A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation Fighting a Ravaging Disease (2018) / Chapter Skim
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4 Notable Outcomes, Pitfalls, and Future Directions
4 Notable Outcomes, Pitfalls, and Future Directions
Pages 113-190
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From page 113... ...
The committee also provided recommendations and considerations for future research (see Boxes 4-2, 4-4, and 4-6 and Future Directions sub sections) for each of the research areas.
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• Asian citrus psyllid (ACP) biology and reproductive behavior have been char acterized extensively.
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• CLas alters the biology of both citrus and ACP, impacting disease develop ment: infected ACP disperse earlier and farther than uninfected individuals, infected females are more attractive to males than uninfected females, and CLas infection alters leaf color and plant volatiles, rendering the plant more attractive to ACP. BOX 4-4 Recommendations for Future Research: Interactions of the HLB Pathogen, Vector, and Host • Continue to characterize CLas effectors, including identification and functional analyses of their host targets, and apply new knowledge to the development of novel HLB management tools.
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• HLB-tolerant citrus rootstock and scion genotypes and HLB-resistant citrus relatives have been identified, providing material for further breeding as well 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 L iberibacter asiaticus (CLas)
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Host Resistance: Genetic Engineering • large number of transgenic citrus expressing genes that may confer resis A tance to HLB have been produced and are being tested. • The genomes of a number of citrus and citrus relatives have been sequenced.
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• Create a strong research effort to assess and model the nature and impacts of sociological and economic factors influencing decision making relevant to HLB management in the Florida citrus industry and among consumers and the general public. • Assess diverse, multifaceted combinations of HLB management approaches under different environmental conditions and production regimes, with the objec tive of designing a flexible system of combinatorial recommendations tailored to the needs of individual growers in different citrus production areas.
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From page 119... ...
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
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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.
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Other microbiome research has focused on the role of diseasecontrolling properties of citrus microbiome communities, or of particular microbial species, as potential approaches to HLB management. These e fforts are described further in the Bacterial Control section of this chapter.
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The research focus on genome sequencing (Recommendation NI-6: E xploit 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.
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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.
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, 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)
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. Dispersal Dynamics of the Asian Citrus Psyllid.
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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)
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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.
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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)
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Host Orientation, Assessment, and Acceptance. Among the most n otable achievements over the past decade with respect to vector–host inter ctions were characterizing ACP behavior and ACP–plant interactions a 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)
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, 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 r esearch 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.
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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
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, 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.
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CLas infection enhances citrus susceptibility to citrus canker and counteracts disease resistance induced by exogenous application of SA, a result attributable
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. Tran t scription 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)
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i 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 inter actions 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.
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, 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.
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, 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)
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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.
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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 pproaches has addressed the 2010 Recommendation NI-8: Support smalla scale studies on the feasibility of alternative horticultural systems suited to endemic HLB.
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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)
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; 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)
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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 12 n index based on the number of species per specified number of individuals, and the A number of species per unit area (species density)
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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.
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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.
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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.
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Pitfalls Pesticides effective against the ACP have been a mainstay for HLB management programs in Florida. However, Hall et al.
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. However, these programs have not provided consistent results or reliable levels of HLB suppression across the Florida citrus industry.
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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.
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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.
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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 indi vidual compounds (Zhang et al., 2010; M
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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.
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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)
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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.
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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 bio technology.
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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)
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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.
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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)
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. In response to the 2010 Recommendation NI-4: Accelerate the sequencng, assembly, annotation and exploitation of a sweet orange i 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)
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. 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)
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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)
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found that a majority of surveyed Florida con umers reported willingness to purchase genetically engineered citrus if s cost incentives were provided (see also Future Directions in this chapter, and Chapter 3)
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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)
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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)
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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)
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. 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)
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Future Directions The choice of diagnostics must reflect the ultimate goal of the diagnostician. Identifying infected trees in commercial settings (orchards and nurseries)
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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.
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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.
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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]
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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.
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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.
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, outcomes, practical applications, and impacts. CRDF should consider hiring a staff person to review and analyze HLB research findings annually.
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2013. Stylet morphometrics and citrus leaf vein structure in relation to feeding behavior of the Asian citrus psyllid Diaphorina citri, vector of citrus huanglongbing bacterium.
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2016. Acquisi tion, replication and inoculation of Candidatus Liberibacter asiaticus following various acquisition periods on huanglongbing-infected citrus by nymphs and adults of the Asian citrus psyllid.
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2015. Chemical control of the Asian citrus psyllid and of huanglongbing disease in citrus.
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2014. Odorants for surveillance and control of the Asian citrus psyllid (Diaphorina citri)
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2013. Silencing abnormal wing disc gene of the Asian citrus psyllid, Diaphorina citri, disrupts adult wing develop ment and increases nymph mortality.
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2016. Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri)
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2013b. Asian citrus psyllid, Diaphorina citri, vector of citrus huanglongbing disease.
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2014. Asian citrus psyllid genome (Diaphorina citri, Hemiptera)
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, for biological control of Asian citrus psyllid, A Diaphorina citri (Hemiptera: Psyllidae)
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2017. Combining 'omics and microscopy to visualize interactions between the Asian citrus psyllid vector and the huanglongbing pathogen Candidatus Liberibacter asiaticus in the insect gut.
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2015. Vibrational duetting mimics to trap and disrupt mating of the devastating Asian citrus psyllid insect pest.
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2014. Insecticide sprays, natural enemy assem blages and predation on Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae)
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2010. Transmission parameters for Candidatus Liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae)
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2014. Insecticidal suppression of Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae, vector of huanglongbing pathogens.
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2016. Quality matters: Influences of citrus flush physicochemical characteristics on population dynamics of the Asian citrus psyllid (Hemiptera: Liviidae)
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2011d. Effect of Candidatus Liberibacter asiaticus infection on susceptibility of Asian citrus psyllid, Diaphorina citri, to selected insecticides.
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2016. Feeding behavior of Diaphorina citri and its transmission of "Candidatus Liberibacter asiaticus" to citrus.
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2011. Chemical compounds effective against the citrus huanglongbing bacterium "Candidatus Liberibacter asiaticus" in planta.
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