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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease Summary Among the many diseases of citrus that have invaded or could invade Florida, greening represents the greatest threat to the industry. Greening has been known internationally, from its first description in the early 1900s in China, as huanglongbing (HLB, translated as “yellow shoot disease”). In Florida, HLB is associated specifically with the bacterium Candidatus Liberibacter asiaticus (CLas), the presumed causal agent. The insect vector, the Asian citrus psyllid (ACP, Diaphorina citri) acquires CLas by feeding on the nutrients carried by phloem cells in the citrus plant and injects the bacterium to other citrus plants. ACP was first detected in Florida in 1998. Shortly thereafter, ACP had spread to the point that eradication became unconceivable. HLB itself was discovered in Florida in August of 2005. HLB is now present in the 34 citrus-producing counties of Florida, but is most prevalent in the southern areas of the state. In April 2009, the National Research Council, at the request of the Florida Department of Citrus (FDOC), formed the Committee on the Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease (Huanglongbing). In developing a strategic plan, the committee was charged to examine: 1) the current citrus disease situation in Florida and the status of public and private efforts to address citrus greening and other diseases; including lessons learned; 2) the capacity of the industry to mobilize a scientifically based response to current disease threats and to translate scientific advances into products and services for the protection of Florida citrus industry in the short and long term, and 3) the relationship of the industry to public, academic, and private research, and to regulatory and funding organizations at the state and federal level, with respect to controlling citrus greening and developing a comprehensive solution to citrus greening and other diseases. In the course of its deliberations, the committee made the following key findings. KEY FINDINGS Citrus products are generally appreciated as being both desirable and healthful, and this appreciation is justified. Without Florida citrus production, orange juice and other citrus products in our stores would be fewer, less diverse, possibly of reduced quality, and likely more expensive. Citrus is emblematic of Florida. There would be great repercussions for Florida’s economy if the estimated $9.3 B annual economic benefit of the citrus industry were to be lost or significantly diminished. Florida’s citrus is an industry very worth preserving.
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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease HLB and HLB control measures reduced Florida orange juice production by several percent by 2008, and losses will likely increase, at least in the immediate future and possibly longer. The two major parts of the Florida citrus industry are the larger juice processing sector and the more profitable, per acre, fresh market sector. The incompletely defined dynamic between citrus production and the processing plants is such that when plants operate below their most productive level, profits are threatened. If a plant closes, producers suffer reduced marketability of their oranges. Thus, declining production can induce a downward spiral. In the Florida situation, HLB is entirely correlated with the presence of, and is presumed to require the participation of, CLas and its insect vector ACP. In Brazil and in Florida, rigorous three-pronged programs have had a demonstrated saving effect in areas not yet severely affected by HLB. The programs rely on (i) production of citrus propagation materials in insect-proof facilities, which has been mandatory in Florida nursuries since January, 2008, (ii) strong reduction of the ACP population, and (iii) identification and removal of infected citrus trees, the principal reservoir of CLas. Although these programs have reduced the percentage of infected trees in some areas, the numbers of infected citrus trees in Florida as a whole continues to increase. Increased use of insecticide sprays, as currently required for successful suppression of ACP populations, brings with it risks of ACP developing resistance to one or more of the most useful insecticides and of adverse affects on beneficial insects. More information on ACP behavior and HLB ecology and new approaches are needed to improve ACP suppression. The identification and removal of CLas-infected citrus trees is currently dependent on scouting for visible HLB symptoms. The process is expensive, not sustainable for many orchards, and very likely would be greatly improved if infected but as yet asymptomatic trees could be identified. The most powerful long-term HLB management tool likely will be citrus cultivars resistant to CLas and preferably to ACP as well. However, there is no clear path by conventional breeding to deliver a robust resistant citrus for many commercial species because these species lack known sources of resistance and a facile breeding system. It is likely that the breeding systems for sweet orange and some other citrus can be greatly enhanced in the long run by capabilities derived from genome sequence analysis and other technologies. Genetic engineering, in the form of transgenic citrus or citrus inoculated with a transgene-expressing virus vector, holds the greatest hope for generating citrus cultivars resistant to CLas and ACP. New information on CLas, ACP and citrus, and the advances of modern biology and chemistry in general, suggest new research directions that may reveal new strategies for HLB mitigation.
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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease The citrus industry and various government agencies have made very significant investments in research on HLB and approaches to HLB mitigation. Results usable in effective HLB mitigation will probably be derived more rapidly from research projects if efforts are more unified than they are at present, on the national and international level, and there is more emphasis on strategic planning. When (or if) HLB no longer is a significant threat, an integrated research infrastructure would continue to serve the industry well. The Florida Citrus Industry The favorable conditions for citrus production in Florida have allowed the industry to grow to the point of having a $9.3 billion annual economic impact on the state. Throughout the Florida citrus industry’s history, this success has been tempered and defined by the adverse effects of the natural calamities of freezes, hurricanes and diseases, and, in more recent times, by urbanization, international competition, and shortage of water. Due to the Great Freeze of 1894–1895, citrus production was abandoned in northern Florida and in other southeastern US states and has become concentrated in mid- and south-Florida. The Florida freezes of the 1960s provided an opening for the expansion of the Brazilian citrus production. Brazil now accounts for about 37 percent of the worldwide citrus production, whereas the United States’ share is about 17 percent. In 2004–2005, hurricanes were largely responsible for the reduction in sweet orange (38 percent) and grapefruit (69 percent) production compared to the year before. Urbanization has displaced citrus production from most of Florida’s coastal areas, leaving the majority of the production in the more freeze-prone inland areas. The seasonality of Florida rainfall means that virtually all Florida citrus is irrigated. A more subtle factor influencing the citrus industry is the interaction between production and processing, as indicated in Key Finding 4, above. Citrus Greening (Huanglongbing) and its Florida Vector, Asian Citrus Psyllid The first observations of diseased citrus corresponding to what is now recognized as HLB were in southern China in the late 19th century. In the mid-20th century, the Chinese researcher K.H. Lin provided a scientific description of HLB and demonstrated its infectious nature. At present, it is estimated that nearly 100 million trees in 40 countries are affected by HLB. In the 1960s–1980s, HLB devastated citrus production in the Philippines, Indonesia, Thailand, and South Africa. In March 2004, HLB was recognized for the first time in the Americas in São Paulo State, Brazil. Subsequently, nearly 3 million HLB-affected sweet orange trees were removed in Brazil. HLB was found in Florida in 2005. In North America, HLB now occurs in Cuba, Belize, Florida, Georgia, Lousiana, South Carolina, and Eastern Yucatan, Jalisco, and Nayarit, Mexico. HLB enters a geographical region in an introduced psyllid vector or in infected live plant material. In the field, psyllid transmission (acquisition, retention through a latent period, then inoculation) is the primary mode of HLB spread. Thus, introduction and establishment of a vector species, which in Florida is ACP , always precedes establishment of HLB. In the principal mode of plant-to-plant transmission with an established psyllid population, older immatures acquire the bacteria, which circulate and propagate in the insects’ bodies while they complete
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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease development to adulthood; then adult psyllids inoculate healthy citrus during feeding. It is also possible for adults to acquire the bacteria. When HLB enters a region hitherto free of the disease, young trees show symptoms earlier and are more severely affected than mature trees. The symptoms of HLB include the appearance of yellow shoots and a blotchy mottle of the leaves. First symptoms may appear in 6–18 months. Severe symptoms may appear as early as 6 months post infection in young trees but more typically at 1–5 years, especially for mature trees. The disease progress in the orchard can be relatively fast, reaching an incidence of more than 95 percent in 3 to 13 years after onset of the first symptoms. Yield per tree is reduced and fruits of affected branches may have color inversion or be abnormally small and lopsided and have aborted seeds. “Off” flavor and small size could cause the fruit to be rejected even for juice production. There are no known citrus species, varieties or combinations of scion and rootstock that are immune to HLB. The Causal Agent of Huanglongbing Almost all HLB-affected citrus trees worldwide are infected with at least one of three recognized bacteria of the genus Liberibacter. In a few cases, HLB symptoms have been associated with phytoplasmas and not Liberibacters, in São Paulo State and China. However, in Florida, the universal association of CLas and ACP with HLB erased virtually all doubt about whether CLas should be a prime subject of research aimed at mitigating HLB. Recently, the complete genome sequence of CLas has been obtained by using deoxyribonucleic acid (DNA) extracted from a psyllid and DNA from infected plants. Detection and Mitigation of Huanglongbing Thermotherapy, shoot-tip grafting or antibiotic treatment can rid budwood of CLas, but antibiotic treatment of orchard trees has not proven to be a practical control method. Vigorous pruning of symptomatic parts of the tree has given only short term relief, if any. Nutritional sprays intended to suppress HLB symptoms have been used in some orchards. However, to date, controlled comparisons have not demonstrated an improvement in crop yield or fruit quality or any long term amelioration of symptoms or reduction in CLas titer from these treatments, and their use can diminish the effectiveness of mitigation measures based on removing sources of inoculum. In the absence of curative treatments or resistant citrus lines, the available method for coping with HLB once the disease has been discovered in a region is the 3-pronged appoach of Key Finding 6. Production of healthy citrus trees in “closed,” insect-proof nurseries is established practice in Florida. The other two prongs of current HLB mitigation are reducing inoculum sources and suppressing ACP. RECOMMENDATIONS Shaped by the key findings and its study of scientific opportunities to addresss HLB, the committee’s strategic plan consists of 23 recommendations, listed in Box S-1, and grouped according to objective: Organizational (O); Informational (In); Near- to Intermediate-Term Research and Technology (NI); and Long-Term Research and Technology (L). The recommendations are listed in approximate order of importance within each group. However, the
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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease success of some recommendations is dependent on other recommendations, requiring coordination in their implementation. BOX S-1 RECOMMENDATIONS Organizational (O) Recommendations O-1. Create “Citrus Health Management Areas” in Florida. O-2. Identify one organization and empower it to have oversight responsibility over huanglongbing (HLB) research and development efforts. O-3. Create a centralized HLB website and data bank that is accessible to researchers and the public. O-4. Commission an analysis of the economics of the citrus industry’s responses to HLB. O-5. Organize an enhanced annual international symposium on all aspects of HLB. Informational (In) Recommendations In-1. Expand extension efforts emphasizing the importance to HLB management of removing infected trees from groves. In-2. Encourage homeowners to remove and properly dispose of backyard citrus trees, particularly HLB-affected trees. In-3. Communicate information on HLB and its potential economic impact to government officials at the federal, state, county, and city level. Research and Technology Recommendations: Near- to Intermediate-Term (NI) NI-1. Improve insecticide-based management of Asian citrus psyllid (ACP). NI-2. Support searches for biomarkers that may be exploited to detect CLas-infected citrus. NI-3. Establish citrus orchard test plots for evaluation of new scouting and therapeutic methods. NI-4. Accelerate the sequencing, assembly, annotation and exploitation of a sweet orange genome to provide a powerful tool for all future citrus improvement research. NI-5. Support development of HLB model systems. NI-6. Exploit the CLas genome sequence for new strategies of HLB mitigation. NI-7. Support research aimed at developing alternative ACP management strategies. NI-8. Support small-scale studies on the feasibility of alternative horticultural systems suited to endemic HLB. NI-9. Support demonstration of RNA interference (RNAi) effects for possible suppression of ACP. NI-10. Develop in vitro culture techniques for Clas to facilitate experimental manipulation of the bacterium for insights into gene function. NI-11. Sequence, assemble and annotate the ACP genome to provide basis for new approaches to ACP management.
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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease Research and Technology Recommendations: Long-Term (L) L-1. Support development of transgenic HLB-resistant and ACP-resistant citrus. L-2. Support development and testing of bactericides, therapeutics or SAR activators. L-3. Support analysis of ACP behavior, ACP-plant interactions and ecology to enhance the knowledge base available for new ACP management strategies. L-4. Explore possible control strategies based on release of modified psyllid males. The scientific basis for each recommendation and its synergy with others are discussed in detail in Chapter 4 of the full report. Near- to Intermediate-Term (NI) research and technology projects are those that can generate proof-of-principle or demonstrate HLB mitigation in less than 5 years. They are most important for the current management of HLB and need to be pursued in conjunction with Organizational (O) and Informational (In) approaches. NI research also can contribute essential knowledge for Long-Term (L) research and technology, which will take more than 5 years to develop, but that offer the greatest opportunity for succesful long-term management. In the sections that follow, several of the key recommendations are broadly discussed (not necessarily in the order presented in Box S-1) in the context of their general roles in the overall strategy, namely: achieving an organized response to HLB, addressing the current, urgent needs of the citrus industry, and developing the technology to sustain the industry in the future. Individual research and technology projects that support these roles are further described in the section on Essential Research Directions. Responding to the Huanglongbing Threat Industry and government both have responded to the HLB threat by significantly increasing the research effort. Citrus research has been funded by several national, regional, state, and citrus industry organizations. Following the detection of HLB in Florida, the Florida Citrus Production Research Advisory Council (FCPRAC), which directed funds to research through competitive grants program, dedicated nearly all of its funds to HLB and canker research. In 2007–2008, the FCPRAC, the state of Florida, and the FDOC, spent $1.5, $3.5, and $2 million, respectively, for HLB and canker research. In June of 2008, the newly launched Florida Citrus Advanced Technology Program (FCATP) issued an Request for Proposal (RFP) which resulted in 205 proposals. These proposals were reviewed by National Research Council-appointed panels. The FCATP awarded over $16M to the funded projects. Currently, the management of industry-connected competitive research grants program is the responsibility of the newly formed Citrus Research and Development Foundation (CRDF). An important goal is improved coordination of research funding and research project monitoring (Recommendation O-2). Chapter 5 of the full report provides an extensive explanation and comparison of four models for support of research and development that may be useful in considering how to fund the needed research. These are research grants, sponsored research, contract research, and inducement prizes. Each of these models has advantages and disadvantages for advancing particular research or development project.
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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease The incursion of HLB has been more effective than any prior event in bringing industry, government, and universities together in the defense of citrus production in Florida. Local, state, and international meetings have been organized. Attendance at such meetings has been excellent, and growers have had numerous opportunities to become current on HLB and recommended practices. Information transfer can be further enhanced by increased use of website-accessible data banks (Recommendation O-3) and an annual international research meeting strategically planned to maximize the possibilities for synergistic interactions among research groups and information transfer among stakeholders (Recommendation O-5). Various advisors hold differing views on the best management practices for HLB. There has not always been a consensus, particularly with regard to the urgency of efforts in discovery and removal of HLB-infected trees. Nonetheless, there is a general recognition of the need to develop regulations that meet the needs of the entire industry without excessively burdening any segment. The Citrus Health Response Program (CHRP) was initiated by the USDA and FDACS, with considerable input from the University of Florida and industry groups. Although aspects of CHRP regulations were considered to be onerous, especially for the nursery industry, the current system of production of all citrus trees in screened enclosures is one result of this effort that has benefited everyone in the industry. A more complete understanding of the already realized and the potential economic impacts of HLB, and of alternative approaches to dealing with the HLB problem, is needed. More complete knowledge of economic impacts will allow fine-tuning of research priorities for the urgent task of keeping the citrus industry viable, while new approaches to longer term HLB mitigation are being advanced (Recommendations O-4 and In-3). Actions Needed to Enhance the Current Management of Huanglongbing in Florida There is reason to be optimistic about the prospects of a future Florida citrus industry that will rely on sustainable methods for HLB mitigation not available at present, but likely to be developed using the concepts and technology of modern biology. In the meantime, Recommendations O-1, In-1, In-2 and NI-1 have a high priority because of their potential for sustaining production until the to-be-developed approaches can be implemented. Citrus Health Management Areas (Recommendation O-1) should be created to facilitate and coordinate control of ACP and removal of affected trees, both in commercial orchards and in residential areas (Recommendations In-1 and In-2). These areas are envisioned to be 10,000–50,000 acres consisting of regions with similar levels of HLB incidence. Each management area would be charged with dealing with threats to citrus production by mandating best management practices for clean-up of abandoned HLB-affected orchards, designing and implementing compensation plans, coordinating area-wide psyllid sprays, and reducing risk from infected urban citrus. Area-wide insect control strategies should utilize “window” strategies where only certain classes of insecticides are permitted during specific periods to minimize the development of resistance. Recommendation NI-1 is intended to empower Recommendation O-1 by promoting more integrated efforts to improve the practices for insecticide control of ACP: (i) improving the surveillance methods for ACP, (ii) registering new insecticides and developing new insecticide application protols, (iii) developing pesticide application protocols informed by ACP behavioral ecology and ACP CLas transmission biology, and (iv) coordinating cultural practices with insecticide applications.
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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease In addition, Recommendations NI-2 (identifying biological markers of infection) and NI-3 (creating test plots to validate biomarkers and new treatments) have the potential to greatly improve the effectiveness of efforts under Recommendation O-1. The new approaches would incorporate biomarkers specific for CLas infection and instrumentation designed to efficiently detect those biomarkers, e.g., plant volatiles analyzers, optical tree analyzers, plant tissue-inserted electrochemical probes. These and other research approaches that can enhance HLB management in the short-term are discussed in the Summary section on Essential Research Directions. Actions Needed for the Sustainable Management of Huanglongbing in the Future The consensus approach to the long-term management of HLB is to deploy citrus lines resistant to CLas and ACP, which can be accomplished in principle by: (i) classical genetics and conventional plant breeding, (ii) genetic transformation to produce transgenic plants, or (iii) introduction of a bacteria- or virus-derived DNA vector bearing a resistance gene of interest. At this time, conventional plant breeding is unlikely to deliver resistant varieties because many commercial citrus species lack a functional breeding system and no genetically compatible source of resistance has been found. This situation renders genetic engineering (options ii and iii) as more viable for developing citrus with resistance to CLas and/or to ACP (Recommendation L-1). A variety of research projects can support achieving this long-term goal, such as completing the sequencing of the sweet orange genome (Recommendation NI-4), the genomes of CLas and ACP (Recommendations NI-6 and NI-11), and exploring the potential to use RNA interference to suppress the psyllid (Recommendation NI-9). These are elaborated further in the section on Essential Research Directions. Should HLB become unmanageable in Florida with current production approaches, it will be necessary to grow citrus differently in the future. Recommendation NI-8 encourages the exploration of new advanced citrus production systems employing high density plantings or screen houses or both, These production systems may be able to reduce infection rates or compress and enhance the citrus production cycle so that economic return can be realized before losses to HLB dominate. The economic viability and manageability of these systems over the long term also need to be investigated (Recommendation O-4). ESSENTIAL RESEARCH DIRECTIONS Discussed below are some of the Committee’s specific recommendations aimed at enhancing the response to HLB and improving the mitigation of HLB through the use of various scientific approaches and technologies. Improving Detection of Huanglongbing Detection of CLas-infected trees for removal is based on visual scouting for HLB symptoms and discovers only symptomatic trees. Often the presence of CLas is confirmed by polymerase chain reaction (PCR) analysis. HLB-infected trees are pulled or cut down, followed by stump treatment to prevent the appearance of infected shoots. If more than a few percent of the trees in
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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease a grove must be removed each year, production will not be sustainable, at least under present conditions of citrus culture. If CLas-infected trees can be detected and removed sooner than visual scouting allows, the numbers of inoculative ACP likely will be reduced significantly due to reduced period of ACP access to infected trees, fewer infected trees and parts of trees, and reduced average CLas titer in the trees. Biomarkers are changes in biochemical, physiological or physical properties of an organism that are indicative of specific biologic states, e.g., asymptomatic CLas infection. Recommendations NI-2 and NI-3 promote a multidisciplinary search for CLas-infection biomarkers in citrus. Biomarkers may be discovered (i) by direct analyses for changes in the prevalence of specific nucleic acids, proteins, other macromolecules or small molecules, including tree volatiles, or in tree optical signals, or (ii) by bioinformatic analysis of changes in gene expression, to predict infection-specific metabolic alterations. Improving Suppression of Asian Citrus Psyllid Populations Several factors challenge efforts to suppress ACP populations in Florida. Compared to temperate climate areas, Florida’s sub-tropical climate allows more generations of insects to be produced and hence requires more intensive management practices. Citrus is a perennial crop, and CLas multiplies in ACP, which can then efficiently inoculate the bacterium for life. Can biological control of ACP work to reduce HLB disease progress? ACP in Florida is attacked by many generalist predators and a number of hymenopterous parasitoids but most effectively by coccinellid beetles. Classical biological control of ACP has been applied in Florida with the release of the tiny wasp parasitoid Tamarixia radiata. This parasitoid became established, but with little affect on ACP. Introduced predators and parasitoids have suppressed several long-established hemipteran species (aphids, whiteflies, and scale insects) over decades in Florida. However, to reduce CLas transmission (i.e. acquisition and inoculation) requires more severe reductions in ACP populations than has been seen for other hemipterans. The only report of successful HLB mitigation through biological control was accomplished in Reunion Island, a small, non-continental landmass, unlike Florida. Thus, biological control alone is not likely to reduce ACP populations sufficiently to provide HLB mitigation. To date, significant reductions in psyllid populations have been achieved in Florida only through the application of insecticides. Recommendations for ACP suppression, based on research, are published in an annual University of Florida management guide. This guide influences grower management practices. However, growers often experiment as well, leading to variable practices in orchards. Presumably, implementation of Recommendation O-1 would result in more uniform practices. The principles of integrated pest management (IPM) are becoming and should become more influential. There are continuing efforts to improve the monitoring of ACP populations, by employing new insect traps, sentinel plants, and strategies for monitoring pest movement through mark and recapture experiments. Generally, production managers are aware of the increasingly important and potentially adverse consequences of inappropriate application protocols and frequent insecticide sprays, including farm worker exposure, the development of resistance to insecticides, reduction in numbers of beneficial insects, and contamination of groundwater. Additionally, the economics of heavy reliance on insecticides must be carefully examined and compared with other methods of control. These comparisons should address not only production costs, but also other costs such as potential harm to the environment (see Recommendation O-4).
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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease Psyllid females strongly prefer to lay eggs in the new flushes of growth that are produced by all citrus, but occur on young trees throughout most of the year. Insecticide applications can be timed to protect flushes, whether they occur as the result of the natural growth cycle of citrus or because of tree hedging operations. Young trees require aggressive ACP suppression involving the soil-applied systemic insecticides aldicarb or imidacloprid. Other recommendations take into account the relatively low psyllid populations that occur during winter, when broad-spectrum foliar sprays may be applied to mature trees to suppress populations of overwintering adults. Production managers and researchers are actively testing modifications to current approaches. Low-volume spraying is gaining widespread use; it allows application of products to large areas in a short time, which is a central component of area wide management approaches to HLB mitigation (Recommendation O-1). New active ingredients and adjuvants are being examined for their ability to provide more effective and more environmentally friendly ACP suppression. The reservoir of scientific knowledge of psyllids has expanded since the discovery of ACP in Florida in 1998. However, additional knowledge about the behavior of and chemical communication by ACP, infected with CLas or not, has potential for revealing points at which the disease cycle might be interrupted (Recommendation NI-7 and L-3). Data support the existence of female sex pheromone(s) and plant volatile attractants. Several observations suggest that specific chemical compounds can attract or repel ACP and could be used in strategies which direct ACP to congregate at sites where their destruction could be more easily accomplished. Knowledge of the ACP and CLas genomes (Recommendations N-11 and NI-6) may contribute significantly to acquiring and interpreting the needed new information. Information on CLas effects on ACP and CLas transmission rates underlies predictions on HLB spread and other HLB investigations. However, this information is very limited, and current estimates of transmission rates vary widely. Methods that potentially could be applied to ACP suppression include the sterile insect technique (SIT) and related genetic technology that would involve release of reproductively debilitated ACP males. However, the dense populations achieved by ACP, the serial mating behavior of ACP females, and the lack of methods for mass rearing of ACP all speak against the possibility of HLB mitigation by altered ACP release. Therefore, Recommendation L-4 advocates limited or no investment in SIT and ACP genetic technology at this time. Genetic Transformation of Mature Citrus Tissue Many protocols for citrus transformation have been published, most initiated from juvenile citrus tissue. For a practical citrus-transformation program with a reasonable time to creation of productive stock, methods for the transformation of mature citrus tissue, or an equivalent process using altered juvenile tissue, will probably be required (Recommendation L-1). Once a system for citrus transformation is developed, several transgenic mechanisms for citrus resistance become possible. Expression of anti-Clas agents is one mechanism. A potential source of anti-Clas agent is the various anti-bacterial peptides produced by both microbial and higher organisms. Many of these peptides are well characterized as to their target and specificity. Various proteins and peptides with specific insecticidal activity could be produced in citrus from transgenes. Systemically acquired resistance (SAR), which is the activation of a systemic defense reaction in a plant, is also currently being exploited by both chemical and transgenic approaches. Another approach has ACP as the target and applies the phenomenon of RNA silencing (see
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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease Recommendation NI-9) activated by double-stranded RNAs that would be phloem-delivered in the transgenic plant to adversely affect feeding insects. A Plant Virus-Derived System for Gene Expression in Citrus The Citrus tristeza virus (CTV), itself a significant pathogen under certain but avoidable circumstances in Florida, has been modified by the laboratory of W.O. Dawson of the University of Florida to create a virus gene-expression vector. Unlike almost all other virus-based vector systems for gene expression in plants, some of the CTV vector constructions were found to maintain gene expression for more than 6 years rather than for days or weeks. The usual application for a transient expression vector such as CTV is as a test bed for evaluating gene constructions intended for later use as transgenes. However, the longevity of expression shown by CTV constructions could allow direct application of this technology to confer commercially useful resistance to CLas and to do so in significantly less time than would be required for creation and deployment of a resistance gene in genetically transformed citrus. Whether new genes are introduced by citrus transformation or by application of a CTV vector, there will be requirements for field testing and to satisfy regulatory requirements. There will also be intellectual property considerations derived from the gene constructions or transformation methods used. These are time-consuming and costly steps that must be taken after the particular approach and construction have passed the proof-of-concept stage. However, there is another important hurdle: public acceptance, which will require public education. Prophylactics and Therapeutics A few current research projects aimed at mitigating HLB use model systems that do not include citrus or CLas or ACP in experiments, or may include only one of them. None of the three, citrus, CLas or ACP, is an easily tractable experimental subject. As the text for Recommendation NI-5 describes, high throughput assays have been or can be developed that use multi-well plates to contain infected or uninfected seedlings or plant stem sections, individual insects, or individual bacterial cultures. Genome sequence information (Recommendations NI-4, NI-6 and NI-11) may guide the selection of chemical libraries to be screened in the multi-well plates in a quest for new bactericides or other therapeutic agents, or prophylactic agents, for use in HLB mitigation (Recommendation L-2) or for the discovery of ACP repellents. The screening and testing of such agents would be materially assisted if in vitro culture techniques for CLas were available (Recommendation NI-10). A model system could be the most direct route to an anti-CLas spray that could protect existing plantings, i.e., a so-called “silver bullet.” CONCLUSION HLB is the latest, and most serious, challenge that nature has presented to the Florida citrus industry. A successful response will require acceptance of the urgency of the situation and willingness to cooperate and persist in managing the disease in the near- and mid-term while new solutions are being developed and implemented. The HLB problem requires an unprecedented degree of cooperation among producers, processors, government officials, and scientists, and of
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Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease course among their respective institutions. However deficient is our current arsenal for fighting HLB, the potential for progress against this disease remains distinctly hopeful.