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Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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4

Notable Outcomes, Pitfalls, and Future Directions

This chapter presents significant research findings and progress, as well as issues of concern, from the past decade of Citrus Research and Development Foundation (CRDF)-funded huanglongbing (HLB) research. For each research area, following the committee’s statement of task (see Chapter 1), the committee highlighted the key research findings (see Boxes 4-1, 4-3, and 4-5), indicated which 2010 National Research Council (NRC) recommendations have been addressed, discussed notable outcomes, and reviewed factors that hamper or present challenges (pitfalls) to the work. The committee also provided recommendations and considerations for future research (see Boxes 4-2, 4-4, and 4-6 and Future Directions subsections) for each of the research areas. However, the committee was unable to define further the extent of research progress or to comment more specifically on the selection of research areas to be continued or discontinued because information available to it on research outcomes, applications, and impacts was insufficient to do so in many cases, particularly for recent projects. At the end of this chapter are the committee’s overarching findings and its conclusions and recommendations for future research efforts in HLB management. The conclusions and recommendations are based upon the information available to the committee from peer-reviewed journal articles whenever possible; other sources included non-peer-reviewed publications, such as trade magazines, conference abstracts, presentations at forums of this committee, and progress reports and final reports from CRDF research.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×
Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×
Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

BIOLOGY AND ECOLOGY OF THE HLB CAUSAL OR ASSOCIATED PATHOGEN AND ITS INSECT VECTOR

Candidatus Liberibacter Species

Notable Research Outcomes

Genome Sequencing, Bioinformatics Analysis, and Genome-Enabled Distribution and Diversity Studies. The completion of the Candidatus Liberibacter asiaticus (CLas) genome in 2009 (Duan et al., 2009) was a major achievement that enabled new research directions in diagnostics, culturing, pathogenesis mechanisms, and bacterial control. Relevant to 2010

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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NRC report Recommendation NI-6: Exploit the CLas genome sequence for new strategies for HLB management, genomic analysis has led to the identification of a variety of candidate proteins (i.e., virulence or other essential proteins) that could be targets for disease mitigation strategies. Many of these genes, some of which have significant effects on plants, have now been cloned and functionally characterized in other organisms. Sequencing of numerous CLas isolates in parallel revealed which candidate genes are highly conserved among strains, allowing researchers to screen out less desirable control targets. The genomes of the additional HLB-causing species Candidatus L. americanum and Ca. L. africanum (Wulff et al., 2014), and of Liberibacter species that cause other diseases were also sequenced. Genomic

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

comparisons among these, and between them and the genomes of related free-living species, including a culturable, nonpathogenic Liberibacter strain, have led to the identification of numerous essential functions likely lost in CLas, those retained, and a few properties predicted to be unique to CLas (Hartung et al., 2011; Kuykendall et al., 2012; Leonard et al., 2012).

The completion of the CLas genome led to the development of several variable genetic markers to improve global surveillance and expand knowl-

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

edge of HLB epidemiology, allowing the rapid differentiation of CLas from different continents (Chen et al., 2010; Katoh et al., 2011; Islam et al., 2012). Advances in sequencing technology also provided a reference for sequencing of nine additional publicly available genomes1 of CLas from infected citrus samples, and roughly 30 more isolate genome sequences are slated for publication in the near future (Duan, 2017). Comparative studies revealed thousands of points of genetic variation among sequenced CLas genomes, revealing new U.S. phylogenetic groupings as well as the presence of mixed CLas populations within a single host plant (Duan, 2017). This information will serve as a resource for tracking the basis and origin of future changes in the location, host range, and severity of HLB.

Functional Comparative Genomics and the Identification of Infection-Associated Genes and Proteins. CLas proteins required for host invasion could potentially be targeted for inactivation through targeted treatments. Analysis of the 1,136 predicted protein-coding genes in the CLas genome revealed numerous candidates for involvement in plant infection based on similarity to known pathogen proteins. Predicted virulence function of several CLas genes, including efficient scavengers of critical plant defense signals, was validated by expression analysis or study of gene function in other organisms (Jain et al., 2015; F. Li et al., 2017). The genome-enabled ability to analyze CLas gene expression in the plant and vector led to the identification of gene activation patterns and regulatory elements in the bacterium (Yan et al., 2013). A LuxR receptor of an unknown citrus signal increased CLas symptoms in transgenic citrus (Gabriel, 2017), and the LdtR transcriptional regulator was shown to be a master regulator of global gene expression, including that of the stress-response gene LotP (Pagliai et al., 2014, 2017; Loto et al., 2017). The genome also revealed that CLas likely has a functional Type II secretion system for transporting proteins from the bacterial cell to the extracellular environment. Sixteen genes encoding potential secreted proteins were identified based on predicted secretion signals,2 and their subcellular localization was characterized in a tobacco leaf expression system (Pitino et al., 2016). One effector induced starch accumulation in tobacco cells, suggesting that this protein could cause the starch accumulation that accompanies symptoms in citrus (Duan, 2017). The protein also triggered cell death in tobacco, leading to the cloning of a tobacco resistance gene that represents the first plant gene encoding resistance to a CLas-secreted element (Duan, 2017).

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1 National Center for Biotechnology Information (NCBI); https://www.ncbi.nlm.nih.gov. Accessed January 23, 2018.

2 A secretion signal is a hydrophobic motif or peptide component at the N-terminus of a newly synthesized protein that directs it to the outer membrane for secretion.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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 Toward Culturing CLas. Many of the advances in understanding described above are still short of being confirmed because the obligate nature of CLas does not allow its phenotypes to be observed directly. The ability to culture CLas sustainably would be a major boon to long-term HLB research and control efforts, allowing dissection of pathogen behavior and facilitating work requiring plant and insect inoculations. Past culturing projects took an iterative trial-and-error approach, initially with plant- or insect-derived culture materials and later informed by improved models of CLas metabolic requirements. Groups have reported progress in the form of growth or prolonged viability of CLas using citrus-based media (Sechler et al., 2009; Parker et al., 2014) or co-cultivation with other bacteria (Davis et al., 2008), with viability lasting for up to 18 days. These outcomes provide a benchmark for progressive improvement.

The identification of the culturable L. crescens has provided an important model system for functional characterization of genes identified in CLas (Recommendation NI-5: Support development of model systems), and tobacco- and periwinkle-based experimental systems have also been extremely useful for bacterial characterization and proxy screening. L. crescens shares 75% genetic identity with CLas (Leonard et al., 2012), and the 76 L. crescens-specific genes essential for culturing are a focus of interest in current CLas culturing efforts (Lai et al., 2016). Other developments include the optimization of protocols to use insect biofilms as a source of culture inoculum and the use of microfluidic devices to replicate the phloem flow environment (Gabriel, 2017). Currently funded efforts to identify possible new medium ingredients that will increase the span of viability (stated goals of the two recently funded National Institute of Food and Agriculture [NIFA] projects) include 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. Thus, culturing projects are designed to yield a wealth of biological information about the vector and pathogen, which may assist strategies other than culturing.

Understanding Antagonistic Phage and Bacteria. The discovery of a probable role of bacteriophage in CLas growth and fitness was an important research contribution suggesting another potential strategy for HLB mitigation. Phage dynamics could impact CLas growth in different host contexts; phage particles were observed microscopically in periwinkle (Catharanthus roseus) phloem, suggesting that they could confer bacterial lysis in this nonhost plant (Zhang et al., 2010). Accordingly, phage lytic genes that were suppressed in psyllid-associated CLas are activated only moderately in citrus-associated CLas, but highly activated in the nonhost periwinkle (Zhang et al., 2010; Fleites et al., 2014; Jain et al., 2015). Understanding the bacterial stresses that cause phage activation could be a route to activating a “self-destruct” mode in CLas or to suppressing it to

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

enhance culturability. On the other hand, several researchers have investigated the potential for CLas phages to be used as a disease management approach (see section on Bacterial Control on page 149 in this chapter).

Impacts of the Citrus Microbiome. Whole-microbiome profiling studies have shown that levels of CLas and other bacteria abundant in the citrus microbiome fluctuate seasonally (Zhang et al., 2013a), that HLB infection is associated with an increase in xylem-inhabiting Methylobacterium spp. (Zhang et al., 2013b), and that HLB decreases the ability of citrus roots to recruit bacterial species from the rhizosphere (Zhang et al., 2017). HLB management treatments such as thermotherapy and antibiotics also exhibit significant effects on microbial composition (Zhang et al., 2013b; Yang et al., 2016). The effects of these changes on disease progression are still unknown. Other microbiome research has focused on the role of disease-controlling properties of citrus microbiome communities, or of particular microbial species, as potential approaches to HLB management. These efforts are described further in the Bacterial Control section of this chapter.

Pitfalls

Due to the continuing inability to culture CLas, the promising target candidate genes revealed by genomics have been characterized in an indirect manner through heterologous expression in other organisms. The 2010 NRC report recommended supporting research into in vitro culture techniques to allow completion of Koch’s postulates and to facilitate genetic studies (Recommendation NI-10: Develop in vitro culture techniques for CLas to facilitate experimental manipulation of the bacterium for insights into gene function). While this area was an intensive focus of CRDF funding related to CLas, the goal of sustainable culturing has remained elusive. Genome-enabled metabolic reconstruction and comparison with culturable Liberibacter have shed light on numerous missing metabolic and culturability genes. In addition to the inability to synthesize critical substrates of primary metabolism, isolated CLas may also require its environment to detoxify methylglyoxal (the non-enzymatically produced byproduct of glycolysis), suppress internal phage, and provide physical and chemical regulatory signals (Project #FLAW-2015-104913). The NIFA Specialty Crop Research Initiative (SCRI) has recently taken over most of the funding of CLas culturing efforts with two large multilaboratory projects.

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3 Project information available at https://citrusrdf.org/wp-content/uploads/2017/06/Cycle-2-2016-NIFA-SCRI-HLB-Project-full-abstracts-CRIS.pdf. Accessed January 23, 2018.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

Future Directions

Understanding the bacterial processes occurring during host–vector interactions has progressed substantially for an unculturable bacterium, resulting in the identification of many known or predicted critical CLas genes. Multiple coordinated projects are now working to validate the function of these genes and to screen for or design interactors or suppressors. In the coming years, these projects could yield technological strategies for bacterial suppression through chemical controls or through citrus gene-editing/transgenic approaches. While any bacterial-targeting approach would ideally be multipronged to avoid evolution of bacterial resistance, the process of advancing each strategy through translation and field evaluation steps will be time consuming and expensive. Although this research is still in early stages, the research community could prepare to maximize funding efficiency by enhancing community coordination on selecting and advancing bacterial targeting strategies as they pass proof-of-concept stages. This process would benefit from the establishment of common benchmarks for evaluating and reporting research outcomes. Continued communication should take place with industry as to which strategy outputs would be preferable to growers and consumers (i.e., synthetic genes,4 transgenes from other plants, or transgenes from other organisms), possibly through the publication in appropriate trade journals of articles describing research outcomes, targeted to growers and the lay public.

The research focus on genome sequencing (Recommendation NI-6: Exploit the CLas genome sequence for new strategies for HLB management) has provided not only an understanding of bacterial genes but also a picture of CLas evolution and community structure in hosts—knowledge important for selecting effective control strategies. Sequencing costs have fallen substantially since 2010 and continue to do so. While whole-genome sequencing is unlikely to yield additional returns in control targets, it will continue to be a cost-effective means of tracking the genetic changes in CLas and associated phages as the bacterium moves into new areas and evolves. Some sequencing efforts have also included taxonomic profiling of the citrus bacterial microbiome or “phytobiome” associated with HLB citrus, identifying some HLB-associated changes and isolating potential antibacterial microbes. The phytobiome is increasingly recognized to play an important role in plant disease (Beattie et al., 2016), and it is possible that other microbes provide signals needed for virulence and transmission of CLas. However, there are few examples of successful biological control against tree pathogens (Cazorla and Mercado-Blanco, 2016), and it is still unclear whether plant community profiling efforts could be translated realistically to HLB control.

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4 Genes constructed artificially from oligonucleotides by chemical means.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

Several research groups have long worked toward the difficult goal of culturing CLas, supported by over $8 million of CRDF and NIFA SCRI funding. Having a singular end goal of maintenance in culture has led to some overlap in specific aims between groups. The absence of means for publishing negative data resulted in there being relatively few publications from these projects, which may have made it difficult for researchers to learn about the many culturing methods that were attempted but unsuccessful. The recent consolidation of funding by NIFA SCRI into two large culturing projects, both with rational and stepwise aims, goes far to address overlap and communication issues and should yield publishable data regardless of culturing outcome. Still, CLas metabolic, detoxification, and regulatory needs are now predicted to be much more complex than anticipated. Future CLas culturing projects might have complex outcomes; for example, culturing methods could require materials and equipment that would be so cost prohibitive as to greatly limit their use. Or, CLas may be obtainable in culture, but extremely difficult to manipulate genetically or to inoculate into vectors and hosts (a large part of the rationale for culturing). While current CLas culturing efforts will continue at peak levels until the end of their funding term (2020), the grower and research communities should evaluate whether to advocate for continued efforts if sustained culture is not attained by that time, taking into account the advances that have been made possible in the absence of culturing. The use of microbiome profiling of diseased and healthy citrus, while providing relevant basic information about the phytobiome, is at this time a developing science that is likely to offer greater returns in the future than it does at present.

HLB Vector: Asian Citrus Psyllid

Notable Research Outcomes

CRDF has supported fundamental investigations into the biology of the Asian citrus psyllid (ACP), especially as it has pertained to vector competence and management of the citrus greening pathogen. Moreover, CRDF has made efforts to implement the findings in terms of the 2010 NRC report (NRC, 2010), with particular emphasis on support of “near- and near- to intermediate-term” recommendations.

ACP Genome Sequencing and Applications of Sequence Information to HLB Management. Progress has been made to address Recommendation NI-11: Sequencing, assembly, and annotation of the ACP genome to provide a basis for new approaches to ACP management. Although support for this approach was only partially funded by CRDF, outcomes have been generated from a combination of these projects along with projects from U.S. Department of Agriculture (USDA) funding sources intended to

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

provide information on which to base future strategies for ACP suppression of or interference with CLas transmission. The first genome draft of ACP was completed initially in 2011 (Hunter and Reese, 2014) and has more recently been amended through support from the Los Alamos National Laboratory, New Mexico, and USDA NIFA. The revised draft genome and transcriptome were assembled and submitted into the public domain at the National Center for Biotechnology Information (NCBI), and genomic resources are currently available from the Genome Sequencing Project5 and the I5K arthropod genome project6 and CitrusGreening Solutions.7 The ACP transcriptome effort has identified over 25,600 predicted genes, supported by an additional 19,598 previous expressed sequence tags, and transcripts were identified for specific life stages, including adults, nymphs, and eggs. The NIFA-funded CitrusGreening Solutions initiative hosts a comprehensive, publicly available website8 that provides a variety of genome resources for data integration, annotation, and other analyses for ACP as well as CLas and citrus.

RNA Interference (RNAi) for Possible Suppression of ACP. The availability of transcriptome data has aided in efforts to develop RNAi targets in ACP (Recommendation NI-9: Support demonstration of RNA interference [RNAi] effects for possible suppression of ACP), and several researchers have used these resources toward the development of strategies to suppress ACP populations using the Citrus tristeza virus (CTV) vector to deliver RNAi molecules into ACP (Hajeri et al., 2014). RNAi, a process in which double-stranded RNA (dsRNA) exerts a silencing effect on complementary messenger RNA (mRNA), has emerged as a promising research tool following functional genomic analyses, and transgenic citrus lines expressing RNAi for ACP control are in the developmental stages (Stover, 20159).

Ease of delivery, high specificity of gene targets, and a lack of environmental persistence are among the benefits of RNAi approaches for crop protection. Recent functional genomic analyses of RNAi-related genes in the ACP genome showed an absence of sequences encoding a dsRNA-binding protein that functions as a cofactor of Dicer (Taning et al., 2016). Since RNAi is an efficient process in ACP after oral delivery of dsRNA, the absent R2D2 cofactor may not be necessary for this process in ACP (Taning et al., 2016). Presumably, a different dsRNA-binding cofactor may compensate for the absence of R2D2 in the ACP. Nevertheless, recent bioassays using both plant-launched systems and feeding delivery have shown that

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5 See http://www.ncbi.nlm.nih.gov/bioproject/PRJNA29447. Accessed June 8, 2018.

6 See https://i5k.nal.usda.gov/Diaphorina_citri. Accessed June 8, 2018.

7 See www.citrusgreening.org. Accessed June 8, 2018.

8 See www.citrusgreening.org. Accessed June 8, 2018.

9 Unpublished reports from CRDF project principal investigators (PIs) are available upon request from CRDF. Accessed June 8, 2018.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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 is very sensitive to ingested dsRNA, demonstrating a strong RNAi response. Small doses of dsRNA (arginine kinase and superoxide dismutase), administered at the time of citrus flush, were sufficient to trigger the RNAi mechanism, causing significant suppression of the targeted transcript, and increasing psyllid mortality (Andrade and Hunter, 2017). Additional studies provide evidence of the functional RNAi machinery present in ACP, which could be further exploited through future research to develop RNAi-based management strategies for the control of ACP (Christiaens and Smagghe, 2014; Killiny et al., 2014a; Taning et al., 2016).

Dispersal Dynamics of the Asian Citrus Psyllid. Understanding of dispersal behavior has been enhanced by CRDF funding. Peak periods of ACP activity in Florida occurred during the spring and summer, whereas activity was lower during the cooler fall and winter seasons (Martini et al., 2014). As reported previously, adult ACPs dispersed relatively long distances (up to 2 km) and the number of immune-marked adults captured increased with “spring-flush” activity in managed groves. When patterns of flight initiation and duration were investigated, nearly one-third of blue ACP morphotypes (having green-blue abdomens) exhibited longer durations of flight, whereas fewer than 5% of gray morphotypes (having gray-brown abdomens) did so. Gray morphotype adults had shorter wings and smaller pronotal thoracic plates than did the blue morphotypes. Furthermore, ACP body size, wing width, and wing aspect ratio differed based on the host plant species on which they were reared, impacting dispersal (Paris et al., 2016). Finally, increases in ACP flight activity corresponded to times of increasing barometric pressure (Martini and Stelinski, 2017). As previously documented, increases in ACP activity and dispersal were also correlated with increasing temperature, but no relationships were observed with relative humidity, whether static or variable. Changes in pressure did influence the responsiveness of ACP, and the magnitude of these impacts corresponded to the degree of overall pressure swings (Paris et al., 2017). Cultural factors and practices, such as row orientation, significantly influenced psyllid density during the winter, and ACP abundance was higher when more than 20% of the surrounding landscape was urbanized (Pelz-Stelinski et al., 2017).

Infection Status and Psyllid Dispersal. When ACP were allowed to develop through immature stadia (stages between successive molts of an insect) on CLas-infected plants, short-distance dispersal of male ACP was greater than that of male counterparts reared on uninfected plants (Martini et al., 2015). The initiation and duration of flight by CLas-infected ACP occurred earlier and for longer time periods than for those of uninfected psyllids. Moreover, the titer of CLas, measured by quantitative polymerase chain reaction (qPCR), was greater in ACPs that performed long flights on the experimental flight mills than in presumably noninoculative psyllids, which flew for shorter periods. Finally, adult female psyllids containing

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

higher titers of CLas were regarded as more attractive to male psyllids than females having lower CLas titers, suggesting that the bacterial pathogen may influence both movement and mate finding of the insect vector.

Determinants of Host Location and Vector Fitness. Increased levels of leaf nitrogen resulting from applications of plant growth regulators (PGRs) to container-grown citrus trees resulted in higher ACP reproductive rates, shorter development time, and larger body mass (Tsagkarakis et al., 2012). Psyllids reared on trees treated with selected PGRs exhibited significant reductions in fecundity, survivorship, and oviposition rates. In general, oviposition occurred later on PGR-treated trees than on untreated controls. The responses of ACP to the selected PGRs were apparently not influenced by access to suitable oviposition sites, nor by direct toxicity of the PGRs, but rather to induced plant biochemical changes that altered host plant quality. Analyses of leaf nutrients and photosynthesis showed no correlation between plant nutrient status or carbon assimilation with changes in psyllid behavior, although it was hypothesized that other changes could have occurred that were not detected in their whole-leaf analyses.

Increasing the Efficiency of ACP Monitoring. Investigations of psyllid vibrational communications in mate attraction (Mankin et al., 2013) revealed that broadcast mimic calls elicited strong female responses comparable to those elicited by a recorded male call, suggesting the potential to develop systems for increased trapping efficiency. Spectral and temporal patterns of calling pairs (duets) have been mimicked using computer-assisted vibrational tools. Other research relevant to increasing ACP monitoring effectiveness is provided in the section Vector–Host Interactions in this chapter.

Pitfalls

A recommendation of the 2010 NRC report was Recommendation NI-1a: Develop new methods to fine-tune field surveillance of ACP via more efficient and consistently applied trapping or other methods, to improve timing and targeting of insecticide applications. CRDF has supported projects to investigate novel trapping techniques together with compounds that could be useful in enhancing trap capture efficiencies. Unfortunately, research has not yet led to standardization of trap selection and optimization of trapping approaches. Populations of ACP continue to be monitored with standard yellow sticky cards, which are not ideally suited for recovery of ACP DNA for CLas detection, and which continue to be collected and replaced according to protocols that differ in different regions of the United States.

Seasonal flight activity of the ACP has been investigated using yellow sticky traps placed in citrus groves and adjacent fallow areas (Hall and Hentz, 2011). These studies have demonstrated that flight can occur at

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

any time of the year, but captures were more consistent during the spring flush of citrus. Except for a modest relationship with relative humidity, the activity of captured adult ACP could not be correlated with any abiotic factor or set of factors, including wind speed, sunlight, and air temperature. Trap color had no influence on the efficiency of ACP captured throughout the citrus production season. Comprehensive field trials over several weeks revealed that three-odor-lured yellow traps caught ~230% more ACP over time than solvent-control yellow traps placed on the same trees (Coutinho-Abreu et al., 2014). Unfortunately, the data from these individual investigations have revealed little about the principal factors that help to explain patterns of ACP capture.

Future Directions

Extensive ACP capture investigations have been supported by CRDF in the past decade. Additional formal efforts to standardize trap types across producers, government agencies, or crop consultants across broader geographic regions are probably not needed at this time, as the various yellow sticky panel traps now available capture adult ACP with similar efficiencies. However, new research is needed to improve the ability to extract DNA from trap samples for use in a number of research areas, and data management and informatics approaches could be used to capture additional value from data collected over years of trapping studies. It would be beneficial to collect and collate the extensive ACP capture data over several locations and years of investigation. Making these digital and georeferenced data more accessible to communities capable of utilizing advanced computational power and sophisticated algorithms for the analysis of large datasets would provide greater opportunities for predicting the complex behavior and patterns of capture of the ACP, especially in agricultural systems. If researchers and funding agencies are to contribute to realizing that promise, however, there is an immediate need to develop research methods that can resolve patterns from these large datasets to aid in limiting risk for producers. Agricultural informatics can be viewed as an offshoot of big data, whereby insights into integrated pest management (IPM) are realized by integrating multiple data streams to create a composite dataset for analysis. This approach can be particularly attractive for researchers wishing to investigate insect or disease management processes that occur at very large spatial or temporal scales not easily investigated through more traditional experimentation. Importantly, agricultural informatics methods are often best used in combination with hypothesis-driven experimentation, and together these can strengthen the potential to draw causal relationships (Boyd and Foody, 2011) as well as to lead to tangible management outcomes.

While researchers and grove managers consider patterns of citrus flush

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

to be a primary cue that triggers ACP movement within groves, the interacting abiotic (or biotic) conditions that would help to describe actual population trends and inform the development of areawide management strategies have defied characterization to date. A more comprehensive ACP trap capture dataset across broad geographic regions in each year would be very important for observing long-term trends and examining their relationships to environmental factors. As discussed in the 2010 NRC report, surveillance should not be limited to commercial orchards but should also include urban landscapes and declining or abandoned groves to identify local ACP populations for treatment before they disperse.

Past and current research, described earlier (Notable Research Outcomes), has shown that novel pest management technologies, such as RNAi, have the potential to provide new strategies for ACP management (Meister and Tuschl, 2004), and research to develop such approaches should be continued.

INTERACTIONS OF THE HLB PATHOGEN, VECTOR, AND HOST

Vector–Host Interactions

Notable Research Outcomes

Host Range of ACP. In screening potential ACP rutaceous hosts, variation in suitability was documented and in some cases was determined to be a heritable trait. Poncirus trifoliata (hardy or trifoliate orange), which is resistant to ACP (Richardson and Hall, 2013; Hall et al., 2015), can hybridize with sweet orange to produce “citranges.” Lower levels of attack by ACP on P. trifoliata appear to result from reduced oviposition. Unlike citrus, P. trifoliata is winter dormant and deciduous, lacking foliage through the winter. In early spring, while P. trifoliata remained in winter dormancy, Citrus and citrange hybrids were producing shoot flushes and supporting populations of ACP. Even after flush shoots were produced on P. trifoliata later in the spring, fewer eggs were laid on this species than on Citrus (Hall et al., 2017). Hybrid citrange cultivars tested, however, lacked resistance to ACP.

A number of native rutaceous host plants were ruled out as alternative hosts outside of managed groves and hence could be considered unlikely reservoirs for psyllid populations (Sétamou et al., 2016); in fact, no alternative hosts capable of maintaining ACP feeding or reproduction during leaf flushes were found, at least in central Florida. In contrast, abandoned groves were identified as important reservoirs for ACP and pathogen populations.

CRDF funding supported work showing that host plant identity influences ACP dispersal potential through changes in wing aspect ratio (Paris et al., 2016). Although all citrus hosts supported ACP development through

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

adulthood, those reared on Citrus taiwanica were smaller and had shorter tibial leg segments, than adults reared on other host plants. Wing aspect ratios varied, with those of ACP reared on Murraya paniculata narrower than those of ACP reared on other hosts.

Host Orientation, Assessment, and Acceptance. Among the most notable achievements over the past decade with respect to vector–host interactions were characterizing ACP behavior and ACP–plant interactions and ecology (Recommendation L-3: Support analysis of ACP behavior, ACP–plant interactions, and ecology to enhance the knowledge base available for new ACP management strategies).

CRDF supported, in part or in toto, research that identified a diversity of signaling modalities associated with host finding and host assessment by ACP. Chemical cues that influence ACP orientation to citrus were investigated by George et al. (2016) and Lapointe et al. (2016), who described chemical blends eliciting probing. In terms of responses to visual signals, in both the field and laboratory, attraction to visual (color) cues varied with time of day, peaking in the afternoon; in the laboratory, ultraviolet (390 nm), green (525 nm), and yellow (590 nm) light elicited the strongest phototactic responses (Paris et al., 2015). The pronounced preference for yellow exhibited by ACP is consistent with orientation behavior of many other phytophagous insects, including other psyllids, and may relate to host leaf reflectance. Flush leaves from HLB-infected plants have higher reflectance in the green and yellow regions of the spectrum than do flush leaves from uninfected plants, which may facilitate orientation to diseased plants by ACP. To some extent, responses to visual stimuli, like responses to olfactory stimuli, are influenced by sex and experience of adults (Stockton et al., 2016).

CRDF-funded research also revealed that multiple forms of sensory stimuli associated with mate finding and reproduction influence host feeding. Documentation of the details of acoustic communication, particularly vibrational duetting, inspired new approaches to ACP trapping involving vibrational signals (Mankin et al., 2015), although they have not yet been field tested. In terms of olfactory stimuli associated with host finding, chemical signals mediating courtship and mating were found to originate in both the insect, in the form of cuticular hydrocarbon constituents, and the host plant. With respect to gustatory stimuli, the identification of phagostimulants that elicit probing behavior provide a foundation for developing attract-and-kill products, which have not yet been optimized for field use (George and Lapointe, 2017). Another important outcome of a range of studies was evidence of effects of experience on many behaviors involved in both mate and host finding and assessment. The finding that ACP nymphs acquire CLas quickly after probing (Ramsey et al., 2015; Pelz-Stelinski and

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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Killiny, 2016) has contributed to appreciation of this life stage as a significant factor in disease epidemiology.

Timing of Attack. Citrus leaves are particularly attractive to ACP during times of flush. Using protein-marked adults, Lewis-Rosenblum et al. (2015) identified leaf flush abundance and availability as primary factors driving dispersal and demonstrated that adult ACP can move 2,000 m or more in under 2 weeks to find new leaf flushes. Movement of marked individuals captured into a central test area of sweet orange trees increased in proportion to the density of emerging young leaves on the trees. The results confirm the threat presented by abandoned citrus groves in Florida by virtue of their status as reservoirs for ACP, which can disperse across long distances despite geographical barriers when leaves flush.

ACP nymphs settle and feed preferentially on the abaxial (lower) side of young leaves on the sides of the midrib, whereas adults settle and feed on adaxial (upper) and abaxial surfaces of either young or old leaves (Ammar et al., 2013). The feeding preference of nymphs is consistent with constraints imposed by morphology; phloem tissue located in young leaves and on the sides, rather than the center, of the midrib is a shorter distance from the leaf surface and thus more easily reached with short mouthparts. As for adults, phloem tissue in mature leaves is surrounded by a thick fibrous ring of sclerenchyma. ACP feeding behavior on young and mature orange leaves, investigated using electrical penetration graph technology (George et al., 2017), showed that, while adults feed on both young and mature leaves the duration of phloem ingestion is shorter on mature leaves; this pattern is consistent with the role of the sclerenchymatous ring as a barrier to feeding in ACP and provides a potential explanation for the vulnerability of young leaves to ACP attack.

In support of the 2010 near-term Recommendation NI-7: Support research aimed at developing alternative ACP management strategies, effects of abiotic factors such as temperature, humidity, wind speed, and barometric pressure on flight and dispersal behavior were documented, in some cases by novel marking methods; these findings have potential applicability for increasing the efficiency of management strategies. Relevant to ACP–host interactions was the finding that windbreaks are significant factors in ACP host finding; fewer psyllids infest edges of groves having windbreaks compared with groves lacking them. As well, replanting patterns influence host finding and infestation rates; ACP were more abundant in groves with solid set replanting compared with resets (trees planted within mature groves to replace dead or infected trees) (Martini et al., 2015).

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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Pitfalls

Unfortunately, virtually all new knowledge gained over the past decade about ACP interactions with citrus hosts has served to underscore the biological recalcitrance of the system to management. Psyllid treatments began in Florida in 2008. Early on the psyllid was not considered a major risk to citrus, but now, a decade later, essentially all citrus groves are infected, and most psyllids carry CLas (Coy and Stelinski, 2015; Stelinski, 2017). The variability of the behavior and ecology of ACP has slowed progress in developing effective and reliable management techniques. Unlike the case for many other pest insects, vibrational, volatile, and phagostimulatory cues influencing ACP courtship and mating are effective over only short distances and thus unlikely to be useful for trapping or monitoring. Moreover, although hundreds of compounds have been screened for ACP attractant or repellent activity, responses to chemical signals, in the context of both mate and host assessment, appear to be labile and subject to influence by prior experience and possibly learning. Similarly, with respect to host finding, although ACP responds to visual signals, including color, responses vary with the developmental state of the host. Any signal modality that elicits responses in laboratory conditions tends to be overwhelmed under conditions of leaf flushes of new growth and, at least in central Florida, flushing occurs continuously through the growing season. Moreover, the health status of the host tree affects attraction, with volatiles produced by infected trees altering relative ACP preferences and possibly accelerating pathogen spread (Mann et al., 2012). Finally, flight activity is influenced by temperature, barometric pressure, and light, even over the course of a single day, introducing variability into the reliability and reproducibility of monitoring by trapping and constraining the development of predictive models. The extreme variability of ACP behavior relative to its interactions with host trees is largely responsible for the fact that, to date, no reliable correlation has been found between trap capture and absolute densities of ACP.

In some ways, the interactions between ACP and CLas thwart conventional approaches to management. As described elsewhere in this report, defining host resistance to HLB is operationally difficult because a reduction in psyllid number is not necessarily reflected by a decrease in disease incidence. Nymphs can acquire the bacterium quickly and, due to their mobility and feeding behavior, they are particularly difficult to control. In Florida, disease spread has outstripped the pace of research aimed at managing psyllids; reducing psyllid numbers by any available means is best considered a stopgap, delaying the spread and reducing multiple infections until the disease can be cured or until disease-resistant citrus trees become commercially viable.

Another pitfall in efforts to combat HLB, not explicitly considered in

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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 2010 NRC report, is human behavior. Groves abandoned for economic reasons support psyllid populations that can reinfest managed groves and circumvent control efforts. Also not directly addressed in the 2010 NRC report was the willingness of consumers to embrace the release of insects altered by new gene editing or modification technologies into the environment, a concept that falls under Recommendation L-4: Explore possible control strategies based on release of modified psyllid males. Even under conditions in which public benefits of such releases are more far reaching and immediate, as in the case of vectors of human pathogens (Ernst et al., 2015), public resistance has been encountered.

Future Directions

Long-term solutions for HLB are likely to be in the areas of citrus variety improvement resulting from new technology. Thus, Recommendation L-1: Support the development of transgenic HLB-resistant and ACP-resistant citrus from the 2010 report remains a goal. Although genetic traits for ACP resistance in Citrus and related genera have been identified, neither traditional plant breeding nor cutting-edge techniques for citrus germplasm improvement have advanced to the point of practicality.

Goals for future research should include significant improvements in genetic transformation, clustered regularly interspaced short palindromic repeats (CRISPR) technology, and transient expression breeding in citrus. The sequencing of the ACP genome has provided new targets for manipulation, including candidate genes associated with host finding (e.g., chemo-receptors for volatile and gustatory stimuli) or feeding efficiency (e.g., salivary sheath formation). The olfactory receptor co-receptor (Orco) gene in other insects has been a target for CRISPR-associated protein-9 nuclease (CRISPR-Cas9) editing (Sun et al., 2017). As well, the availability of the ACP genome will facilitate the exploitation of the CYPome (the genomic inventory of detoxification genes in the cytochrome P450 monooxygenase superfamily) to interfere with growth and development, targeting genes encoding enzymes that detoxify both phytochemical and pesticide toxins. Tiwari et al. (2010, 2011a,b,c,d) documented stage-specific differences in activity in both cytochrome P450 monooxygenases and glutathione-S-transferases; transcriptomic analysis across developmental stages could aid in identifying suites of genes associated with xenobiotic metabolism,10 and characterizing the specific chemical substrates for these differentially expressed genes could provide new targets not only for counteracting insecticide resistance but also for interfering with host use efficiency by

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10 A set of metabolic pathways that modify the chemical structure of xenobiotics, which are compounds foreign to an organism’s normal biochemistry, such as any drug or poison.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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developing inhibitors to block metabolism of host plant phytochemicals. Other future research directions are in the realms of sociology and economics, with the goal of identifying obstacles to grower participation in area-wide management efforts and public acceptance of management tactics. These sociological and economic considerations are addressed in more depth in Chapter 3.

Pathogen–Host Interactions

Notable Research Outcomes

Identification of Pathogenesis Factors Involved in Pathogen–Host Interactions. The NRC (2010) recommended NI-6: Exploit the CLas genome sequence for new strategies of HLB mitigation. Relevant projects funded by CRDF and other funding agencies have significantly advanced the basic understanding of pathogen–host interactions in HLB. Systematic and comparative analyses among genomes of CLas and other Liberibacter spp. strains resulted in the identification of a number of pathogenicity-implicated Liberibacter effectors derived mainly from type I and general secretory systems (T1SS and Sec, respectively). Using bioinformatics tools and seven well-annotated Liberibacter genome sequences, varying numbers (133 to 214) of proteins in those genomes were predicted to contain Sec-dependent signal peptides (Prasad et al., 2016). Of a total of 166 such predicted proteins from one CLas strain (Las-psy62), 86 were experimentally confirmed, using the Escherichia coli-based PhoA assay to measure acid phosphatase activity, to be secreted into the extracytoplasmic milieu. Due to the conserved nature of the Sec system among microbes, it is likely that those effectors are also translocated into CLas extracytoplasmic spaces and interact with host components and biological processes (Prasad et al., 2016). Pitino et al. (2016) examined 16 putative CLas effector genes for their subcellular localization and host cellular responses in the nonhost plant Nicotiana benthamiana through ectopic expression. These effectors exhibited diverse subcellular location patterns when fused with green fluorescent protein reporter genes under the strong promoter CaMV 35S. One of them, Las5315mp (mature protein), induced hypersensitivity (rapid cell death at the infection site) and associated plant defense responses (Pitino et al., 2016). J. Li et al. (2017) have identified a CLas effector that functions as a salicylic acid (SA) hydroxylase. The encoded protein (SahA) enzymatically degrades SA and its derivatives. Ectopic expression of SahA in tobacco plants abolishes SA accumulation and consequently interferes with hypersensitive response caused by nonhost pathogens. CLas infection enhances citrus susceptibility to citrus canker and counteracts disease resistance induced by exogenous application of SA, a result attributable

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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to SA degradation by SahA. When SA analogs BTH and INA were used to treat HLB, significant reduction of disease severity and pathogen growth were observed, suggesting the possibility of using SA analogs to control HLB (J. Li et al., 2017). An effector protein (SC2-gp095) encoded from a CLas prophage, shown to be a reactive oxygen species (ROS) scavenging peroxidase, may be implicated in scavenging host-generated ROS and suppressing ROS signaling events during CLas infection (Jain et al., 2015). Through computational analyses of the CLas proteome, Cong et al. (2012) curated all CLas proteins having predicted subcellular localization, structure, and function.11 These effectors represent potential targets for disease mitigation.

Host Genes and Gene Activation Involved in Pathogen–Host Interactions. Significant breakthroughs have been made in identification and characterization of host genes and signaling pathways activated in response to CLas infection. Several groups funded by CRDF used transcriptomic, proteomic, and metabolomic approaches to characterize host responses to HLB and the molecular basis of citrus–CLas interactions. Transcriptomic research on infected versus healthy citrus fruits and leaves revealed significant differences in gene expression, mainly reflecting suppression of immunity (e.g., systemic acquired responses) and metabolic dysfunction (sugar biosynthesis, transportation, and metabolism) attributable to source-sink disruption (Kim et al., 2009; Martinelli et al., 2013). Similarly, mRNA profiling from healthy and CLas-infected fruit revealed significant changes in transcription, particularly in biological processes, such as the light reactions of photosynthesis and in adenesine triphosphate synthesis, protein degradation and misfolding, source-sink pathways, and phytohormone-mediated pathways (Martinelli et al., 2012). When citrus roots and stems of infected versus healthy citrus plants were analyzed for changes in gene expression, the numbers of genes affected were greater in stems than in roots. The affected genes were found to be involved in cellular functions, such as sugar metabolism, cell wall biogenesis, stress responses, signaling, and protein modification and degradation (Koh et al., 2012; Aritua et al., 2013). Transcription profile comparisons showed more genes expressed differentially in tolerant host plants than in susceptible ones during CLas infection, likely reflecting a molecular basis of host sensitivity and basal resistance to HLB (Albrecht and Bowman, 2012; Fan et al., 2012; Nwugo et al., 2013; Wang et al., 2016). Using meta-analysis and gene co-expression network modeling on 22 transcriptome datasets, Rawat et al. (2015) identified the 65 most common probe sets regulated by CLas infection; these represent transcriptional modulations by CLas in sugar metabolism, nutrient transportation, and stress responses. The identification of resistance-specific probe sets that

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11 See http://prodata.swmed.edu/citrusgreening/index.html. Accessed June 8, 2018.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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represent leucine-rich repeat proteins, chitinase, miraculins, and constitutive disease resistance (CDR) elements are of significance (Rawat et al., 2015). The identification and molecular characterization of CDR genes from Citrus and its close relative, HLB-tolerant Poncirus trifoliata, may provide a useful resource for producing HLB-tolerant citrus (Rawat et al., 2017). Finally, proteomic analysis of CLas-infected leaves from susceptible and tolerant citrus revealed that four glutathione-S-transferases were upregulated in the tolerant cultivar Volkameriana but not in the susceptible navel orange (Martinelli et al., 2016). These findings may yield prospects to boost host defense mechanisms or alter host susceptibility for mitigation of HLB.

Pitfalls

Despite considerable research effort, much remains to be learned about the mechanics of CLas–citrus interactions at the molecular level. Genetic and functional analyses of pathogen metabolic and effector genes, and their in vivo roles in the biology and pathogenesis of CLas and its close relatives, have been hampered by the inability to culture HLB-causing Liberibacters (Recommendation NI-10: Develop in vitro culture techniques for CLas to facilitate experimental manipulation of the bacterium for insights into gene function). Understanding of in planta interactions has also been limited by the need for effective tools for genetic manipulation of host genes relevant to HLB, another important aspect that requires citrus genetic engineering technologies, which remain elusive or very limited.

Future Directions

Ongoing research, including studies funded by CRDF, the NIFA SCRI Citrus Disease Research and Extension (CDRE) program, and the Citrus Research Board, characterizing the components of pathogen–host interactions continues to yield advances in the areas of functional characterization of bacterial effectors, identification and functional analysis of their host targets, and exploitation of molecular host–pathogen interaction for novel strategies to manage HLB. Identification and utilization of resistance (R) genes from within or beyond citrus that counteract bacterial effectors should be a priority of future research. Furthermore, identification of critical genes, proteins, or metabolites in hosts that are targets of Liberibacter spp. and more comprehensive characterization of their interactions will provide keys to interfering with the disease processes, resulting in both genetic and chemical means to control the disease.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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

Notable Research Outcomes

Considerable new information regarding ACP–CLas interactions has been generated since the release of the 2010 NRC report. Investigators have followed to some extent the 2010 NRC Recommendation NI-1c: Investigate the behavioral ecology and CLas transmission biology of ACP to improve timing and other aspects of insecticide application, and Recommendation NI-7: Support research aimed at developing alternative ACP management strategies.

CLas Transmission Parameters. While the general characteristics of CLas transmission by ACP, e.g., the duration of acquisition, latent, retention, and inoculation periods, have long been known (da Graça, 1991), the 2010 NRC report listed several deficiencies in an understanding of ACP–CLas interaction that would affect transmission and HLB epidemiology. These included the role of ACP nymphs in CLas transmission and HLB epidemiology, effects of CLas acquisition and infection on ACP biology and fitness, and the potential for vertical transmission of CLas through ACP. CRDF funded several projects to investigate several of these aspects of CLas transmission by ACP.

While early reports suggested that ACP nymphs were not important in the transmission of CLas (da Graça, 1991), more recent studies have shown that early instar nymphs acquire CLas more efficiently than do adults (Pelz-Stelinski et al., 2010; Grafton-Cardwell et al., 2013; Pelz-Stelinski, 2014; Wu et al., 2016; Canale et al., 2017). Furthermore, transmission efficiency from nymphs reared on CLas-infected plants is higher than that from adults, although long feeding times by adults can negate these transmission efficiency differences (Pelz-Stelinski et al., 2010). Initial studies suggested that CLas does not replicate in the ACP (Pelz-Stelinski et al., 2010), but more recent studies have shown that CLas titer increases over time (Ammar et al., 2016), suggesting that CLas replicates in the insect and is a pathogen of both plant and insect. Efficient CLas replication may occur in nymphs but not in adults (Inoue et al., 2009), possibly due to the fact that more ACP proteins involved in immunity to bacteria were differentially expressed in adults than in nymphs (Ramsey et al., 2017). Several CLas genes are differentially expressed depending on whether the bacteria are associated with plants or with ACP (Yan et al., 2013). While the bacteria may not replicate efficiently in all life stages of psyllids, it is clear they survive for long periods in the insect and can be vertically transmitted, albeit at a low frequency (Mann et al., 2011; Grafton-Cardwell et al., 2013). The efficiency of acquisition of the CLas pathogen by ACP is influenced by plant age and maturity (Luo et al., 2015; Hall et al., 2016), and pathogen acquisition can have profound effects on ACP behavior and life parameters, including

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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, reproduction, and gene expression (Martini et al., 2015; Ramsey et al., 2015; Pelz-Stelinski and Killiny, 2016).

As documented in many other studies on pathogen transmission by insects, all of the measured parameters can vary widely depending on pathogen and insect populations (Coy and Stelinski, 2015), pathogen source materials, experimental design and methods, and environment. Nonetheless, the new information on CLas transmission by ACP is being used to develop and modify HLB management recommendations (Ukuda-Hosokawa et al., 2015; Udell et al., 2017).

Disrupting CLas Transmission. While not specifically detailed in any of the 2010 NRC report recommendations, several of the recommendations mentioned various aspects of replication and pathogenicity of the bacteria in the insect, as well as the molecular and cellular mechanisms that allow the bacteria to move and infect different tissues in the ACP. More information on these topics would provide the basis for interdiction strategies to prevent ACP from becoming a vector. There has been progress in characterizing CLas movement in the ACP (Ammar et al., 2011a,b, 2016) and in identifying molecules in the ACP that are involved in CLas infection and transmission and examining the potential methods to disrupt these interactions (Yan et al., 2013; Hoffmann et al., 2014; Killiny et al., 2014b; Pelz-Stelinski, 2014; Kruse et al., 2015, 2017; Ramsey et al., 2015, 2017; Ghanim et al., 2016; Arp et al., 2017; Gill et al., 2017). A key factor in this work has been research efforts toward the 2010 NRC Recommendation NI-11: Sequence, assemble and annotate the ACP genome to provide a basis for new approaches to ACP management. Initial seed money from CRDF, USDA Agricultural Research Service (ARS) and the Los Alamos National Laboratory provided preliminary ACP sequence data and led to a USDA NIFA-funded project to develop comprehensive bioinformatics resources for ACP (NIFA, 2018) as well as interdiction molecules targeting various ACP–CLas interactions and interfering with either CLas infection in ACP or CLas transmission by ACP. A key component of this work is the ability to test these molecules for biological activity either through the development of transgenic citrus expressing them, the use of CTV vectors to transiently express them (El-Mohtar and Dawson, 2014; Hajeri et al., 2014; Dawson et al., 2015), or the use of RNAi to directly target genes in the ACP (El-Shesheny et al., 2013; Hajeri et al., 2014; Andrade and Hunter, 2017; Galdeano et al., 2017). Several laboratories are identifying genes, proteins, and metabolites that are being tested for activities. Intellectual property concerns have kept many specific details out of this review. The expression and quantification of compounds, either in transgenic plants or transiently using the CTV vector, are in progress (El-Mohtar and Dawson, 2014; Hajeri et al., 2014; Dawson et al., 2015).

The 5-year $9 million NuPsyllid project formulated by CRDF and

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

funded by USDA NIFA concluded in 2017. The very ambitious overall goal of the project, to develop a modified psyllid that would not transmit CLas and could be deployed in the field to displace the natural ACP population, was not achieved, but the project did contribute significant advances to the fundamental knowledge of ACP–CLas biology (Soderlund et al., 2014; Cicero et al., 2015; Ding et al., 2015, 2016, 2017; Yuan et al., 2015, 2016; Brown et al., 2016; Stover et al., 2016; Liu et al., 2017).

Pitfalls

A major concern in this research area is the apparent lack of communication among members of the research community investigating how to control ACP and those investigating how to reduce transmission of the pathogen. There is a disconnect between insect control and vector control with respect to management of HLB in the field. Insect control efforts should be integrated with studies on how those strategies will impact CLas transmission among hosts, within and between citrus groves. Reduction in ACP populations alone may not significantly slow the spread of the pathogen. Similarly, studies on host response to CLas infection as well as the progress and virulence of the infection are not always linked to studies on pathogen transmission. Furthermore, while there are active and productive collaborative groups working on CLas–ACP interactions, the groups themselves would benefit from frequent discussions and sharing of information to minimize redundancy and facilitate progress.

The ongoing studies focused on CLas–ACP interactions would be facilitated by an expansion of efforts to generate high-quality genomic data for the pathogen and insect vector, to characterize more extensively, and to inform research on host responses and better understand the genomic diversity of CLas (including bacteriophage genes) and ACP (including mitochondrial and endosymbiont genes). A NIFA-funded project has developed extensive ACP genomic data and analysis tools (NIFA, 2018), but it is unclear how these resources are being used by the greater HLB research community. It is also unclear how these resources will be supported beyond the life of the grant. Furthermore, intellectual property concerns hinder the sharing of some of the research results in this area.

Future Directions

That transmission is influenced by CLas titer and distribution in trees, as well as by differential host responses to infection that influence vector attraction, feeding, and dispersal, are all fruitful areas of research that can lead to predicting with greater accuracy how types and levels of host resistance or tolerance are likely to influence the epidemiology of HLB.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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 is considerable effort to discover and develop molecules that interfere with some aspect of the CLas life cycle and lead to a reduction in CLas titer or distribution in the plant or insect host. Molecules are also being examined for their ability to reduce CLas transmission by ACP. Ultimately these molecules, along with durable host resistance, will be the bases for sustainable interdiction strategies that can slow or prevent the expansion of HLB. Promising preliminary results are still scientifically far from being translated to effective field management tools, a process that will necessitate regulatory and intellectual property considerations. With this in mind, the industry may want to consider how these hurdles specifically influence the selection of molecules for development. How each molecule would be classified by the regulatory agencies will affect what types of data will be needed to satisfy permitting decisions, which in turn may affect how the industry and funding agencies prioritize project proposals aimed at short- to mid-term field management tools. An overarching panel (see Recommendation 4.6 in the section Overarching Findings, Conclusions, and Recommendations for Future Research Efforts in HLB Management in this chapter) reviewing all research and extension proposals would help facilitate this decision making, especially if members had a presence on the advisory boards of all of the large multidisciplinary and multi-institutional projects. While intellectual property concerns must be acknowledged and respected, they are slowing progress and hampering data sharing. A respected review committee would be aware of the total research activities supported by all funding agencies and familiar with information and data that, if shared, would facilitate other research activities. With the appropriate confidentiality agreements in place, information could be shared, and management opportunities translated to the industry more quickly.

HLB MANAGEMENT

Cultural Control

Notable Research Outcomes

Research over the past decade on HLB management using cultural approaches has addressed the 2010 Recommendation NI-8: Support small-scale studies on the feasibility of alternative horticultural systems suited to endemic HLB. Relevant CRDF-funded projects have investigated strategies that could reduce the immigration or inoculating efficiency of ACP, retard disease development in inoculated trees, and improve tree health and productivity.

ACP Immigration and Inoculation Efficiency. Several projects evaluated cultural strategies for reducing or eliminating ACP populations in

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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citrus groves. Full-coverage screen houses prevented ACP access to grapefruit trees, while environmental parameters (such as air temperature, cumulative rainfall, and solar radiation) and tree development factors (including canopy surface area, water use efficiency, and leaf area index) remained suitable for grapefruit production (Ferrarezi et al., 2017a,b). The possibility that ACP could be diverted from citrus trees by interplanting them with alternate ACP hosts known to be highly attractive to the psyllid was investigated in field studies. Modest reductions in ACP numbers on citrus were noted after the first year of interplanting with orange jasmine (Murraya exotica L.) but did not continue into the second year (Hall et al., 2013a), and although intercropping citrus with pink guava (Psidium guajava L.) resulted in lower ACP numbers it did not prevent the introduction and spread of HLB (Gottwald et al., 2014). On the other hand, the use of metalized polyethylene mulch in citrus groves repelled ACP, resulting in significant reductions in both ACP populations and the incidence of HLB (Croxton and Stansly, 2014).

Data from microsimulation modeling led Lee et al. (2015) to conclude that entire citrus groves can become infested with up to 12,000 ACP per tree in under a year, prior to the appearance of HLB symptoms. However, the model showed that disease could be delayed significantly by applying control measures that reduce ACP numbers by 75% during leaf flush. Reduction of local inoculum by removal of HLB-infected trees has been studied primarily in Brazil, where experimental data and epidemiological modeling results confirm that immigration of inoculative ACP from source trees is a significant factor in epidemics and suggest that area-wide inoculum reduction strongly affects HLB control (Belasque et al., 2010; Bassanezi et al., 2013a,b; Bergamin Filho et al., 2016). However, these results have not led to any widely adopted management recommendations in Florida, due primarily to the high levels of CLas-infected trees throughout all production areas and the constant movement of CLas-infected ACP into and within citrus production areas (Gottwald, 2010; Gottwald et al., 2012, 2014; Hall et al., 2013b; Lee et al., 2015).

Slow Disease Development in Inoculated Trees. A number of cultural management approaches target CLas within plant phloem, either reducing existing titers or impacting bacterial reproductive rates.

Thermotherapy. The development of citrus thermotherapy to reduce or eliminate CLas in infected citrus trees has yielded beneficial results in greenhouse applications and some limited field tests in which trees have been enclosed for treatment (Ehsani et al., 2013; Hoffman et al., 2013; Fan et al., 2016; Pelz-Stelinski, 2016). In growth chamber experiments, Hoffman and coworkers (2013) measured lower CLas titers in 3-year-old citrus treated at 40–42°C for 48 hours than in control trees, while tree growth was unimpaired. Similar to the findings of Hoffman et al. (2013), but funded by

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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other sources, Brazilian scientists Gasparoto and colleagues (2012) found that young citrus trees grown at 27/32°C did not become infected after graft inoculation in the greenhouse, in contrast to those maintained at lower temperature regimes. However, results are more variable and less beneficial when trees are more mature or are field grown or when solarization is used as the heat source, and the effect of thermotherapy on the acquisition efficiency of ACP feeding on treated trees is mixed (Yang et al., 2016; Doud et al., 2017; Pelz-Stelinski, 2017). Pelz-Stelinski (2017) reported mixed results depending on the experimental setup. Limited field experiments (Ehsani et al., 2013), in which single mature trees were enclosed in clear plastic tents, showed that trees heat treated in midsummer (but not in late summer) had yields and Brix content indistinguishable from those of untreated controls. The success of thermotherapy in the field may be hampered by natural weather conditions, such as cloudy or rainy days lowering the temperature or solar radiation resulting in much higher temperatures near the upper sections of the plastic enclosures (Ehsani et al., 2013).

Improve Tree Health and Productivity. There is increasing interest in supporting and extending tree health in the presence of HLB infection through advanced production systems that supplement water, macro-nutrients, and related materials both to foliage and to soil (Campos-Herrera et al., 2013, 2014; Kadyampakeni et al., 2014, 2016; Ferrarezi et al., 2017a,b). Potential benefits for growers include extending the productive lifespan of diseased trees and shortening the time required for new trees to become economically productive.

Advanced citrus production systems manage tree health in a “fertigation” approach by constraining root growth into small clumps that can be supplied directly and daily, through drip or microsprinkler irrigation, with predefined water and nutrient supplements (Kadyampakeni et al., 2014); such intensive fertigation resulted in higher nitrogen accumulation in plants as well as increases in growth rates (as much as 330%) and canopy area (Kadyampakeni et al., 2016). The same principle is being applied by some growers using intensified foliar-applied nutritional supplements (Ingram, 2017). High-density planting in new or replanted groves is also becoming more popular in Florida (Black, 2017; Ingram, 2017); growers anecdotally report that when this practice is performed in combination with intensified nutrient and water supplementation trees apparently do not suffer from competition, and the result can be greater per-acre productivity and greater economic gain.

While the successes of the projects are often difficult to measure in terms of contribution to slowing disease progress or improving fruit yield and quality, many of the outcomes from these studies are contributing to the apparent grassroots efforts in tree health management and intensive orchard management in Florida (Campos-Herrera et al., 2014).

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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Pitfalls

Although thermotherapy has shown promise in reducing or eliminating CLas populations within infected trees (Ehsani et al., 2013; Hoffman et al., 2013) and could be useful in greenhouse operations or for valuable individual trees, the requirement that plants be enclosed for treatment presents challenges for achieving positive cost–benefit ratios. Further economic analyses are needed to determine whether investment in additional research is likely to lead to effective and affordable disease management.

Intense efforts to nurture infected trees for extended productivity have led to mixed results and may have some unintended consequences. After 2 years of treatment, Gottwald et al. (2012) found no difference in bacterial titer between a number of different enhanced nutritional programs and control treatments. That result, combined with other findings from the same study, led to concern that such approaches, in the absence of other measures, may allow for inoculum buildup and spread.

In contrast, Shen et al. (2013) (with funding from the Emerging Pathogens Institute at the University of Florida and the Smallwood Foundation) found higher population levels of CLas after supplemental nutrient treatments compared to those without. The Ct (cycle threshold) value of CLas was positively associated with leaf contents of several elements, although no significant association was observed with leaf contents of nitrogen, phosphorous, and potassium (N, P, and K). Furthermore, the richness index12 of endophytic α-proteobacteria was significantly greater for leaves that had received the nutrient treatment than the insecticide treatment. In particular, calcium and manganese (Ca and Mn) content and nutrient management were important environmental variables controlling the endophytic α-proteobacteria community structure (Shen et al., 2013). Some growers feel that the use of reflective mulches has an added benefit of stimulating tree development along with reducing numbers of alighting ACP (WBUR, 2017). Unfortunately, scant data have been generated on the utility of these nutritional approaches, and some reports are based on relatively short-term studies that would not reveal longer-term benefits. It will be important to continue to monitor, and in fact to design new and comprehensive field testing approaches to evaluate, the efficacy of these tree health practices so as to provide growers with robust research-based data with which to make sound management decisions.

Although most of the recent research on the citrus microbiome has focused on possible impacts on maintaining the health and productivity of infected trees, there is preliminary evidence that applications of beneficial

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12 An index based on the number of species per specified number of individuals, and the number of species per unit area (species density).

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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bacteria may also lead to lower CLas titers (Wang and Pelz-Stelinski, 2017). While this observation advances understanding of the system, the microbiome communities of citrus are so complex and variable that other CLas-directed management strategies are likely to be simpler and more effective.

It will likely be several years before the benefits of different therapies on trees can be assessed, and these will all need to be validated on a range of tree ages relevant to commercial production.

Future Directions

Elimination of abandoned groves currently serving as refugia seems to be absolutely essential to managing HLB. Research could explore simple and inexpensive ways to “neutralize” abandoned groves that can be accomplished with minimal landowner involvement. In some ways the HLB situation resembles the challenge of eradicating vector-borne human diseases, such as yellow fever from Havana and Panama—efforts to stop the disease were unsuccessful until breeding sites for the mosquitoes were eliminated.

While the intensive management of trees to reduce stress appears to improve overall tree health and productivity it is not clear if the pathogen populations are negatively affected or if the improved tree health will have any effects on reducing pathogen acquisition or transmission. If the infected trees remain as reservoirs of CLas for ACP, then the benefit of improved health and fruit production will need to be evaluated against the longer-term effects of the tree as an inoculum source for future dissemination of the pathogen. It is also not clear if fruit quality is correlated with improved tree health and yield. Commercial production groves that are practicing various components of enhanced citrus management are becoming potential laboratories for replicated trials that could provide the data needed to tease out the individual contributions of these new cultural management programs. While conducting research in commercial settings can be challenging, it may be possible to rent large blocks and adequately compensate growers to ensure their full cooperation in conducting stringently designed experiments.

The 2010 NRC report identified efforts in advanced production systems and recommended new cultural practices be evaluated for their effectiveness against HLB epidemics. While there are many examples mentioned above that have addressed alternative or enhanced management strategies, there is no evidence of a concerted and coordinated effort to consolidate information so the contributions of the individual and combined components can be experimentally evaluated and validated. This was a concern mentioned in the 2010 NRC report. Rather than the science providing impetus and supporting data to drive changes in cultural practices, the grower commu-

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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nity through grassroots efforts has begun to experiment with and adopt a diversity of measures to enhance tree health and fruit production.

ACP Control

Notable Research Outcomes

The status of chemical and biological control in the Florida citrus industry was quite different in 2017 than it was at the time of the 2010 NRC report. From 2000 to 2009 scientists and growers worked to document integrated control strategies that could suppress the vector, reduce the numbers of infested trees or at least slow the spread of the pathogen, and support the long-term goal of managing the damaging effects of the pathogen (NRC, 2010). In 2017 the industry was in decline (Farnsworth et al., 2014), with packing sheds closing (Browning, 2017) and the 57,000+ hectares of abandoned orchards providing overwhelming amounts of inoculum and infected psyllids (Lewis-Rosenblum et al., 2015). Growers who spoke at the committee webinar held on November 20, 2017 (see Appendix B for agenda and speakers) expressed the belief that vector control is no longer useful since every tree in the state is considered infected with the Ca. Liberibacter pathogen and virtually every psyllid is assumed to carry the pathogen. However, continued psyllid control is still needed, and removal of abandoned trees is still of value since multiple or repeated inoculations result in faster spread within large trees and subsequently greater losses (Pelz-Stelinski et al., 2010). Thus, controlling the vector can provide benefits even for an infected tree. At the same webinar the committee heard that not all growers were interested in integrated control strategies because these programs did not always effectively manage HLB or provide a level of control that allowed citrus to continue as an economically viable industry.

Chemical Control. Recommendations in the 2010 NRC report relevant to chemical and biological control of ACP include Recommendation N1-1: Improve insecticide-based management of Asian citrus psyllid, and Recommendation NI-7: Support research aimed at developing alternative ACP management strategies. Researchers have investigated which insecticides could provide control and if biological control agents could be used effectively to improve vector suppression. These goals have been largely met, at least with respect to performing and completing funded research.

Chemical control strategies for pests in most agricultural crops rely on sampling to determine if populations have reached an economic threshold13 that justifies pesticide application. Considerable progress has been made to understand and optimize sampling and counting strategies that accu-

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13 The level of crop damage at which the lost value exceeds the cost of pest management.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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rately reflect numbers of both nymphs and adults on trees so that pesticide applications are applied only when warranted by numbers, thereby reducing environmental impacts and likelihood for the development of insect resistance. Sampling plans for ACP and pesticide treatment thresholds have been developed for both young and mature trees, although these plans have not been widely adopted.

Nearly every pesticide available for insect control has been tested against psyllids in Florida (reviewed in Grafton-Cardwell et al., 2013; Qureshi et al., 2014; Boina and Blumquist, 2015), addressing Recommendation N1-1: Improve insecticide-based management of Asian citrus psyllid. Psyllid behavior, population size, and life stage at the time of acquisition influence pesticide effectiveness (Pelz-Stelinski et al., 2010). Psyllids infected with CLas are more susceptible to at least five commonly applied pesticides than those that are not infected (Tiwari et al., 2011d), possibly because esterase activity in infected psyllids is reduced (Tiwari et al., 2012). Not surprisingly, growers have focused on the most effective pesticides and applied them more frequently and at higher rates, actions that quickly led to resistance to organophosphates and carbamates (Tiwari et al., 2012), followed shortly thereafter by resistance to the most effective systemic pesticides, the neonicotinoids (Tiwari et al., 2011d). In response, researchers have designed and implemented a resistance assessment program to assist growers in documenting the effectiveness of key pesticides in their fields (Kanga et al., 2015). Most researchers have reported that timing of applications is critical for population reduction. One practice that has potential for organic farms in particular is to combine oils with insecticides, although this would not meet organic requirements if the pesticide is not organically certified. The only material reportedly being developed for commercial use is dimethyl disulfide, which has been incorporated into a slow-release matrix to repel adult psyllids (Mafra-Neto et al., 2013).

Researchers and growers alike are aware of the risk that psyllids can readily evolve resistance to pesticides that are applied repetitively. Pesticide resistance management plans have been developed and are being used in some area-wide management programs, such as Citrus Health Management Areas (CHMAs).

Biological Control. The benefits of indigenous predators and parasites of the psyllid have been evaluated and quantified, constituting progress on Recommendation NI-7: Support research aimed at developing alternative ACP management strategies. A wide variety of indigenous natural enemies attack ACP (Chong et al., 2010; Hall et al., 2012; Juan-Blasco et al., 2012). Their effectiveness has not been evaluated on the basis of individual species, but from cage experiments. Monzo et al. (2014) estimated that the collective suppression of immature psyllids in untreated orchards ranged from about 15% to 91%. These approaches may be most effective in abandoned

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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orchards and in urban and suburban citrus because pesticides are applied intensively in active commercial groves. Considerable field research effort has been focused on the imported psyllid parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) (Qureshi et al., 2009), which has been reared in large numbers and released throughout Florida. Again, the utility of this technique was limited by pesticide application in commercial groves, but it may have considerable value in reducing immigration from abandoned orchards and urban citrus. T. radiata is now established in at least 16 Florida counties, with levels of parasitism generally below 20% (Michaud, 2004; Chong et al., 2010), but as high as 56% in southwestern Florida in November (Qureshi et al., 2009). This parasitoid apparently spread through Florida, Texas, and Mexico (De Leon and Sétamou, 2010) and, in 2008, reached California (Gomes, 2008). A combination of host feeding and oviposition in the laboratory has been reported to kill about 50 nymphs per female (Skelley and Hoy, 2004), although Chien et al. (1993) stated that female T. radiata could kill between 16 and 245 nymphs, depending on the temperature. Entomopathogenic fungi have also been tested for psyllid management; most of the fungal isolates tested provided some suppression, but certain environmental conditions, such as low humidity and the application of fungicides for citrus pathogens, limit commercial control potential.

Pitfalls

Pesticides effective against the ACP have been a mainstay for HLB management programs in Florida. However, Hall et al. (2013a) suggest that the threshold for treatment is fewer than one per tree, so finding even a few psyllids results in a recommendation for treatment, and sampling strategies can vary in accuracy depending on environmental and host tree variables. Regardless of the sampling technique employed, psyllid reproduction in abandoned, untreated, or organic orchards can lead to their detection throughout the year in numbers that can trigger treatment (Tiwari et al., 2010; Lewis-Rosenblum et al., 2015). This has caused many growers to increase pesticide applications, which, in turn, has increased psyllid insecticide resistance levels, leading to further increases in pesticide use (Alverez et al., 2016). This “pesticide treadmill” has put growers in the economically difficult position of spending more money on pesticide application at a time when crop productivity (along with income) is declining due to HLB. Unfortunately, psyllid population suppression within active commercial groves requires nearly 100% control throughout the season even though the infection rate in psyllids averages about 70% (Coy and Stelinski, 2015). Missing even a few psyllids per tree can result in transmission of the pathogen. As a result, some growers either treat frequently, assume all

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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plants are infected and do not spray, or spray on a schedule rather than as guided by insect surveillance.

Of the available insecticides, pyrethroids (synthetic compounds that are related to pyrethrins and have similar insecticidal properties), organophosphates, and neonicotinoids work reasonably well and can last several weeks (Qureshi et al., 2014; Boina and Blumquist, 2015). However, by the time psyllids are again detected feeding on the plants, transmission will have occurred and the action threshold will have been passed. So, although many of the pesticides initially tested offered excellent control, the short residual control provided by many of the insecticides, the occurrence of multiple flushes requiring additional pesticide treatments, the development of resistance by psyllids to the longer-lasting systemic insecticides, and the need for nearly 100% psyllid suppression in order to stop transmission of the HLB pathogen have led growers to apply pesticides more frequently and at maximum rates. Overapplication of pesticides not only increased costs but also led to additional resistance development. As a result, several CRDF projects were funded to examine how resistance could be managed within an IPM framework. Researchers implemented a resistance assessment program to assist growers in documenting the effectiveness of key pesticides. In a committee meeting on July 24, 2017 (see Appendix B for agenda), Stelinski (2017) stated that although some of the CHMA (UF IFAS, 2018) programs delayed resistance development in 2013–2014, resistance evolution began to accelerate again.

The implementation of additional IPM programs has resulted in partial success, particularly when used in large-scale area-wide programs (Grafton-Cardwell et al., 2013; Qureshi et al., 2014; Boina and Blumquist, 2015). However, these programs have not provided consistent results or reliable levels of HLB suppression across the Florida citrus industry. While the suppression of psyllid populations by both indigenous and imported biological control agents is undoubtedly useful in minimizing immigration into commercial orchards from urban trees (dooryard citrus) and abandoned groves, the level of control is not commercially adequate as a stand-alone practice. While useful, indigenous predators alone will not provide adequate suppression in commercial orchards. Given the extensive acreage of abandoned orchards in Florida and the large numbers of untreated trees in urban areas, this level of suppression will help to reduce the overall environmental load of inoculum and psyllids; but because the movement of psyllids from abandoned orchards to active commercial groves can be substantial (Lewis-Rosenblum et al., 2015), suppression will be inadequate unless additional control strategies are employed. Furthermore, use of biocontrol agents in active citrus orchards is severely limited by the frequent application of insecticides toxic to the beneficials. A persistent problem with biocontrol is that many pesticides effective against the ACP are also

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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lethal to beneficial organisms. Much of the potential benefit of biocontrol agents in commercial citrus groves is likely lost because of application patterns that use multiple pesticides in rotation. This approach, while critical for preventing or slowing resistance development by the psyllid, essentially guarantees that insecticides toxic to the beneficials will be applied.

The parasitoid T. radiata attacks nymphs in the third to fifth instar (Skelley and Hoy, 2004), but trees can become infected by adult feeding well before any subsequent nymphs develop to an acceptable stage for parasitoid oviposition. As a result, the primary long-term benefit of psyllid suppression by T. radiata in commercial orchards would be to reduce the production of infected adults developing from nymphs within the orchard. While this outcome would clearly provide some benefit, a reduced adult population would not eliminate the loss of the infected trees or the eventual spread of the pathogen.

Future Directions

Despite the concern that most citrus trees may be infected by CLas, there is still a need for vector control using chemical, biological, and cultural control strategies. For example, while breeding for resistance or genetically engineering citrus may offer the best long-term strategy for control, even these programs require pathogen-free plants. The simplest strategy is to exclude the psyllid vectors (for example, in greenhouses) and chemically control any that escape the exclusion defenses. At this time, it is not known whether any resistant germplasm or genetic modification that may be found will result in 100% suppression of HLB or ACP. Therefore, as for many crops that have partial resistance, pesticide use may be required to prevent repeated inoculum transfer that can overwhelm the defenses (Prioul et al., 2008; Hariprasad and van Emden, 2010). Because the HLB pathogen spreads faster with repeated inoculations (Pelz-Stelinski et al., 2010), any strategy that reduces repeated inoculation will help slow the spread of the pathogen. Biological or other controls that can result in reduction in psyllid populations can help to reduce the overwhelming numbers of psyllids moving from abandoned orchards or urban environments to newly planted trees or to research facilities. In California, where HLB has not yet caused significant losses in commercial orchards, a reduction in inoculum (from infested psyllids and trees) could serve to slow the spread of the pathogen, allowing more time for scientists to explore other management strategies. The usefulness of most of the older pesticide chemistries has been well established. The focus should be on the newest chemistries—how to get them into or onto the trees and how they interact with changing cultural strategies, such as increased tree density.

Like nearly all members of the Hempitera, ACP possess a unique,

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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highly developed microbial endosymbiotic system involving a highly specialized organ containing cells necessary for the endosymbiosis. The microorganisms have been hypothesized to compensate for the nutritional deficiency resulting from the imbalance of nutrients in plant phloem sap. Investigations on such systems can increase insights into the mechanisms allowing the psyllid to cope with infection of the CLas organism, the pathways mediated by endosymbiont associates resulting in vector competence, and ultimately the proteins and pathways that can be targeted for disruption of transmission.

There are many proposed treatment strategies, and determining which is best for a particular citrus grove is not a simple process. The distance from an abandoned orchard could easily change the effectiveness of a management approach. Most of the IPM research reported to date is accomplished by rotating chemical applications. Nonetheless, a number of strategies (such as reflective mulches, which work reasonably well with younger trees integrated with insecticide programs; Croxton and Stansly, 2014) have not been fully tested in conjunction with biocontrol agents or high-density plantings. Documenting which strategies can be combined and will provide cost-effective control (if any) will require cost–benefit analyses. Unfortunately, generating these analyses can be difficult (Farnsworth et al., 2014) and may be hampered by concerns about patent disclosure by researchers and by the need for chemical use strategies, yield data, and other information considered proprietary by the growers.

Bacterial Control

Notable Research Outcomes

There have been numerous research efforts over the past decade in response to the 2010 Recommendation L-2: Support development and testing of bactericides, therapeutics, or SAR [systemic acquired resistance] activators. A number of research projects have identified useful enhancements for the use of, and application approaches for, chemical and antibiotic substances to control the pathogen.

Antibiotics and Chemotherapy. With increasing bacterial resistance to copper-containing formulations for bacterial diseases such as HLB, zinc (Zn) has been explored as an alternative. In a past CRDF-funded project a compound designated “TSOL,” containing chelated Zn, was taken up into stems and roots of sour orange seedlings, and spraying grapefruit trees every 21 days with TSOL formulations reduced disease incidence by 34% (Santra et al., 2017). CRDF also funded the development of the Zn-based nanoparticle treatment, Zinkicide, which has undergone initial testing in the field. The product showed initial promise in field tests (Johnson, 2015),

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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but the data have not been published. A project related to this formulation is now being supported through NIFA SCRI.

Sulfonamide antimicrobials (sulfadimethoxine and sulfathiazole) were highly effective in eliminating or suppressing CLas, with low infection rates and bacterial titers in scions and rootstocks (Zhang et al., 2014). Cell-wall inhibitors in the beta-lactam group (ampicillin, carbenicillin, penicillin, and cephalexin) were highly effective using a graft-based greenhouse evaluation assay on rootstocks, as was the transcriptional inhibitor rifampicin (Zhang et al., 2014). Effectiveness of sulfonamide antibiotics was increased when combined with thermotherapy (Yang et al., 2016).

Penicillin was more effective in controlling the CLas bacterium and decreasing disease severity than the antimicrobials EBI-602, silver nanoparticles, or carvacrol (Powell et al., 2016). Three of the beta-lactam antibiotics eliminated detectable CLas from infected scions, prevented HLB symptoms, and prevented transmission to the rootstocks (Zhang et al., 2014). Less effective were peptide antibiotics (vancomycin and colistinmethanesulfonate), the trehalase inhibitor validoxylamine, the cell wall inhibitor cycloserine, the quinolone ciprofloxacin, and the translation inhibitor chloramphenicol. Aminoglycoside antibiotics had either moderate effectiveness (zhongshengmycin, hygromycin B, spectinomycin, and streptomycin) or no effect (amikacin, gentamicin, kasugamycin, neomycin, and tobramycin). Lincomycin, the peptide polymixin B, and the quinolone cinoxacin had no effect on CLas (Zhang et al., 2014). Several tetracycline derivatives ineffective against human bacterial pathogens showed greater potency than oxytetracycline against strains of Liberibacter (Nelson, 2014). Formulated for delivery to the tree bark, two of the most potent compounds, EBI-601 and EBI-602, penetrated and translocated throughout the citrus tree. Both compounds showed the ability to suppress CLas growth in infected citrus, but as yet there have been no publications from this work. In some cases, combinations of antibiotics have been more effective than individual compounds (Zhang et al., 2010; M. Zhang et al., 2011). Antibiotic mixtures containing either penicillin and streptomycin or kasugamycin and oxytetracycline resulted in reduction of both the number of microbes on leaf midribs and the diversity of microbial taxa (Zhang et al., 2013a).

A number of research efforts have focused on improving bactericide treatment effectiveness by optimizing the method of plant delivery. Hu and Wang (2016) identified optimum parameters for trunk injection of oxytetracycline to achieve uniform antibiotic distribution in planta, thereby enhancing tree productivity (Hu and Wang, 2016). Others showed improved outcomes by adding adjuvants, creating nanoemulsion formulations (Yang et al., 2015; Powell et al., 2016), or using “soft” or nonclumping nanoparticles (SNPs) (Moudgil et al., 2015). Moudgil and coworkers (2014) are working to develop SNP nanoemulsions of two essential oils, EO A and EO B, that

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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effectively inhibit bacterial growth. Project reports indicate that their characterization is progressing, but no data have yet been published. Even lasers can improve the uptake of foliar sprays (Etxeberria et al., 2016).

New approaches such as chemical genomics for identifying novel chemicals that are potentially antimicrobial (Patne et al., 2011), the use of Zn to replace Cu, which has been applied since the 1940s and to which bacterial resistance is a growing problem (Johnson, 2016; Santra et al., 2017), and the development of nanoparticle and nanoemulsion formulations that promote uptake into the plant (Moudgil et al., 2015; Yang et al., 2015; Powell et al., 2016) have produced promising preliminary results. Another area of significant progress has been the identification of factors, including chemical stimuli and pathogen effectors and signal molecules, of both pathogens and beneficial microbes that trigger pathogen-associated molecular pattern host defense induction in the citrus host resulting in suppression of disease progress, disease severity, or bacterial multiplication (Patne et al., 2011; Lu et al., 2013; Moore and Febres, 2014; Roose et al., 2014; Shi et al., 2015; Li et al., 2016; J. Li et al., 2017). Several new host defense molecules have also been identified and could be the target of research to develop new antimicrobial compounds. Hundreds of new chemical formulations have been tested in an effort to identify novel anti-CLas compounds (Triplett et al., 2017), and predictions were made about which of these would likely be phloem mobile, but these results have not yet been published. Through mutagenesis of L. crescens (Lai et al., 2016) 314 genes essential for growth in culture have been identified; 238 of these have homologs in Ca. Liberibacter asiaticus and could potentially be targeted through development of novel antibiotics. Furthermore, Pagliai et al. (2014, 2015) showed that a CLas transcriptional regulator (ldtR) and a transpeptidase (ldtP) affected CLas viability; such novel approaches may be useful as pathogen-targeted control elements.

Bacteriophage Therapy. Significant research efforts have led to the identification of bacteriophages present in CLas and its Ca. Liberibacter relatives, and their roles in the survival and fitness of their bacterial hosts (S. Zhang et al., 2011; Fleites et al., 2014; Zhang et al., 2014; Gabriel et al., 2017; Jain et al., 2017). Since CLas prophages were discovered through annotation of the CLas genome, these projects are relevant to both Recommendation L-2 and Recommendation NI-6: Exploit the CLas genome sequence for new strategies of HLB management. CRDF funding has led to the characterization of regulatory factors mediating phage existence in and, in some cases, transition between lytic or lysogenic phases. Unexpectedly, phage status (prophage or lytic) differs depending on the host; some phage regulatory elements found in citrus are absent in the ACP. More about the biology of CLas phages can be found in section Candidatus Liberibacter asiaticus in this chapter. One of the CLas phage encodes a

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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secreted peroxidase that suppresses plant defense and could support infection (Jain et al., 2015). An in vitro reporter assay has been developed in L. crescens to measure the suppressive activity of host extracts on phage lethal genes. That work has enabled screening for specific phage-inducing stresses and treatments (Fleites et al., 2014; Jain et al., 2017), resulting in the identification of a psyllid endosymbiont protein that strongly represses phage gene activation (Jain et al., 2017). Suppression of phage promoters is a heat-labile phenomenon, providing one potential explanation for the efficacy of thermotherapy treatments in suppressing CLas in citrus (Fleites et al., 2014). Ongoing work includes the attempt to engineer culturable L. crescens to take up phage in order to facilitate study in a living system, and to identify compounds or organisms that can trigger the phage lytic cycle or suppress entry into the lysogenic state (Gabriel, 2017).

Biological Control. Bacterial-based biocontrol is a desirable control method for many growers as it is generally compatible with organic production. A screen of 327 citrus-inhabiting bacteria uncovered 21 that have antimicrobial properties (Riera et al., 2017). The abundance of the beneficial bacteria Bradyrhizobium and Burkholderia, the two most dominant rhizoplane-enriched genera, was lower in HLB rhizoplane samples than healthy samples, suggesting that plant health might be enhanced by supplements containing these microbes. The production of antimicrobial compounds by beneficial bacteria isolated from HLB-escape citrus trees had antagonistic activity against several citrus root pathogenic fungi and oomycetes (Riera et al., 2017). Application of four antimicrobial species to infected citrus resulted in some slowing of disease progression, but only if applications were made during very early infection (Wang and Pelz-Stelinski, 2017). Beneficial microbes may suppress infection and slow symptom development by triggering host plant defense responses (Wang et al., 2017). Application of Burkholderia spp. also induced natural plant defenses (Zhang et al., 2017). Because biocontrol efforts can be hampered by low microbial survival, several candidate beneficial bacteria are being analyzed for antimicrobial compounds that could be extracted for concentrated application (Wang and Pelz-Stelinski, 2017). In as-yet unpublished work, these beneficial bacteria slowed HLB disease index increase and CLas titers for the asymptomatic or mildly symptomatic trees compared to the control, but they had no effect on severely symptomatic trees (Wang and Pelz-Stelinski, 2017).

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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Pitfalls

For HLB, every component of the disease triangle14 (or pyramid, counting the insect vector) presents unusual challenges for research and applications. Intensive research and development efforts funded by CRDF and other agencies have yet to result in a robust set of strategies for managing HLB in Florida citrus groves and nurseries. The phloem habitat of the pathogen in citrus provides a safe haven for the bacteria from many bactericides and antibiotics, and the inability to culture CLas hampers screening of such compounds in the laboratory.

There are mixed opinions about the utility of bactericides and chemical sprays for CLas control, particularly in relation to recent legislation permitting the emergency use of tetracyclines in Florida groves (M. Zhang et al., 2011) or the applications of novel compounds generated through chemical genetic approaches. Concerns about chemical residues in fruit and juice, the likelihood of development of bacterial resistance, and even the ability of such applications to reduce or eliminate bacteria in the field influence whether such approaches will be widely adopted in the future. Other concerns include the high cost of application, the need for repeated applications since the compounds are bacteriostatic rather than bactericidal, the risk of phytotoxicity, and, as microbiome-related issues are revealed and characterized through ongoing research, deleterious effects on nontarget (and possibly beneficial) bacteria.

Much remains unknown about the ultimate impact of phage therapy, and researchers are far from translating current knowledge into an effective product. Even if it is possible to manipulate CLas phages to enter the lytic cycle and kill a bacterial host cell, it is not clear whether this action could be managed in such a way as to eliminate all of the bacteria in an individual host plant and, further, in all of the citrus trees in a grove or region.

Future Directions

Major approaches for managing an insect-transmitted plant pathogen are to enhance host resistance, control the vector, and reduce the inoculum. Thus, killing or significantly reducing pathogen populations is an important component of an overall disease management strategy. Research-based evidence has shown that some approaches for doing so are likely to be more effective than others. A recent review of challenges in the management of HLB (Blaustein et al., 2018) included thoughtful comments on pathogen-targeted approaches.

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14 A conceptual model that shows the three factors required for disease development: a susceptible host, a disease-causing organism (the pathogen), and a favorable environment for disease.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

It will be important to continue to explore the potential of novel chemicals such as those involved as regulators or intermediates in key host plant defense response pathways, which might be exploited in the development of resistant citrus cultivars, as well as substances identified through chemical genetics. Research to identify and develop strategies to enhance chemical uptake and translocation within the plant should continue, as they will help to reduce usage rates and increase effectiveness of formulations applied to plant surfaces. Combinations and seasonal rotations of materials should be explored further, for both their bactericidal and bacteriostatic impacts and their potential to reduce the development of bacterial resistance. Just as important will be verifying the overall effectiveness of any chemical or application strategy through controlled field testing over multiple years and in a variety of environmental conditions.

Promising current research is exploring possible applications of phages or their regulators, including some factors found in the ACP endosymbiont Wolbachia. Preliminary results from research on bacteriophage regulatory factors, and the potential applications of such regulators as antibacterials, should be continued.

CLas management strategies that target the pathogen operate under the premise that reduction of bacterial numbers will result in less disease, less pathogen transmission, and more productive trees. However, data to support these assumptions are scant. It will be important to investigate further whether trees currently receiving tetracycline applications are, in fact, more productive, and at what bacterial titers ACP acquisition or transmission rates are prevented or lowered to an acceptable level.

Host Resistance and Conventional Breeding

Notable Research Outcomes

The citrus breeding programs in Florida prior to HLB were innovative and advanced the knowledge of citrus genetics, breeding, and biotechnology. Over the years of breeding and selection these efforts produced a range of promising advanced selections and cultivars (Cooper et al., 1962; Gmitter et al., 2007; McCollum, 2007; Grosser et al., 2010).

After the arrival of HLB in Florida CRDF funding helped bring the conventional breeding programs through the first phases of work on identifying HLB-resistant or -tolerant genotypes. Some level of apparent tolerance in existing citrus fruiting varieties and rootstocks, such as Persian and Mexican limes, Eureka and Volkemer lemons, and Carrizo citrange and Poncirus trifoliata rootstocks was detected but most commercial citrus grown in Florida was found to be quite susceptible (Folimonova et

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

al., 2009). Although high-level resistance was not detected in commercial-ready genotypes, useful levels of tolerance were found in both rootstock and fruiting selections that had been developed prior to the detection of HLB in Florida. These included rootstocks US-942 (USDA, 2010), US-802 (Bowman et al., 2016a,b; Albrecht et al., 2012), US-1516, 896, 1503, and 1524 (Bowman et al., 2016b), and US-1279, 1281, 1282, 1283, and 1284 (Bowman and McCollum, 2015) and fruiting varieties such as “Sugarbelle,” “Tango,” and “Bingo” (Grosser et al., 2015; McCollum and Stover, 2017; UCR, 2018). CRDF supported the testing of these materials, and they have been either released to growers or are undergoing advanced field trials (Albrecht, 2017; Grosser and Gmitter, 2017).

The committee noted that none of the recommendations in the 2010 NRC report addressed conventional breeding.

Pitfalls

Citrus breeding is a long-term activity marked by challenges such as long juvenility periods, high levels of heterozygosity, and large tree size, requiring large and expensive field test sites. Polyembryony in some citrus cultivars and germplasm makes it difficult or impossible to produce new genetic combinations through conventional hybridization, although it facilitates clonal propagation through seeds (Kepiro and Roose, 2007).

New cultivars of citrus that had been developed through breeding programs in place prior to HLB were only slowly, if at all, accepted by the industry because profitable production depends upon market acceptability, uniformity of product, and acceptable levels of fruit production. Acceptance levels changed dramatically after the destruction caused by HLB in Florida after 2005 (McClean and Schwarz, 1970; Miyakawa, 1980; Nariani, 1982; Miyakawa and Zhao, 1990; Lopes and Frare, 2008; Folimonova et al., 2009). Also on the rise is interest in the testing of advanced selections toward commercialization. Chaires (Giles, 2017) noted that, “Whereas there used to be one release every 20 years or so, we have seen approximately 22 fresh selections made available through the accelerated programs and a number of private or proprietary selections come into Florida for trial.” Unfortunately, the most important commercial orange and grapefruit cultivars currently grown in Florida appear to be among the most susceptible to HLB (McClean and Schwarz, 1970; Miyakawa, 1980; Nariani, 1982; Miyakawa and Zhao, 1990; Lopes and Frare, 2008; Folimonova et al., 2009).

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

Future Directions

Apart from HLB, citrus trees face many challenges, biotic (including, but not limited to, Phytophthora, CTV, canker, and nematodes) and abiotic (including cold damage, flooding, and hurricanes), that must be considered along with productivity and fruit quality. That there is industry demand for new genotypes provides an opportunity for both breeders and growers. Although evaluations are being done, they need to be ramped up. The standard definitions of resistance, tolerance, and susceptibility for HLB may not follow those for other plant diseases. Instead, the community might be well served to regard host responses based on whether trees become unproductive or continue to thrive and produce marketable fruit in spite of HLB infection. A community-accepted standard for evaluation of host resistance may be accompaniedby or correlated with omics and molecular bases for host responses. Evaluations must take place in the field and must continue for multiple years before recommendations can be made with confidence. With that said, growers and industry supporters of research will need to be willing to take some risks with new citrus genotypes.

Citrus trees used in commercial production are composed of two interacting biological systems: the fruit-producing scion and the soil-inhabiting rootstock. Each must be evaluated for reaction to HLB and other biotic and abiotic factors. While fruit quality is not a factor for rootstock selection, ease of vegetative propagation and seed production, if polyembryonic, are important selection factors. Rootstock-scion combinations must be evaluated first for graft compatibility and then for overall tree health, abiotic stress resistance, effects on tree architecture, productivity, and fruit size and quality. Tolerance to HLB can be affected by environment and pathogen load, and perhaps by pathogen genetics. Therefore, considering the investment in developing a new grove and the time that young trees are nonproductive, growers need to have some assurance that they will profit, in the long run, from investment in a tolerant rootstock or scion.

Tolerant genotypes such as “Sugar Belle” mandarin, “Temple” tangor, and “Triumph” and “Jackson” grapefruit hybrids, released prior to the incursion of HLB into Florida, and a number of advanced selections (Stover et al., 2012; Gmitter et al., 2017; FFSP, 2017) will require focused investigations to ascertain the mechanisms of resistance or tolerance and guide the development of new resistant or tolerant material (Albrecht et al., 2016; Killiny and Hijaz, 2016; Killiny et al., 2017). Evaluation of the current collection of tolerant material should be shifted toward extension and industry, and field data and critical observations provided to breeders to assist in guiding next generation cultivar development. To some extent this ramped-up field testing is already under way; currently, through projects supported by CRDF and Multi-Agency Coordination (MAC) Group fund-

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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ing, more than 50,000 trees, including approximately 100 rootstocks, 50 scions, and 70 rootstock–scion combinations, are being field tested. These tests provide opportunities for the release of new HLB-tolerant rootstock and scion cultivars but also present challenges. The material being developed by the breeding programs can help provide industry with direction for the future, but it can do so only with strong industry collaboration in advanced selection and new cultivar testing programs. Mechanisms already in place for such collaboration, leading to rapid evaluation and distribution of promising new citrus selections, include the New Varieties Development & Management Corporation (NVDMC) and the Fast Track (NVDMC, 2015, 2017; Chaires, 2016) mechanism co-developed by NVDMC, University of Florida (UF) Institute of Food and Agricultural Sciences (IFAS), and Florida Foundation Seed Producers Inc. The latter program is strengthening connections between breeders and the grower community for their mutual benefit (UF IFAS, 2018). In the long term, these types of research and development efforts, managed collaboratively by the breeding community and the industry, will increase the likelihood of survival and perhaps healthy evolution of the Florida citrus industry.

A critical area of collaboration is that between breeding programs, especially between UF and USDA breeding programs in Florida (but extending also to California-based and other citrus breeding programs worldwide). The industry is depending on these programs for long-term solutions to HLB. Considering the challenges in citrus breeding described above, breeding programs must be fully integrated at the scientist and institutional levels. Funding, facility sharing, and intellectual property issues must be worked out by the respective institutions to facilitate multi-institutional breeding efforts. Certainly, the physical proximity of the Florida programs to each other and to the industry create a natural environment for close cooperation that must be fully exploited.

Host Resistance and Genetic Engineering and Editing

Notable Research Outcomes

Genetic resistance to HLB has been considered key to the survival of the Florida citrus industry and hence was a primary focus of research funding, but hybridization and selection, identification of naturally occurring sports (segments of the plant that are distinctly different from the parent plant in both phenotype and genotype), mutagenesis, and somatic hybridization15

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15 The fusion of enzymatically isolated somatic cell protoplasts (naked cells) from primary tissues, circumventing complex sexual incompatibility and enabling important traits to be transferred from donor to recipient species.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

have not yet led to highly resistant or immune commercially acceptable citrus varieties. Unfortunately, no suitable sources of high-level resistance have been identified in citrus for use in breeding programs, but promising genes from other plants may be used in genetic engineering (GE) approaches (NRC, 2010).

In response to the 2010 Recommendation 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, the genomes of several citrus cultivars and species have been sequenced16 (Xu et al., 2013; Wu et al., 2014). Gene identification, now under way, must take into account the large and complex genomes of citrus species and cultivars.

The terms “resistance” and “tolerance” are rather loosely applied in relation to HLB. The term “tolerance,” as discussed by Castle et al. (2015), is a “concept . . . of endurance . . . or thriving in the presence of the pathogen or pest.” Horns and Hood (2012) state that “Resistance traits reduce the harm caused by disease by preventing infection or limiting subsequent pathogen growth and development within the host through avoidance or clearance of infection.” In many cases for citrus it is not yet clear if particular genotypes express tolerance or some level of resistance.

Citrus Transformation. The 2010 NRC Recommendation L-1: Support development of transgenic HLB-resistant and ACP-resistant citrus, has been very well addressed by CRDF funding. Many new transgenic lines have been developed in a number of projects; of these, most possess resistance to CLas, although a few are targeted for ACP control.

Transformation of citrus has been accomplished through Agrobacterium-mediated transformation of protoplasts (plant cell protoplasm whose cell wall has been removed), leaves, stem segments, seeds, epicotyls, and embryogenic cell suspensions (Dutt and Grosser, 2010). Particle bombardment has also been successful. Factors influencing transformation efficiency include the genotype, age and type of explant, Agrobacterium strain, inoculation procedure, preculture conditions, and selection regime (Febres et al., 2011). Transformation, which is specific to genus, species, and cultivar (Dutt and Grosser, 2009), has been successful in relatively few genotypes (Febres et al., 2011; Donmez et al., 2013). P. trifoliata, used for decades as a source of cold-hardiness genes for transfer to citrus germplasm, is at least tolerant of, if not moderately to highly resistant to, HLB. Unfortunately, while P. trifoliata is readily transformed, it generates offspring having poor quality or inedible fruit so it is of interest to the industry primarily for rootstock use. Transformation has generally been undertaken with explants from juvenile tissues

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16 Citrus genome database URL: https://www.citrusgenomedb.org/. Accessed December 18, 2017.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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such as epicotyls and nucellar embryos produced from parent tree tissues. While these explants reproduce a tree having the original genotype, the time to fruiting is extended due to juvenility. Juvenile tissues have been used to produce transgenic rootstocks, sweet oranges, grapefruits, mandarins, limes, and lemons (Dutt and Grosser, 2010; Orbović and Grosser, 2015). Rates of transformation are still quite low, in the range of single digits, for many commercial genotypes (Hu et al., 2016).17 The rootstock cultivar “Carrizo,” a hybrid of P. trifoliata, and “Duncan” grapefruit, has among the highest transformation rates using juvenile tissues (~40%) (Dutt and Grosser, 2009). There are relatively few reports of mature citrus transformation (Cervera et al., 2008; Khan et al., 2012; Marutani-Hert et al., 2012; Orbović et al., 2015; Wu et al., 2015). When it does occur, transformation rates from mature tissue explants are low (1% or less), but the time to fruiting is shorter than when using explants from immature tissues. In general, major issues keeping transformation rates low are low rates of Agrobacterium infection and low integration efficiencies (Febres et al., 2011). The use of the knotted1 (kn1) gene in transformation significantly increased transformation and transgenic plant recovery rates using juvenile tissue explants, but its effect on mature explants has not yet been fully evaluated.18 While the kn1 gene did not appear to affect the transgenic shoots, the effects on tree development, flowering, and fruiting remain to be investigated (Hu et al., 2016). Investigation of the kn1 gene and other potential regeneration-inducing genes may be useful in increasing levels of regeneration, a critical process for most methods of stable genetic transformation (genetic engineering).

Low transformation rates make it difficult to reliably evaluate the effect of a particular gene since many factors influence transgene expression, including the site of insertion, copy number of the insert, the arrangement of copies, and base pair deletions in the gene of interest or promoter. Large numbers of plants from each transformation event are important for determining the effect of a particular gene and/or promoter. In the short term, testing of genes and constructs may also be accomplished through the use of virus-based vectors, which would not require transformation or regeneration of plants for evaluation.

Transgenic Plants. Hundreds of independent citrus lines have been produced despite challenges associated with citrus transformation; these transgenic plants express antimicrobial proteins (Stover et al., 2013b; Dutt et al., 2015b), disease resistance or R genes from citrus or other plant species (Lu et al., 2013; Dutt et al., 2015a,b), antibodies against CLas proteins,19

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17 M. Dutt, Citrus Research and Education Center (CREC), Lake Alfred, Florida, personal communication, December 12, 2017.

18 J. Zale, CREC, Lake Alfred, Florida, personal communication, December 12, 2017.

19 E. Stover, USDA ARS, personal communication, December 20, 2017.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

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).

These plants, which represent the culmination of years of work, are a notable outcome that must be well cared for and critically evaluated in the long term for their impact on HLB resistance. They will provide a basis for future work and may provide the genetic material that the industry will need to prosper. Work with transgenic HLB-resistant citrus expressing a defensin gene from spinach is well advanced; field tests have been ongoing in Florida since 2009. The groups supporting this work (Texas A&M and Southern Gardens Citrus) have obtained permits from Animal and Plant Health Inspection Service (APHIS) Biotechnology Regulatory Services and the Environmental Protection Agency for more extensive field tests in both Florida and Texas. Methods of selection of transgenic shoots are improving (Acanda et al., 2017). Gene expression in particular tissues, such as phloem, can now be targeted using transgene promoters (Dutt et al., 2012; Benyon et al., 2013; Belknap et al., 2015). Development of transgenic plants using anthocyanin markers rather than antibiotic resistance (Stover et al., 2013a; Dutt et al., 2016) could facilitate selection of plants already containing another marker for use in retransforming, or stacking, transformation events.

CRDF funding has supported research efforts to identify genes that are expressed differentially in susceptible and tolerant citrus cultivars, and to produce transgenic HLB-resistant or -tolerant citrus20 (Dutt et al., 2015b; Wang et al., 2016). These studies have moved forward the fundamental knowledge of the Citrus and Poncirus genomes with respect to how plants react to HLB infection and how they might be manipulated to provide resistance (Rawat et al., 2015). These, as well as mapping studies, have demonstrated that naturally occurring tolerance and resistance in citrus relatives is complex and is likely the result of multiple genes and multiple pathways (Wang et al., 2016). This apparent complexity makes it critical to have open communication between genomic researchers, breeders, and researchers studying the disease process and reactions to inoculations in order to identify genes that are responsible for resistance or tolerance.

Resources Required for Genetic Engineering. Resistance screening of research-derived HLB-resistant genetically engineered citrus is a bottleneck in delivery of new varieties to the industry. CRDF supported research on high-throughput screening using ACP, an approach that is more similar to the natural infection pathway than the use of graft inoculation, but that may require additional greenhouse capabilities in which to select a smaller advanced set for field testing. Using new flushes of leaves significantly in-

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20 Z. Mou, University of Florida, Gainesville, Florida, personal communication, December 4, 2017.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

creased rates of disease transmission (Hall et al., 2016), and ACP nymphs were more efficient transmitters of CLas than adults (Ammar et al., 2016). It is critically important that resistance screening be supported and that vetted data be meticulously collected in order that decisions can be made on what material is to be moved to the next stages of replicated testing. Data from these tests will also be useful for regulatory submissions.

Acceptance of Genetic Engineering Traits. CRDF funding has supported investigation of many potentially useful R genes from a variety of sources that may be candidates for gene editing. Many in the field assume that gene-edited products will be readily accepted by consumers. Ishii and Araki (2016) reported finding no basis at this time to support such a claim; however, House (2017) found that a majority of surveyed Florida consumers reported willingness to purchase genetically engineered citrus if cost incentives were provided (see also Future Directions in this chapter, and Chapter 3). The citrus industry would do well to keep informed and be proactive in interacting with the public on all of the technologies that are being developed (genetically modified or non-genetically modified) to save U.S. citrus production. Additional discussion of sociological and economic factors influencing public and market acceptance of genetically engineered citrus is provided in Chapter 3.

Pitfalls

In addition to the scientific and technical challenges of citrus transformation, progress is hampered by a reduction, in severe cases, in transformation rates due to low seed quality from HLB-infected trees,21 and this issue is holding back the development of transgenic lines. While the use of a more tractable model system, such as Arabidopsis, or the use of viral-based vectors can provide preliminary data to guide further research it will be important to move the work into more relevant systems as quickly as possible. Sourcing disease-free citrus trees will be essential for transformation work.

Despite optimism that improvements to HLB resistance produced through genetic engineering technologies will ultimately provide solutions for HLB management, there has, as yet, been only very limited interaction between citrus developers and representatives of the regulatory agencies whose approvals will be required for dissemination of new genetically engineered and gene-edited citrus cultivars. The regulatory process can be both challenging and time consuming, and Florida growers have little time to spare in implementing industry-saving measures.

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21 V. Orbović, CREC, Lake Alfred, Florida, personal communication, December 1, 2017.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

Future Directions

Many potentially useful transgenic lines developed over the past decade must be fully tested so that only the most promising material, selected based on reliable, replicated data, moves to intense field testing. Field, and in some cases greenhouse, testing will require sufficient and long-term funding in order to achieve practical results for the industry. Preparation for release of enhanced citrus varieties must include consideration of intellectual property and regulatory issues.

There is a critical need for a coordinated effort to develop highly efficient transformation systems for citrus genotypes of commercial interest. In the meantime, it may be useful for the community to select a susceptible citrus host—not necessarily of commercial interest—that provides consistently high levels of transformation as a model that could be used as a community-wide standard for evaluating genes of interest. Such a cogeneric model may be more useful than an Arabidopsis system, or it may be used to follow up on Arabidopsis rapid screening. For example, if a transgene is effective in “Duncan” grapefruit (now being used in work at UF Citrus Research and Education Center), a genotype having a transformation rate of 40% or greater (Dutt et al., 2009), that gene would be a good candidate to move into a transformation-recalcitrant but commercially important genotype. A constitutive promoter such as 35S should be considered for the initial tests of a candidate gene because constitutive expression may provide a more robust assay; tissue-specific expression could miss important tissue targets for providing resistance.

Long-term solutions to the citrus greening crisis will involve a combination of tactics, and disease management will likely depend on reducing inoculations by ACP coupled with durable host resistance. The citrus industry (juice and fresh) can work to eliminate barriers to the implementation of genetic solutions, including the incorporation of disease resistance genes into desirable citrus varieties and modification of ACP to render them incompetent vectors.

The Picos Farm and other citrus field test sites are critical for the pipeline from gene discovery, transformation, and greenhouse testing to field testing and cultivar release. Although these screening goals are different from those mentioned in the 2010 NRC report (Recommendation NI-3: Establish citrus orchard test plots for evaluation of new scouting and therapeutic methods), such field plots are critical for real-world assessment of genetic improvements as well as therapeutics and scouting approaches. These sites will need long-term CRDF, institutional, and industry support.

More rapid advancement in citrus transformation should be a major goal of all teams working on the genetic engineering projects, and more robust communication on methodologies and strategies—both successful

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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 unsuccessful—would be beneficial. Once efficient productive transformation systems are developed, all research groups will benefit, as will the growers who are waiting for a breakthrough in transgenic citrus highly resistant or immune to HLB. Information on genes of interest, promoters, and selection strategies should be shared as soon as possible throughout the CRDF-supported community.

In summary, the following are important steps for this stage of the research and development (R&D) pipeline:

Screening plants that have already been developed for resistance and moving the promising clones into replicated field test for critical evaluation by researchers and industry. This work will ultimately provide valuable information and guidance for the ongoing search for useful genes, native to citrus and related species, or from unrelated organisms. It could also lead to a major breakthrough in HLB resistance. Support is needed for long-term evaluation of field tests for all promising plants (whether GE or conventionally bred) developed through CRDF funding. MAC grants are recognized as a great benefit in this area.

Improving transformation. Transformation efficiency is a major bottleneck. There seems to be a rather wide range in transformation efficiencies within the community, particularly with the more recalcitrant genotypes. It is not clear if the more successful approaches are being effectively shared; information on regeneration protocols, the effects of promoters, and genes of interest could help to prevent duplication of efforts and increase research efficiency. Communication could be supported by having frequent principal investigator–sanctioned communications among technical personnel, graduate students, and postdocs within and between research groups.

In addition to generally improving citrus transformation efficiency, a model citrus system should be developed that can be readily transformed, with a consistently high transformation rate, for use in citrus genetic engineering and/or gene-editing research. It would also be wise to come to a consensus on a model vector and transgene cassette.

Understanding the regulatory and intellectual property landscape of the genes, promoters, and vectors being used in CRDF-funded projects. There should be a clear understanding of the regulatory potential of any gene to be used for resistance studies. If a gene is unacceptable for regulators it should be avoided and its use not funded by CRDF. Communicating with genetic engineering plant regulators is critical. The citrus industry can learn from the experience of others who have already received regulatory approvals for crops including apples, plums, and papayas, or are currently going through the process (chestnuts). Ideas on public outreach, website development, and the use of consumer focus groups can also be obtained from developers of other GE fruit and nut crops. The interactions of Texas A&M and Southern

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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Gardens Citrus with regulatory agencies and with consumers can serve as additional examples. By sharing their experiences these groups can learn from one another the best approaches to constructing a successful regulatory dossier and perhaps, when appropriate, could present a unified voice on issues pertaining to regulatory oversight of transgenic tree crops.

While there will always be consumers opposed to the use of biotechnology, there are some who will benefit from factual information presented in lay terms in order to make an informed decision on the use of the technologies.

Diagnostics

Notable Research Outcomes

Significant advances have been made in HLB detection technologies, notably those that detect biomarkers produced by the infected host, a biomarker secreted by the pathogen in the host, or volatile organic compounds (VOCs) released by an infected host (Cevallos-Cevallos et al., 2011, 2012; Aksenov et al., 2014; Ding et al., 2017; Pagliaccia et al., 2017; Slupsky, 2017). These results have addressed the 2010 Recommendation NI-2: Support searches for biomarkers that may be exploited to detect CLas-infected citrus.

It is unlikely that further development of conventional diagnostics will result in increased pathogen sensitivity, although gains are possible in adapting some of the technologies to field-based assays that may help growers conduct diagnostic tests on the farm rather than sending samples to accredited laboratories. The primary continuing need for CLas diagnostics is for methods that detect infection shortly after inoculation, well before any symptoms of disease can be observed. The diagnostic breakthroughs are likely to come from alternative technologies that are geared toward detection of disease rather than the pathogen: to identify unseen symptoms rather than signs or to look for markers of disease at the tree level rather than the leaf, branch, or root level (or by extension the vector population level rather than the individual insect). These approaches, described collectively as early detection technologies (EDTs), will detect the pathogen indirectly by detecting subtle changes in the plant triggered by pathogen invasion, prior to the onset of obvious visual symptoms of the disease.

One promising detection method is based on the chemical analysis of VOCs that are released by HLB-infected trees. Biomarkers specific to CLas have been found and could be analyzed using gas chromatography/mass spectrometry and gas chromatography/differential mobility spectrometry (Aksenov et al., 2014). Greenhouse tests showed that a mobile differential mobility spectrometry system was able to distinguish volatile differences

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

between closely related citrus cultivars and to show volatile-profile differences between healthy and infected citrus (McCartney et al., 2016). The use of canines to sniff out these volatiles specific to infected trees has been an effective strategy for citrus canker detection, and preliminary observations show promise for dogs to be able to detect HLB infections before symptom expression (Berger, 2014).

Detection of HLB through optical sensing has also been explored, but this technology is still being optimized and tested. Field experiments have demonstrated that optical sensors, such as a rugged, low-cost multiband active sensor that measures reflectance of tree canopy in two visible bands at 570 and 670 nm, and two near-infrared bands at 870 and 970 nm (Mishra et al., 2011), and a field portable spectroradiometer that collects the spectral reflectance data in the range of 350 to 2,500 nm (Sankaran and Ehsani, 2011) can be used to identify HLB-infected trees from healthy trees. Differentiation of infected and healthy trees based on tree canopy reflectance was also achieved using airborne multispectral and hyperspectral images (Kumar et al., 2012). These types of field imaging also yielded useful classifications of disease status if coupled with high-quality ground truth records (Li et al., 2012). Unmanned aerial vehicle imaging in combination with remote sensing and support vector machine technology produced higher classification accuracies than images obtained from aircraft (Garcia-Ruiz et al., 2013).

The data presented by invited speakers at the committee webinar22 on omics biomarkers are impressive and appeared to minimize the effects of cultivar, environment, time after infection, and CLas strain, but it is not clear if there are enough data to draw broad conclusions. Furthermore, whether the uneven distribution of the pathogen in trees, especially large field trees, affects biomarker quantity and quality is as yet undetermined.

Pitfalls

CRDF has not been at the forefront of funding research on CLas diagnostics, presumably due to the high incidence of HLB in Florida and the emphasis on disease management rather than disease detection. A basic tenet of plant disease management is to remove or abate sources of inoculum. This principle was discussed in the 2010 NRC report (Recommendation NI-2: Support searches for biomarkers that may be exploited to detect CLas-infected citrus.) along with the need to improve CLas diagnostics so the early infections could be detected and trees removed. The 2010 report did elaborate on the need to look beyond diagnostics that detect the

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22 Webinar on Citrus Greening (HLB) Diagnostics and Detection, August 24, 2017. Agenda and speaker information are located in Appendix B of this report.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

pathogen directly and to consider biomarkers that would allow detection of disease well ahead of detecting the pathogen.

Research results on remote optical sensing clearly identified differences in reflectance spectra between healthy and infected trees, but the differences were not detectable much before the onset of symptoms or detection by conventional pathogen diagnostics. An apparent shortcoming of efforts to employ canines for detection is that much of the canine training is done using potted, young trees rather than mature trees in a commercial grove setting. Nevertheless, canines may be useful for a quick preliminary screening of large acreages, and initial detections can be supported using laboratory instrument–based HLB diagnostics. Presentations at a committee webinar revealed data supporting the hypothesis that VOCs are cultivar independent (Davis, 2017), but it is not clear if there are enough data to indicate that VOC profiles of cultivars do not differ significantly.

Identifying ACP carrying CLas is more a problem of sampling than of detection since qPCR and other conventional assays can easily detect small copy numbers of bacterial DNA if it can be recovered from the insects. Because only a relatively small proportion of the ACP individuals are actually competent vectors (Coy and Stelinski, 2015), the insect sample size must be large enough to determine infection pressure accurately. Furthermore, the trapping method used to collect ACP must allow quality DNA to be recovered from the insects. Sticky cards are effective for trapping insects but not for the recovery of quality DNA. Alternative traps are being studied, but it is important to consider trap designs that will not only accurately estimate ACP seasonal phenologies and numbers but will also allow for accurate determination of CLas infection in the ACP population.

Future Directions

The choice of diagnostics must reflect the ultimate goal of the diagnostician. Identifying infected trees in commercial settings (orchards and nurseries) may demand different diagnostics than identifying CLas-infected ACP. Furthermore, as already mentioned, the major issue is not the sensitivity of pathogen detection but finding the pathogen within large trees occupying extensive acreages, or in large ACP populations. Continued efforts to improve the sensitivity of CLas diagnostics should be minimized with the possible exception of optimizing reliable, user-friendly, cost-effective, field-based diagnostics that can be used on the farm to test ACP immigrating into the orchards or trees in later stages of infection. These are unlikely to be effective early detection methods. The most critical and time-sensitive diagnostic need is to find and validate methods of detecting infection during the latency period when CLas titers are, for the most part, undetectable.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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These will be methods that indirectly detect disease (i.e., HLB) rather than the CLas pathogen.

In the area of volatile sensing, a comparison of canines and laboratory instruments to detect the disease in the same trees would be informative, as would parallel studies to see if the same trees can be identified on subsequent days, weeks, or months. It is important to know whether there are diurnal or seasonal patterns to VOCs and, if so, how they affect diagnosis using VOCs. Also important is whether infection by other pathogens or other stresses or farm inputs affect VOCs and HLB detection. For all of these parameters, cost–benefit ratios are needed to support decision making by growers.

Exploration of the use of biomarkers should continue, with comparisons of omics biomarkers and VOCs. Adding other diseases to the analyses seemed to lessen the separation between data clusters in the principal component analyses, which could be a major issue for the validation of omics biomarkers. Furthermore, additional biotic and abiotic stresses or farm inputs may have effects on the ability of biomarkers to separate HLB-infected and non-HLB-infected trees. All of these issues should be addressed in continuing research.

Although remote optical sensing has the potential for monitoring large acreages for symptomatic disease, it may not be an effective early detection diagnostic. Additional research is needed to address whether the reflectance spectrum in a large tree has a uniform distribution or whether it reflects the nonuniform distribution of the pathogen.

While Florida citrus is effectively 100% infected and early diagnostics might seem irrelevant to some, this view is short sighted because a future citrus industry in Florida will need to minimize CLas inoculum sources before starting over with either noninfected or HLB-cured trees that must be monitored for new or resurgent CLas infection. Such monitoring will require early detection technologies so this avenue of research should be a high priority for the entire U.S. citrus industry. Furthermore, the 2010 NRC report recommended the establishment of field test plots of trees whose health status is controlled so that any detection technologies could be validated on trees of different ages representative of trees in commercial orchards. This critical need has not been addressed anywhere in the United States and represents a significant impediment to the testing and validation of any EDT.

It is unlikely that “any one diagnostic” approach for HLB, targeting either the disease or the pathogen, will be sufficient. More likely, grower decisions on treatment will be based on results from a series of diagnostic assays. As well, diagnostics differ with respect to economics and throughput, increasing the likelihood that a variety of assays will be required in the future.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

Pertinent to the recommendations to pursue HLB rather than CLas diagnostics is the adoption of these technologies by the state and federal regulatory agencies responsible for delineating quarantine and regulated areas, certifying testing laboratories, and approving eradication and compensation programs. Currently, qPCR followed by conventional PCR and DNA sequencing are the approved methods that must be used by laboratories accredited under the National Plant Protection Laboratory Accreditation Program.23 A diagnostic result that will result in federal regulatory action must be confirmed by an appropriate federal laboratory, and currently federal confirmation requires direct detection of the pathogen. This means that any of the EDT diagnostics could not be used by regulatory agencies to make decisions, but research laboratories and, more importantly, growers and grower organizations are free to use information from the EDTs to make disease management recommendations and decisions. With this distinction in mind, one approach moving forward could be to work with the industry and growers to adopt EDTs as the primary tools for making management decisions, downplaying the regulatory aspects of diagnostics. The qPCR test is unsatisfactory from a plant pathology and disease management standpoint since trees are a source of inoculum long before the regulatory-approved diagnostics can provide information on the infection status. Data available from Brazil clearly support the basic tenets of vector-borne plant disease management—minimize inoculum and reduce vector pressure (Belasque et al., 2010; Bassanezi et al., 2013a,b; Ayres et al., 2015; Bergamin et al., 2016). While current strategies are attempting to reduce vector pressure, little is being done to identify and remove inoculum sources early on.

OVERARCHING FINDINGS, CONCLUSIONS, AND RECOMMENDATIONS FOR FUTURE RESEARCH EFFORTS IN HLB MANAGEMENT

Since 2010 the impact of HLB on the U.S. citrus industry has continued to worsen, not only in Florida but, as the disease moved westward through the southern citrus-producing states into California, in an increasing diversity of environments. Disease management approaches and research priorities for rejuvenating the heavily damaged citrus industry in Florida will differ in significant ways from those aimed at preventing similar devastation of the industries in other U.S. states which have yet to feel the full brunt of HLB. However, some of the research projects funded by CRDF (and other agencies) have led to a greater understanding of the interactions between

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23 Philip Berger, USDA Animal and Plant Health Inspection Service (APHIS) PPQ, Raleigh, North Carolina, personal communication, September 19, 2017.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

citrus and CLas and citrus and ACP, and the biology of CLas and ACP at the organismal and molecular levels, while others have yielded results that have led or may eventually lead to more effective HLB management strategies in any location. Some, despite investment and effort, have been unsuccessful, but even negative findings can be helpful in guiding future research directions.

Finding 4.1: Although research supported by CRDF and other agencies has advanced our knowledge of HLB since 2010, the disease remains an intractable threat to the Florida citrus industry and has progressed from an acute to a chronic disease present throughout the state.

The establishment of the Citrus Research and Development Foundation, with its formalized structure and framework for identifying, prioritizing, and allocating funding for research on HLB in Florida, was a response to a major organizational recommendation in the 2010 NRC report (O-2: Identify one organization and empower it to have oversight responsibility over huanglongbing [HLB] research and development efforts). The organizational framework of the CRDF has supported significant improvements to the effectiveness of HLB research prioritization, fund distribution, and communication within the Florida citrus production community.

Recognizing the threat of HLB to the U.S. industry, other research funding agencies (California’s Citrus Research Board, the Texas Citrus Producers’ Board, USDA NIFA and MAC competitive grants programs, as well as USDA ARS and APHIS, and others) are also supporting efforts to combat the disease. The disease in California and the southern United States is still considered acute, rather than chronic, and therefore requires site- and situation-appropriate management approaches that differ from those in Florida. Stronger communication and research linkages among those in the citrus-producing states will benefit all involved. Both the administrators and the researchers involved in these programs acknowledge the importance of communication, cooperation, and information and resource sharing at the interagency, interinstitutional, and interlaboratory levels, and a number of interactive planning meetings have taken place to address overarching needs.

Finding 4.2: A number of technical obstacles have been addressed by funded projects but continue to represent significant barriers to research progress and the generation of HLB solutions. Technical hurdles that remain unresolved include the following: transformation of citrus, in vitro cultivation of CLas, the need for optimizing nutritional approaches for maintaining tree health and productivity in the presence of HLB, the development of a transformable citrus model system, the development of diagnostic advancements addressing early detection (whole tree diagnostics) and improved

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

tree sampling, and the development of standardized methods for research protocols, controls, assay validation, and assessment, for enhanced reliability and comparison across studies. Resolution of any one of these issues would constitute a significant step, supporting, in turn, the advancement of a number of other research fronts.

Conclusion 4.1: Citrus growers, particularly in Florida, still need short-term solutions for the industry to remain viable while researchers continue to generate longer-term approaches for managing HLB.

Recommendation 4.1: Continue support for both basic and applied research and both short- and long-term research efforts.

Conclusion 4.2: Longer-term solutions to HLB are likely to involve citrus variety improvement, much of it derived from new molecular techniques.

Recommendation 4.2: Continue to support the development and application of gene modification, including gene editing, focusing on targets mediating molecular interactions among plant, bacteria, and vector.

Conclusion 4.3: HLB research is hampered by the lack of standardized methods and parameters for measuring, evaluating, and analyzing factors including vector transmission rates, fruit yield, plant tolerance and resistance, citrus variety performance, antibacterial compound effectiveness, and diagnostic assay evaluation. Inconsistency in experimental designs, sampling methods, and field investigations limit the ability to compare findings and use previous research as a springboard for further exploration.

Recommendation 4.3: Support the development of community-accepted standards for the conduct, evaluation, and assessment of research to facilitate comparisons of research results across teams and institutions.

Finding 4.3: Novel approaches to foster communication, collaboration, and innovation among HLB researchers and representatives of funding agencies and the citrus industry may advance research progress and facilitate solutions to HLB.

The committee discerned that, at a practical level, both the planning and the execution of HLB research still operate primarily within individual states, programs, projects, institutions, and laboratories. Although opportunities to share information have increased through periodic conferences such as the International Research Conference on Huanglongbing (most recently held in Florida in March 2017, sponsored by CRDF, California Citrus Mutual, California Citrus Research Board, Cutrale Citrus Juices USA, Fundecitrus, KeyPlex, SunKist, and Texas Citrus Mutual), these events have

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

focused primarily on brief, formal presentations of research results rather than futuring or brainstorming discussions to garner diverse community expertise and foster visionary thinking about HLB solutions. Furthermore, the confidentiality required by the development of patent applications has, to some extent, interfered with the sharing of information.

A number of researchers and funding agency representatives were invited to share their HLB expertise, experiences, research directions, and accomplishments in National Academies of Sciences, Engineering, and Medicine forums and webinars on HLB by addressing a set of focused, objective-based issues (see Appendix B). The committee noted that, in this setting and framework, most addressed the problem from broad and overarching perspectives and engaged in innovative and synergistic discussion of challenges, priorities, and solutions. Providing future opportunities in which members of the research community and the industry move beyond presentations of individual research outcomes and encouraging direct community effort to generate fresh, broad-based thinking could be a good investment.

Finding 4.4: There are inconsistencies in the format, content, and frequency of CRDF-funded research progress reporting by researchers, as well as in the inclusion of specific outcomes, impacts, and products.

With respect to the CRDF funding program in particular, this committee found a lack of consistency in regular reporting, by principal investigators, of research progress, including outputs, outcomes, and impacts, presentations at conferences, and publications. A number of projects have resulted in no, or relatively few, publications in peer-reviewed journals, and midterm and final research progress reporting to CRDF was inconsistent in both compliance and the amount of detail provided. For example, for 75 completed CRDF-funded research projects related to the Asian citrus psyllid, comprehensive final reports were available from only 11, of which only 8 listed publications. Of the 38 publications mentioned in those reports, 18 were published, peer-reviewed journal articles; 4 were articles in preparation or in press in peer-reviewed journals; 2 were articles to be published after a patent was received; 10 were abstracts or papers presented at conferences; and 1 was a dissertation. The requirement for annual reporting, using a standard, thoughtfully developed template that ensures the reporting of both research accomplishments and their current or expected impact on HLB solutions, could be a condition for continuation of funds in multiyear projects. Report information, like the scientific literature mentioned above, could then be used to populate a database for use by the research community.

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

Conclusion 4.4: Improved reporting consistency is needed to reduce constraints in reviewing research progress and delays in applying new information to HLB solutions.

Recommendation 4.4: CRDF should develop a standardized format, procedure, and timeline for mandatory reporting of midterm project research progress and final reports, to include outputs (publications and presentations), outcomes, practical applications, and impacts. CRDF should consider hiring a staff person to review and analyze HLB research findings annually.

Finding 4.5: More timely publication of research results in refereed scientific journals and trade journals would facilitate communication among the research community and between researchers and growers and support research assessment efforts.

Finding 4.6.1: Engaging with a disease that threatens the survival of an industry and requires a short-term and sustainable solution could benefit from a nonacademic research model or approach.

The committee observed that a majority of current HLB research efforts are taking place in academic settings in which the tenure and promotion system emphasizes publication and funding, training students and postdocs, and strengthening the careers of individual investigators, while discouraging out-of-the-box thinking and high-risk research. In this model, individuals or small groups typically compete for the same funding with relatively little coordination. It is a model that works in some cases but does not appear to be the best approach for dealing with HLB. With this in mind, CRDF might consider looking into adopting a product-focused industry-model of R&D and best practices. In the case of HLB research, this model could facilitate cross-institutional collaboration/teaming of multidisciplinary investigators; creating research teams (consortia) for each of the HLB research areas, with clear responsibilities set out for team leaders to review research and to communicate with other team leaders; and the setting aside of funds for innovative and/or high-risk projects.

Finding 4.6.2: Despite the commendable efforts of multiple funding agencies to coordinate funding and encourage appropriate interstate, interagency, and interdisciplinary collaborations, decisions about research funding priorities and allocations occur largely within the domain of each agency.

Conclusion 4.6: The committee concludes that the current system of research prioritization and funding, accomplished primarily within each relevant funding agency, is not optimally efficient and has not led to the

Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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.
×

development of an overarching master plan for HLB research and its translation to management solutions.

Recommendation 4.6: CRDF should consider working, together with representatives of other agencies at the national and state levels, to create an overarching HLB research advisory panel to develop a fresh, systems approach to HLB research prioritization and the strategic distribution of resources for research leading to effective HLB management.

This new approach could involve national-level and multiagency research prioritization and emphasize a strategic distribution of resources for research leading to effective HLB management. It could also encourage and facilitate timely and effective information sharing as well as translation of research outcomes to field-deployable actions. Finally, a multiagency approach could facilitate the achievement of coordination between regulatory, research, and production priorities.

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Suggested Citation:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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:"4 Notable Outcomes, Pitfalls, and Future Directions." 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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Next: Appendix A: Committee Biographies »
A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease Get This Book
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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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