host, pathogen, and vector, as well as interactions of each of these with other members of the trophic level and with the physical environment (including climate, soil qualities, and agronomic practices).

Most scientific research, by its nature, is conducted within a limited set of parameters (controlled experimentation) to facilitate the attribution of research findings to the particular experimental variables employed. However, applicability of research findings to real-world problems requires a systems approach in which, ideally, all parameters pertaining to all of the participants (host, pathogen, and vector) should be considered. Because this level of inclusion is not possible, as many of the factors as possible should be incorporated. Modeling should be further developed as a systems approach to HLB management, in which it can be used in a myriad of applications, for example, to evaluate risk, to predict how best to incorporate HLB-tolerant groves in relation to susceptible neighboring fields, to provide guidance on how to deploy the CTV vector to express AMPs and RNAi and potential benefits of doing so, to help assess replant strategies, to compare the effectiveness of different single and combinatorial HLB management practices, and to inform economic decision making.

Genetically Modified HLB-Resistant Citrus

Most of the strategies discussed here for managing HLB in Florida are considered by growers, processors, and consumers to be stop-gap measures to preserve as much as possible of the state’s citrus industry while awaiting the development and release of HLB-resistant cultivars that still retain desirable characteristics such as yield, flavor, size, and shelf life. Despite long-term intensive breeding programs, no such resistance is yet available. During the same period, however, significant advances have been made in crop improvement through genetic manipulations, including genetic engineering and CRISPR technology; these approaches are being explored by

Suggested Citation:"3 HLB Research and Development Efforts." 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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a number of researchers in Florida and elsewhere for the development of acceptable varieties having resistance or high levels of tolerance to HLB.

A critical question about whether genetic modification will provide the ultimate solution to HLB is whether the technology will be accepted by growers, processors, and consumers. Singerman and Useche (2017) surveyed19 citrus growers from different production areas in Florida about their perceptions and concerns about the use of this technology. Fewer than half (40%) of respondents felt that they were reasonably well informed about genetic modification technology. Approximately 75% of growers indicated willingness to plant genetically modified (GM) trees if they were resistant, but 12% of these were concerned about consumer and processor acceptance, both necessary to assure their market. The most important specific concerns about GM HLB-resistant citrus in particular included, in order, (1) consumer acceptance, (2) processor acceptance, (3) production issues, (4) cost of acquiring and planting the new trees, (5) safety issues related to the origin of the added genes, and (6) environmental concerns (Singerman and Useche, 2017).

On the consumer side, House (2017) conducted three online surveys (2012, 2013, and 201620) of the general public about their attitudes toward genetic modification technology. Consumers indicated concerns about its use, particularly in foods such as fruits and juices. Asked about hypothetical scenarios involving their attitude toward purchasing GM foods, a small proportion of respondents indicated that they would not purchase them at all. Most, however, were willing to purchase GM foods if they were priced at a significant discount. The amount of the discount required was influenced by factors such as (1) the type of food, (2) the country from which the technology originated (they were more likely to accept technology from their own country), (3) the type of institution from which the technology arose (academia, large industry, or government), and (4) the rationale for using genetic modification technology (“saving an industry” was partial justification but did not completely overcome their objections).

Ironically, the loss of a portion of consumers due to genetic modification concerns would add to an already-lowered consumption of citrus juices that is due in part to public concerns about its sugar content and in part to the decreased production of fruit for juice in Florida, which is outpacing the decline in consumption (Zansler, 2017).

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19 This survey was not funded by CRDF.

20 These surveys were not funded by CRDF.

Suggested Citation:"3 HLB Research and Development Efforts." 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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OVERALL FINDINGS, CONCLUSIONS, AND RECOMMENDATIONS

Finding 3.1: CRDF support for the multiple HLB research projects is responsive to several recommendations contained in the NRC 2010 report, particularly to 8 of the 11 near- to intermediate-term (NI) research and technology recommendations:

  • NI-1: Improve insecticide-based management of Asian citrus psyllid
  • NI-4: Accelerate the sequencing, assembly, annotation, and exploitation of a sweet orange genome to provide a powerful tool for all future citrus improvement research
  • NI-5: Support development of HLB model systems
  • NI-6: Exploit the CLas genome sequence for new strategies of HLB mitigation
  • NI-7: Support research aimed at developing alternative ACP management strategies
  • NI-8: Support small-scale studies on the feasibility of alternative horticultural systems suited to endemic HLB
  • NI-9: Support demonstration of RNA interference (RNAi) effects for possible suppression of ACP
  • NI-10: Develop in vitro culture techniques for CLas to facilitate experimental manipulation of the bacterium for insights into gene function

CRDF also funded research efforts that address three of the four 2010 NRC long-term research and technology recommendations: L-1: Support the development of transgenic HLB-resistant and ACP-resistant citrus; L-2: Support development and testing of bactericides, therapeutics, or SAR activators; and L-3: Support analysis of ACP behavior, ACP–plant interactions, and ecology to enhance the knowledge base available for new ACP management strategies.

Finding 3.2: Other agencies are funding efforts to address Recommendation NI-11: Sequence, assemble, and annotate the ACP genome to provide a basis for new approaches to ACP management as well as NI-2: Support searches for biomarkers that may be exploited to detect CLas-infected citrus, although there are CRDF-funded projects that could provide useful data leading to improved HLB detection.

Finding 3.3: Recommendation NI-3: Establish citrus orchard test plots for evaluation of new scouting and therapeutic methods remains to be addressed.

Suggested Citation:"3 HLB Research and Development Efforts." 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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Conclusion 3.3: Although CRDF supports Picos Farm, a secure transgenic field test site in Florida, additional sites are needed for assessment and validation of scouting and therapeutic approaches for HLB management with significant impacts on Florida as well as California, Texas, and Arizona.

Recommendation 3.3: CRDF should consider establishing an infrastructure enhancement project to assess field testing needs for all citrus disease and insect research and validation activities and to design plans to meet those needs by enhancing current field test sites, establishing new field test sites, and/or developing collaborations with citrus growers to use production orchards for testing.

Finding 3.4: A single breakthrough discovery for managing HLB in Florida in the future is unlikely, since intensive research efforts over almost 20 years have not led to this result.

While it is possible that genetic engineering or gene editing will lead to the availability of HLB-resistant citrus cultivars, it is likely that such cultivars will also benefit from reduced inoculum loads, ACP control to reduce vector populations, and improved cultural management practices. Currently, each of the IPM strategies tested offers a partial solution, and combining these approaches is providing the best management solution available at present.

Conclusion 3.4: Finding the best combinations of control strategies suited to different environmental and growing conditions, vector and pathogen pressure, tree cultivars, and tree health would help growers in Florida and in other states where HLB is not yet a chronic problem but soon could be, especially if HLB infection is not detected quickly and if there is reluctance to remove inoculum sources.

Recommendation 3.4: Consider specific funding for the development of sets of management approaches that can be combined in different ways, optimized, and validated for use in different locations and conditions.

Finding 3.5: Plant pathologists, sociologists, and economists are using modeling to assess the complex interactions that characterize HLB; however, no CRDF funding has directly supported research on economic and sociological factors that impact decision making and behaviors of growers, processors, and the public and can influence the adoption and success of future HLB management efforts.

Conclusion 3.5: Greater investment and researcher involvement are needed to develop and apply modeling technologies for analysis and prediction of the effects of economic and sociological factors on the acceptance and application of HLB management practices.

Suggested Citation:"3 HLB Research and Development Efforts." 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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Recommendation 3.5.1: CRDF should consider adding these research areas to its funding portfolio.

Recommendation 3.5.2: CRDF should consider creating centralized, researcher-accessible databases to support sociological and economic modeling of HLB-related research outcomes and application projections. It should support systems approaches for field testing combinations of the most promising developments in replicated field studies, emphasizing the need for collecting research data sufficient to inform and support model training and applications to effectively predict cost–benefit ratios of all HLB management strategies.

Finding 3.6: Recent and growing interest in research using genetic modification to develop HLB-resistant citrus, and concerns about stakeholder acceptance of these technologies, indicates that expanded efforts in educational outreach to growers, processors, and consumers could facilitate eventual deployment of new citrus lines. Data from previous advertising strategies directed at adjusting consumer attitudes about citrus consumption have demonstrated that such communication with the public can change behaviors.

Conclusion 3.6: Further research is needed to assess the level of current understanding of genetic modification technology, and the factors that influence their willingness to purchase genetically modified foods.

Recommendation 3.6: CRDF should consider funding research to assess consumer attitudes toward genetic modification technologies for producing HLB-resistant citrus cultivars and their willingness to consume genetically modified citrus, as well as how targeted advertising campaigns could be effective outreach strategies.

REFERENCES

Albrecht, U., D. G. Hall, and K. D. Bowman. 2014. Transmission efficiency of Candidatus Liberibacter asiaticus and progression of huanglongbing disease in graft- and psyllid-inoculated citrus. HortScience 49(3):367-377.

Ammar, E., R. G. Shatters, C. Lynch, and D. G. Hall. 2011a. Detection and relative titer of Candidatus Liberibacter asiaticus in the salivary glands and alimentary canal of Diaphorina citri (Hemiptera: Psyllidae) vector of citrus huanglongbing disease. Annals of the Entomological Society of America 104(3):526-533.

Ammar, E. D., R. G. Shatters, and D. G. Hall. 2011b. Localization of Candidatus Liberibacter asiaticus, associated with citrus huanglongbing disease, in its psyllid vector using fluorescence in situ hybridization. Journal of Phytopathology 159(11-12):726-734.

Ammar, E. D., J. E. Ramos, D. G. Hall, W. O. Dawson, and R. G. Shatters. 2016. Acquisition, replication and inoculation of Candidatus Liberibacter asiaticus following various acquisition periods on huanglongbing-infected citrus by nymphs and adults of the Asian citrus psyllid. PLoS ONE 11(7):e0159594.

Suggested Citation:"3 HLB Research and Development Efforts." 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:"3 HLB Research and Development Efforts." 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:"3 HLB Research and Development Efforts." 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:"3 HLB Research and Development Efforts." 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:"3 HLB Research and Development Efforts." 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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