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3
Citrus Greening Research and Development and Industry Preparedness

This chapter is concerned with research results from recently completed and ongoing research projects and their potential contributions to huanglongbing (HLB) mitigation. We begin with a discussion of research on the presumed causal agent of HLB, Candidatus Liberibacter asiaticus (CLas), and then turn to an analysis of research that is related to and supportive of current HLB mitigation practices, principally identifying and removing HLB-affected trees and reducing Asian citrus psyllid (ACP) populations in orchards. Since it is generally agreed that citrus varieties resistant to CLas or to ACP and preferably to both would provide the most sustainable HLB mitigation tool, we summarize research on citrus breeding, genetics, transgenics, including the potential of Citrus tristeza virus (CTV)-derived vectors for introduction of resistance traits into citrus. Possible contributions of new citrus cultural practices and research model systems to HLB mitigation are considered. Finally, we summarize the history of funding support for pest management and for citrus research and development generally and the importance of establishing communication channels for citrus researchers. An inventory of HLB research projects that are ongoing can be found in Appendix J and a list of HLB research milestones from 1956–January 2009 is provided in Appendix K.

CLAS GENOMICS AND CULTURE

The demonstration by researcher H. K. Lin in China in 1956 that the HLB agent was transmissible by graft inoculation established the infectious nature of this pathogen. As is described in Chapter 2, by 1970 electron microscopy had revealed bacterial cells with cell walls to be residing in the phloem tissue of citrus showing HLB symptoms. By 1974, the bacteria were shown to be Gram-negative. This bacterium, the only viable candidate as the causative agent of HLB in Florida, is designated CLas. See Chapter 2 for a discussion of the currently known HLB-associated agents and other information on CLas.



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3 Citrus Greening Research and Development and Industry Preparedness This chapter is concerned with research results from recently completed and ongoing research projects and their potential contributions to huanglongbing (HLB) mitigation. We begin with a discussion of research on the presumed causal agent of HLB, Candidatus Liberibacter asiaticus (CLas), and then turn to an analysis of research that is related to and supportive of current HLB mitigation practices, principally identifying and removing HLB-affected trees and reducing Asian citrus psyllid (ACP) populations in orchards. Since it is generally agreed that citrus varieties resistant to CLas or to ACP and preferably to both would provide the most sustainable HLB mitigation tool, we summarize research on citrus breeding, genetics, transgenics, including the potential of Citrus tristeza virus (CTV)-derived vectors for introduction of resistance traits into citrus. Possible contributions of new citrus cultural practices and research model systems to HLB mitigation are considered. Finally, we summarize the history of funding support for pest management and for citrus research and development generally and the importance of establishing communication channels for citrus researchers. An inventory of HLB research projects that are ongoing can be found in Appendix J and a list of HLB research milestones from 1956–January 2009 is provided in Appendix K. CLAS GENOMICS AND CULTURE The demonstration by researcher H. K. Lin in China in 1956 that the HLB agent was transmissible by graft inoculation established the infectious nature of this pathogen. As is described in Chapter 2, by 1970 electron microscopy had revealed bacterial cells with cell walls to be residing in the phloem tissue of citrus showing HLB symptoms. By 1974, the bacteria were shown to be Gram-negative. This bacterium, the only viable candidate as the causative agent of HLB in Florida, is designated CLas. See Chapter 2 for a discussion of the currently known HLB- associated agents and other information on CLas. 67

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68 STRATEGIC PLANNING FOR THE FLORIDA CITRUS INDUSTRY CLas Genomics The complete genome sequence of CLas has been obtained by using DNA extracted from a single psyllid carrying a high titer of CLas (Duan et al., 2009). Metagenomic analysis of DNA from phloem of CLas-infected citrus revealed more than one CLas genome per phloem cell and only trace representation of any other bacterial cell (Tyler et al., 2009). Although the analysis was not capable of revealing viruses, the result suggests very strongly that CLas alone is the causal agent of HLB in Florida. Recent work by Wulff et al. (2009) and Duan et al. (2009) have shown that the genomes of both Clam and CLas appear to be circular with 3 ribosomal ribonucleic acid (rRNA) operons and similar size. Analysis of the Clas genome has revealed many other properties of the organism, particularly the absence of pathogenicity systems involving toxins, enzymes or specialized secretion systems. The absence of such specialized secretion systems (referred to as Type III secretion systems or T3SS), which are common in Gram-negative bacteria that are pathogenic to humans, animals, insects, and plants, had led to speculation that other mechanisms of pathogenicity may be involved (Bové and Garnier, 2003). The gene for a bacteriophage DNA polymerase, discovered in 1993, has now been shown to be part of a bacteriophage DNA genome associated with HLB on citrus (Gabriel and Zhang, 2009). These results raise the question of a possible role of liberibacter phages in HLB. Sequencing also has revealed genetic diversity among CLas isolates collected in Southeast Asia (Tomimura et al., 2009). Obtaining the CLas genome sequence has added to our knowledge of HLB, but how this information can contribute to HLB mitigation remains to be demonstrated. The genomes of several other bacterial plant pathogens have been sequenced. These include the genomes of Xylella fastidiosa, causal organism of citrus variegated chlorosis (and Pierce’s disease of grapevine), Xanthomonas axonopodis pv. citri, causal organism of citrus canker, and Spiroplasma citri, causal agent of citrus stubborn disease. These bacteria have been cultured on synthetic media. The genomes of several phytoplasmas, which have not been cultured, have also been completed. In each case new candidate pathogenicity genes have been discovered and in some cases those genes have been functionally confirmed to contribute to pathogenicity. An interesting example is the onion yellows phytoplasma. Its genome sequence revealed that it possesses specific genes that allow it to send protein molecules out of the sieve tubes to carry out functions beneficial to the phytoplasma. This protein, called Tengu, is believed to inhibit an auxin-related pathway which affects plant development (Hoshi et al., 2009). CLas Culture When HLB was first detected in Florida in 2005, the three citrus liberibacters, CLas, Candidatus Liberibacter africanus (CLaf), and Candidatus Liberibacter americanus (Clam), were known, but no evidence indicates any agent for HLB in Florida other than CLas (Tyler et al., 2009). In 2008, co-cultivation of the Asian HLB-associated bacterium (CLas) with Gram- positive actinobacteria was reported by a research group in Florida, but a pure culture of CLas could not be obtained (Davis et al., 2008). In 2009, a report on cultivation of all three HLB- associated Liberibacters and fulfillment of Koch’s postulates was published by Sechler et al. (2009). However, neither the Sechler et al. (2009) result or previous reports of successful axenic

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CITRUS GREENING RESEARCH AND DEVELOPMENT AND INDUSTRY PREPAREDNESS 69 culture of Liberibacters (Garnett, 1985; Whitlock and Chippindall, 1993) have been repeated in other laboratories. RESEARCH SUPPORTING CURRENT HUANGLONGBING MITIGATION PRACTICE: REMOVING HUANGLONGBING-AFFECTED TREES A major problem in managing HLB is the long latent period between inoculation of CLas by ACP and the appearance of symptoms visible to scouts or the accumulation of CLas in a manner conducive to its ready detection by polymerase chain reaction (PCR). Firstly, the long latent period affects the thinking of growers who embark on a program of infected tree removal because, in the first 2 to 3 years of the program, the fraction of trees that is found to be HLB infected may increase even though the reservoir of inoculum actually is decreasing, i.e., the program is successful but appears to be not successful. Secondly, CLas-infected trees may may remain in the grove for months or years without apparent symptoms but with the ability to serve as a source for acquisition of CLas by ACP. At present, there is no practical method for detecting infected trees before they show visual symptoms. The problem is accentuated by the uneven distribution of CLas in the tree, which manifests itself in sectored development of symptoms, with some branches remaining asymptomatic. If infected but asymptomatic trees can be rogued, the result may be far more effective at CLas inoculum reduction than is possible with current scouting practice, if for no other reason than a reduction of months in the time of interaction between infected tree and psyllid. Also, the extent of CLas spread within the tree can be expected to be less for an asymptomatic tree than a symptomatic tree. Irey et al. (2006) tested trees for CLas by PCR by using plots of about 190 trees. Leaf sampling was from the two most recent flushes and from 3 of the 4 sides of each tree, but was otherwise undirected. DNA was recovered from petioles and midribs. PCR detected about 60 percent additional HLB-infected trees beyond those detected by visual assessment alone (6.5 percent of the trees in the plots were visually HLB positive; 4.1 percent were PCR positive but visually negative). Given the difficulties of detecting infected trees by PCR analysis, the above results suggest that for every symptomatic tree in an orchard there will be at least one infected asymptomatic tree. Among the separate plots, the numbers of PCR-identified infected trees was well correlated with the total number of infected trees, R2 = 0.89, suggesting a natural progression of infected but asymptomatic trees to symptomatic trees. Although the average CLas titer in asymptomatic trees was significantly less than the titer in symptomatic trees (M. Irey, United States Sugar Corp., Clewiston, FL, personal communication), some asymptomatic trees showed a high titer, reinforcing the importance of identifying and removing infected but asymptomatic trees. More than 80 percent of the PCR-positive trees were within 25 m of a symptomatic tree (Irey et al., 2006), indicating significant secondary, short-distance spread (Gottwald et al., 2007) but also long-distance spread not revealed by symptom development at the time of sample taking. Presumably, the distant infected but asymptomatic trees will become foci for future local HLB spread. These observations suggest that:(i) the pathogen is readily spread by the vector to adjacent or nearby trees; and (ii) spread over longer distances also occurs, possibly caused by dispersing vectors. Alternatively, dispersal over long distances could result from farming practices. Pre-symptomatic trees have been reported to be sources for CLas acquisition by ACP, but with reduced efficiency compared to acquisition from symptomatic plants (Coletta-Filho et al., 2009).

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70 STRATEGIC PLANNING FOR THE FLORIDA CITRUS INDUSTRY Sweet orange maintained in a greenhouse developed symptoms at about 90 days after being graft-inoculated with CLas-infected tissue. However, CLas was detected in extracts of leaf midribs at 30 days after inoculation. The CLas DNA accumulation was about 50,000-fold greater at 90 days than at 30 days (Li et al., 2006; Alvarez et al., 2007), suggesting that at least some CLas-infected but asymptomatic orchard trees will have low CLas titers and a significantly diminished ability to serve as HLB sources compared to symptomatic trees. Detecting a CLas-infected tree by analysis for CLas is the obvious but not necessarily the most efficient approach to discovering infected trees. Perhaps the asymptomatic but CLas- infected tree is altered sufficiently, even in parts of the tree not yet infected, to develop a specific altered-state signal, a biomarker, that would telegraph the presence of infecting CLas. A CLas- infection-specific, or even general infection-specific, biomarker may be based on changes in tree volatiles production, optical properties, or chemical properties, including accumulation or loss of specific messenger RNAs (mRNAs) (see section on the citrus transcriptome, below), proteins or metabolites. These changes may be exploited as disease-specific biomarkers either directly or may be used to predict the identities of biomarkers after intensive bioinformatics analysis that can document changes in metabolic pathways and anticipate alterations in the concentrations of specific small, including volatile, molecules. Even a biomarker that was not activated by asymptomatic infection but would efficiently reflect a symptomatic infection could be of value by allowing scouting to become partially automated. Some plants have been reported to release biologically active volatile organic compounds (VOCs) in response to infection by a specific pathogen, including fungi, bacteria and viruses (Huang et al., 2005; Cardoza and Tumlinson, 2006; Medina-Ortega et al., 2009; Werner et al., 2009). Profiling of volatile metabolites has been used to discriminate among pathogens infecting apples (Vikram et al., 2004). Production of a new, citrus canker-specific volatile by infected and symptomatic grapefruit leaves has been reported (Zhang and Hartung, 2005). Preliminary results suggest that CLas-infected and uninfected citrus could be distinguished based on VOCs analysis (Dandekar et al., 2009). Modern analyzers, such as the differential mobility spectrometer (DMS), have the capability of detecting VOCs in the parts-per-trillion range and the potential to be reduced to portable, field-mobile devices suitable for use in the orchard (Davis et al., 2010; Hao et al., 2009). Should these results be validated, the availability of a practical VOC-based detector of CLas-infected trees nevertheless is likely to be several years away. In hyperspectral imaging, data from a broad section of the visible or visible-plus-infrared spectrum are collected across the image of an object and analyzed to produce a signature of the object that, in the case of a plant, may reflect its physiological state (Aleixos et al., 2002; Du et al., 2004; Blasco et al., 2007; Mishra et al., 2007; Nicolaï et al., 2007; Du et al., 2008; Lee et al., 2008; Qin et al., 2009). Hyperspectral data have been reported to detect changes in the chemical composition of plants (Ferwerda, 2005; Tilling et al., 2007) revealing, for example, the nutrient and water status of irrigated wheat (Tilling et al., 2007). Spectral differences have been detected in comparisons of healthy and HLB-affected citrus using detached leaves and leaves of intact citrus trees (Mishra et al., 2007; Tilling et al., 2007; Lee et al., 2008; Poole et al., 2008). Hyperspectral imaging is very data intensive, and its practical application in an orchard would presumably require the use of multiple detectors, field-rugged computers and sophisticated software packages that may require significant money and time to develop. However, if a few essential components of the hyperspectral image can be identified, far less elaborate spectral analyses may be sufficient to provide biomarkers for HLB. Surface-enhanced Raman spectroscopy (Zeiri, 2007; Kiefer, 2008; Driskell et al., 2009) and laser-induced fluorescence

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CITRUS GREENING RESEARCH AND DEVELOPMENT AND INDUSTRY PREPAREDNESS 71 (Panneton et al., 2010; Malenovsky et al., 2009) are other spectral methods that have found application for detection of plant biomarkers. In current practice, symptomatic leaf samples are collected from orchard trees suspected of being CLas-infected, and the samples are transported to a laboratory for analysis. There is a critical need for compact, user friendly, low-cost, field-deployable instruments or devices for rapid and high-throughput identification of CLas-infected trees, symptomatic or asymptomatic, in the orchard. Devices under consideration may rely on simple preparation of extract to be analyzed or on a microelectrode to be inserted into tree tissue. Classes of compounds to be detected include nucleic acids, antigens and small molecules. For example, a research project is aimed at developing a novel oligonucleotide microarray technology that will generate a colorimetric signal after amplification and rapid nucleic acid hybridization. Nucleic acid purification, amplification and hybridization occur in a device about the size of a credit card by lateral flow chromatography on a nitrocellulose membrane (Carter and Cary, 2007). This technology is similar to the home pregnancy test kit, and a prototype was tested in a grove in the fall of 2009 where it detected CTV, the target to which it was designed . Other devices are being developed that are designed to provide cost-effective surveillance for several pathogens or traits simultaneously by relying on multiplexed analyses and computational methods. In higher organisms, small RNAs (miRNA) are often induced rapidly and specifically by invading pathogens, making miRNAs attractive potential biomarkers for early and specific disease diagnosis (Zhang et al., 2006). The Citrus Transcriptome The citrus genome sequence, when it becomes available, will provide a list of all of the candidate genes of the citrus tree and the complete amino acid sequences for almost all of the proteins those genes encode. Part of the key to understanding the function of a given gene resides in the molecule that serves to transfer the information encoded in genomic DNA to the cell machinery that synthesizes the protein, i.e., the mRNA molecule. Many of the genes on the candidate list will be so well known from prior genomic and biochemical work with other organisms that functions can be assigned to the corresponding gene products (proteins) with a high degree of certainty. The functions of many other genes will remain a mystery. What will also be almost entirely unknown will be what genes are active in what tissues of the tree and at what stage in development or disease state of the tree. Although protein synthesis is not strictly proportional to mRNA synthesis in any cell, a specific protein will not be synthesized without the corresponding mRNA being present. Thus, identifying what mRNAs are accumulating in what tissues and at what time and under what conditions of stress, i.e., characterizing the transcriptome, can be very informative for various approaches to combating disease or improving productivity. Analysis of the transcriptome and the corresponding global accounting of the proteins of the organism, designated as the proteome, can result in significant new understanding of the etiology of HLB. Changes in gene expression due to bacterial infection have been observed for citrus, including a few infection-related changes that precede symptom development (Albrecht and Bowman, 2008; Cernadas et al., 2008; Kim et al., 2009), suggesting that it may be possible to develop the desired biomarkers. The technology used in these investigations is the DNA array, or gene chip. Tens of thousands of synthetic short DNA molecules, corresponding to known mRNA sequences of an organism, are immobilized on a surface in a regular array of spots. Fluorescently

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72 STRATEGIC PLANNING FOR THE FLORIDA CITRUS INDUSTRY labeled molecules corresponding to RNA extracted from specific tissue of the organism are exposed to the array. Hybridization reactions result in fluorescent molecules being bound to the array. The pattern of fluorescent spots on the array is interpreted to discover what mRNA molecules accumulate in enhanced or depreciated amounts under specific conditions in specific tissue. The DNA arrays used in these experiments would not have been possible without earlier work by members of the International Citrus Genome Consortium (http://www.citrusgenome.ucr.edu) that identified tens of thousands of “expressed sequence tags” (ESTs, i.e., sequences from mRNAs). More sensitive and comprehensive methods have recently been developed for identifying differences in mRNA accumulation between infected and healthy tissue. In the “RNA-seq” approach, cell RNA is fragmented and the fragments are copied into DNA. High throughput sequencing methods are applied to millions of these DNA molecules to give an in-depth picture of the identities and relative amounts of the RNA molecules of the sample (Wang et al., 2009). One example is gene silencing, wherein a particular gene is "switched off" by machinery in the cell in response to changes in the environment or pathogen invasion. RESEARCH SUPPORTING CURRENT HUANGLONGBING MITIGATION PRACTICE: REDUCING ASIAN CITRUS PSYLLID ACCESS TO CITRUS Suppression of ACP by Insecticide Application Currently much effort is being expended by university, government and private industry researchers with the goal of developing new, more effective and safer insecticides and insecticide application technologies. Equally important is the goal of maintaining the effectiveness of valuable insecticides by delaying or avoiding the appearance of insect resistanct to the insecticide. Costs, effectiveness, run-off in the environment, applicator safety and effects on non- target organisms are other important considerations affected not only by the choice of insecticide but also by application methods. Among the many insecticides registered and recommended for ACP, the only listed soil-applied insecticide that reduces ACP populations on large trees is aldicarb. Reliance on a single insecticide or class of insecticide will increase the likelihood of insect resistance. Furthermore, reliance on aldicarb is also of concern from an environmental and health standpoint. Other alternative insecticides should be used and, in recent years, a suite of insecticides with novel modes of action have been developed. Insecticides are now formally classified into groups based on mode of action (IRAC, 2009). There are newer insecticides (e.g. spinetoram) from new insecticide classes that are recommended for ACP. These products have restrictions on the number of times they may be used so that resistance does not evolve rapidly occur. Other products such as cyazypyr and its derivatives also hold some promise for control of ACP. These groups and still others present many options for controlling ACP, and collecting new information about how they can be most effectively deployed to control ACP is likely to be a major research goal for the foreseeable future. Success in ACP management will require improvements in monitoring of ACP populations and of the development of ACP resistance to insecticides. Importantly, more extensive incorporation of IPM principles is needed, including development of appropriate insecticide resistance management (IRM) programs. There are programs (such as in cotton in Australia) where good IRM programs have been practiced continuously with the result that some

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CITRUS GREENING RESEARCH AND DEVELOPMENT AND INDUSTRY PREPAREDNESS 73 insecticides remain effective for decades. In a “window strategy,” application of only insecticides of a specific class is allowed within a given Citrus Health Management Area during a given time period (e.g., a 2-month window), followed by rotation to an insecticide of another class. Such rotations have been the foundation of the IRM program. Rotation delays the evolution of resistance to any of the insecticides used (Roush, 1989; Forrester et al., 1993). There may be concerns about implementing such a strategy (e.g. several companies promoting their products simultaneously in the same location), but history has shown that a window strategy is effective in countering development of insect resistance to insecticides. Some insecticides have long residual action, so there is increased concern about the development of insecticide resistance, because insect populations are exposed to sub-lethal doses of the insecticide over a longer period of time. However, Boina et al. (2009) have shown that when citrus is treated with imidacloprid, ACP adults and larvae that feed on the plant may become exposed to sublethal concentrations, and these can have negative developmental and reproductive effects which nevertheless could lead to population reductions over time. Cocco and Hoy (2009) conducted laboratory studies showing that certain adjuvants had the effect of increasing ACP mortality while reducing the mortality of its parasitoid. Sterile Insect Technique The sterile insect technique (SIT) applies to pests that reproduce sexually and works effectively if sterilized males are sexually aggressive and successfully compete with indigenous males for mating with females (Knipling, 1955; Knipling, 1985). This method should be considered for use only with a serious pest where thorough knowledge exists of its biology, ecology and behavior and methods have been developed of its mass rearing. The most commonly used method for inducing sexual sterility in insect pests is by radiation emitted from radioisotopes such as caesium-137 or cobalt-60 (Bushlad and Hopkin, 1953; Lindquist, 1955). The dosage of radiation applied must have no significant adverse effect on the male longevity, searching behavior or mating ability. Matings between sterile males and wild females do not yield offspring. Thus, if sufficient numbers of sterile males are released, most indigenous females will mate with sterile males, the number of individuals in the wild population will be reduced in the next generation. Thus, the concept of SIT uses continued releases of high- quality sterile males to overwhelm wild-type males over successive generations and result in progressively reducing the indigenous population to levels of extinction. If SIT can be developed for a pest, it is the key component of an area-wide integrated pest management program. SIT has been used successfully with a variety of pests. The most well-known is the screwworm fly, a pest that feeds in open wounds of cattle and other animals. Screwworms were mass-reared and irradiated. Sterile male screwworm adults were then releeased by the millions into populations of indigenous screwworms reduced in size by insecticides (Knipling, 1955; 1985). Pesticide application is necessary to reduce the feral population and increase the proportion of sterile males to indigenous males. The SIT has now also been used against the pink bollworm moth codling moth, false codline moth, painted apple moth, cactus moth and others. Unfortunately, current SIT technology does not appear to be appropriate technique for suppression of ACP. No mass-rearing program has been developed to produce ACP in large enough numbers to make irradiation and SIT feasible. Furthermore, nothing is known about

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74 STRATEGIC PLANNING FOR THE FLORIDA CITRUS INDUSTRY radiation methods to sterilize psyllids or what consequences irradiation and sterility may have on male longevity, flight capacity or mating behavior. ACP Trap Plant A trap plant may be of a distinct species from the crop plant, or it could be a peripheral planing of the crop plant. In an example of the latter, a 10-m zone of papaya trees is planted as a trap crop around the main papaya groves, which helped reduce damage from the papaya fruit fly, Toxotrypana curvicauda (Aluja et al., 1997). The ornamental citrus relative, Murraya paniculata (orange jasmine, mock orange), is known as a preferred host plant for ACP. M. paniculata has been considered as a candidate psyllid trap plant but actually is unsuitable because it is also a host for CLas. Transformation of this plant with a gene that encodes an ACP feeding toxin has been proposed. However, it is likely that under orchard conditions the proportion of ACP on M. paniculata or any other known candidate trap plant would be small relative to the proportion on the commercial citrus trees, which are themselves good hosts of the psyllid. This difficulty might be overcome by pairing trap plants with the use of repellents on citrus (see section below on Distraction of ACP by Chemical Attractants and Repellants). Guava as ACP Repelling Plant Several groups of scientists from Australia, the United States, Brazil, France, and Spain that visited the Mekong Delta of South Vietnam have noted that mixed orchards of guava (Psidium guajava, Xa Li and Bom cultivars) and citrus (mostly King mandarin, and pummelo cultivars Nam Roi, Da Xanh, and Doan Hung) near My Tho, Vinh Long, and Can Tho have very low populations of ACP and show very few HLB symptoms. In contrast, nearby guava-free citrus orchards have high psyllid populations and high percentages of HLB-affected trees. These observations have been made in what are essentially commercial orchards in collaborations between growers and the South Vietnam Fruit Research Institute (SOFRI) at My Tho. Other plant species (i.e. banana, longan, mango, and durian) have not demonstrated this effect. The guava effect is pronounced only under the following conditions: (i) the main crop and citrus is only used as an intercrop (i.e., typically one citrus tree surrounded by four larger guava trees); (ii) guava trees have been planted and become well-established one year before the citrus trees were planted; and (iii) guavas and citrus are planted close together. The guava affect occurs year- round, which suggests that leaves rather than fruit are the source of protection. The guava- induced protection is not sufficient to keep the psyllids completely out of the orchard, especially once trees become two to three years old, an age when they must be supplemented with one or two insecticide treatments per year. Protection is diminished once the citrus trees are taller than the guavas. The citrus trees themselves may exhibit symptoms of HLB , and the number of HLB-affected citrus trees increases as the mixed orchard reaches three to four years of age. The high ratio of guava trees to citrus trees needed to achieve protection does not seem compatible with current Florida citrus production, but guava trees could find application in orchards following the Advanced Citrus Production System (described below). Field experiments have been set up to test the protective power of guava under Florida conditions.

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CITRUS GREENING RESEARCH AND DEVELOPMENT AND INDUSTRY PREPAREDNESS 75 Distraction of ACP by Chemical Attractants and Repellents Research at the University of Florida is aimed at identifying and developing attractants for ACP, for both monitoring and management. Male ACP colonize in great numbers on those citrus plants that are currently or were previously infested with virgin or mated female ACP, suggesting that female ACP may produce a sex pheromone that attracts conspecific males, a suggestion supported by olfactometer results (Wenninger et al., 2008). Results of behavioral tests and electrophysiological experiments indicate that the ACP uses both visual and olfactory cues in orientation to host plants (Wenninger et al., 2009b). Analyses by gas chromatography and mass spectrometry indicated that both sexes of ACP produce several volatile compounds, with some compounds specific to each sex. Research is underway to develop methods to use attractants to recruit parasitoids into groves to improve biological control. These results have not yet been published and should be considered preliminary. Infection by specific aphid-transmitted viruses has been shown to produce apparently aphid-attracting VOCs (Medina-Ortega et al., 2009; Werner et al., 2009), suggesting another approach to identifying ACP-attracting volatile compounds. Protection of citrus from ACP in mixed guava-citrus orchards is described above, research is also underway to develop a repellent for ACP, based on the volatiles released by guava and their effects on ACP behavior. Results from laboratory experiments suggest that the guava effect is due to volatile compounds produced by the guava plants, which may be acting as psyllid repellents, or by masking or counteracting the citrus volatiles that attract the psyllids. Young and old guava leaves are equally active as a source of repellents and male and female psyllids are similarly repelled. It has been shown that crushed guava leaves were more repellent to ACP than intact guava leaves. Analyses of volatiles from crushed and intact “white” guava leaf flushes were compared (Rouseff et al., 2008). Undamaged guava leaves yielded five sulfur volatiles, and crushed leaves yielded an additional sulfur volatile, dimethyl disulfide (DMDS). Leaves of sweet orange, grapefruit, and rough lemon, crushed or intact, did not give off DMDS, and DMDS is highly repellent to ACP. Sulfur compounds are highly toxic to most insect species due to the disruption of the cytochrome oxidase system of the insects’ mitochondria (Dugravot et al., 2004). In olfactometer studies by Noronha and Bento (2008), intact guava plants without crushed leaves repel psyllids, so the protective effect of guava in mixed orchards likely is not due to DMDS. This observation does not diminish the merits of DMDS as a potential psyllid repellent. Repellent effects of available DMDS formulations have remained for 3 to 4 weeks after distribution of dispensers, which should be long enough for preliminary field studies to assess exclusion of ACP from the groves. If DMDS is effective and new formulations, perhaps with other active ingredients included, can extend the longevity to 15 weeks or more, field trials could lead to registration with the US EPA. Registration will be costly and may require from 8 to more than 55 months, but could result in an elegant short-term form of HLB mitigation. Trap plants have been proposed for use in conjunction with feeding repellents of ACP compounds such as DMDS in a “push-pull” strategy to concentrate ACP populations into a zone for eradication by insecticide application. Potential feeding repellent compounds include pymetrozine and Flonicamid (Harrewijn and Kayser, 1997; Bedford et al., 1998; Polston and Sherwood, 2003; Bextine et al., 2004; Morita et al., 2007), which have worked for suppression of

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76 STRATEGIC PLANNING FOR THE FLORIDA CITRUS INDUSTRY other pathogen vectors. If feeding on citrus can be sufficiently disrupted, ACP transmission capability may be reduced along with ACP numbers. The push-pull strategy has been considered as a component of an area wide ACP-HLB management program. HUANGLONGBING EPIDEMIOLOGY AND ASIAN CITRUS PSYLLID BEHAVIORAL ECOLOGY There remains a great need for more information on HLB epidemiology under Florida conditions; such information could provide the basis for creation of management/mitigation programs or regulatory programs in the state. Some current information on CLas spread in the orchard is presented above in the above section on research and mitigation practices. The currently funded projects on HLB epidemiology are listed in Appendix J. Several of these projects are ongoing in Brazil, while others are being conducted in Florida. The specific research topics include transmission (i.e. via seed), effects of control measures on HLB spatio-temporal progress, comparative epidemiology of HLB caused by CLas and Clam, and alternative hosts/host range of CLas. Understanding and Exploiting ACP Biology “Psyllids are probably the most benign of the Sternorrhyncha and therefore the least well studied” (Percy, 2010). That is, compared to other hemipterans such as aphid, whitefly, mealy- bug and scale insects (all phytophagous sucking insects), few psyllid species are significant pests of crop and ornamental plants. Moreover, most psyllids have tropical distributions and are found on woody plants (few herbaceous crops and no grains are hosts to psyllids), and therefore tend to receive less attention from entomologists than do non-pest species. What was known about ACP prior to 1998 is from studies of the insect in Asia and islands in the Indian Ocean (Halbert and Manjunath, 2004). Subsequently, entomologists, primarily in Florida, took up the study and the number of publications on ACP expanded exponentially. In his review, Hall, (2008) remarks that “entomologists are in the discovery phase of research detailing information on the biology, behavior, ecology and biological control (a topic of Chapter 2) of D. citri in hopes of finding weak points in psyllid populations that could be exploited to help curb disease transmission and reduce the need for chemical control.” Research topics have included life history, biology and behavior, host plant relationships, sampling, vector/pathogen relationships, and vector control strategies. Nevertheless, some aspects of ACP have been neglected or have received insufficient attention, as indicated below. Behavioral Ecology Study of adult and immature ACP behavior, movement, feeding, CLas transmission and other interactions with plant hosts (Wenninger et al., 2009b) could yield information applicable to reducing HLB spread. Spread of HLB in Florida occurs primarily by the dispersal of psyllid vectors, but could potentially be spread by citrus nursery stock including ornamental citrus. Relatively little is known about the flight behavior of ACP. Some reports indicate limited, short- distance dispersal; others suggest longer migratory flights. It is likely that both occur. Extensive

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CITRUS GREENING RESEARCH AND DEVELOPMENT AND INDUSTRY PREPAREDNESS 77 information about aphid flight behavior (Vialatte et al., 2007; Lankin-Vega et al., 2008; Klueken et al., 2009) could guide ACP investigations. Information on psyllid flight could facilitate improved sampling methods for monitoring ACP with benefits to management approaches. Although the technology would not be applicable to citrus, an example of an approach that has been successful in reducing transmission of virus diseases has been the development of reflective mulches to repel viruliferous aphids from vegetable crops (Brown et al., 1993; Stapleton and Summers, 2002). Research on the behavior of ACP adults has revealed aspects of probing (stylet penetration) behavior and of communication by means of substrate vibration and semiochemicals (Wenninger et al., 2008; Wenninger et al., 2009a; Wenninger et al., 2009b; Bonani et al., 2010). The nymphs deserve more attention than they have received because their unique and specialized lifestyle may make them more vulnerable to manipulation than adults. As with other psyllids, ACP nymphs are highly specialized. They are relatively immobile, live closely aggregated and are seemingly vulnerable. Fourth and fifth instar nymphs have been found to acquire CLas efficiently and become, as adults, inoculators of HLB. ACP nymphs almost always are found on the new flush of growth of their hosts. Psyllids likely have evolved behavioral tactics to protect themselves in the developing colony. Two strategies employed by other hemipterans whose nymphs are closely aggregated are alarm pheromones (Nault and Phelan, 1984) and myrmecophily (beneficial association with ants) (Hölldobler and Wilson, 1990). Aphid alarm pheromones have been used to manage aphid pest populations (Dawson et al., 1990; Guedot et al., 2008), but no studies report a search for alarm pheromones in any psyllid species. There are a few anecdotal reports of ants collecting honeydew from ACP nymphs and from the nymphs of other psyllid species, suggesting the possibility of facultative myrmecophily. Chemical communication between ants and their attendant hemipterans (Nault et al., 1976) could lead to the discovery of new semiochemicals and novel approaches to reducing ACP populations. There may be predator and parasitoid avoidance vibrational signals (Cocroft, 2001; Wenninger et al., 2009a) and evasive behaviors that are critical to nymph survival. Modifications of host leaves induced by salivary secretions (Luft et al., 2001), recruitment of ants by nymphs to avoid predators (Novak, 1994) and chemical (or tactile) communication between ants and psyllids are other aspects of nymph behavior that could inspire new control measures in the longer term. Two aspects of ACP nymphal development deserve close attention. The first is the ability of the immature psyllids to alter the biochemistry and morphology of their hosts and create the protective niches in which they live. Developing plant tissues may be responding to compounds in ACP saliva. Isolation and characterization of these putative chemicals could lead to new ways to alter psyllid populations as has been suggested by Miles (1999) for aphid salivary enzymes. The second subject concerns ACP nymphs’ incorporation of their honeydew into non-sticky, waxy filaments that are readily carried away from colonies by wind and rain. Learning more about the chemistry of ACP excreta would be worthwhile. In addition to protecting nymphs from fouling their environment, the excreta may contain compounds that protect them against predators, parasites and pathogens, or conversely volatiles that attract the enemies of predators and parasites. Such compounds could lead to new ACP management strategies. Vector-pathogen Interactions Information on CLas transmission rates and CLas effects on ACP is needed to provide the foundation for epidemiological models and predictions of HLB spread and other HLB

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82 STRATEGIC PLANNING FOR THE FLORIDA CITRUS INDUSTRY starting explants from vigorous, optimized citrus trees having a very low population of contaminating bacteria and fungi. A coordinated and focused effort to develop mature citrus transformation systems is underway in Florida in several institutions or laboratories. Emphasis is put on citrus plant pre- conditioning and parameters affecting transformation and regeneration. Also, to gain time, the transformation technology for mature citrus tissues, which has been developed in Spain, will be transferred to Florida, where new laboratory and greenhouse facilities are being established. Because mature tissue transformation is not yet available in Florida and juvenile citrus transformation is more efficient and less cultivar specific than mature citrus transformation, a project on accelerating the production and commercialization of transformed juvenile citrus is also being developed. Several approaches have been taken to minimize or eliminate the problem of juvenility. These include use of high-quality sweet orange clones selected for reduced juvenility and development of a universal transgenic rootstock that promotes flowering by expression and transport into the scion of a flowering-inducing protein. Another strategy for curtailing the maturation period of juvenile citrus is transformation with genes that accelerate flowering, such as the Arabidopsis LEAFY or APETALA (Peña et al., 2001). Attention is being given to another approach to introducing resistance to CLas into varietal scions: genetic transformation of citrus rootstock. If expression of the transgene results in the production of a resistance-mediating molecule that can be secreted in the rootstock and effectively mobilized across the graft union, the rootstock could be used to confer resistance or tolerance to a variety of non-transgenic, fruit-producing citrus scions. Use of mature scions would avoid the long juvenility phase. TRANSGENES FOR RESISTANCE TO CLAS In order for GE of citrus to contribute to HLB mitigation, transgenes capable of conferring resistance to CLas or ACP or tolerance to CLas must be identified and incorporated into constructions suited for expression and proper targeting in citrus. Several research groups are working on the isolation and testing of candidate genes. Anti-bacterial Peptides Anti-bacterial peptide (ABP) genes have received early attention because this class of molecules represents one of the few known with members capable of acting against CLas and other bacteria. ABP genes can be found in humans and animals (Myeloid antimicrobial peptides, human beta-defensin-1, Polyphemusin-1, Tachyplesin, Protegrin-1, magainin, indolicidin, and others), insects (cecropins, Sarcotoxin IA, pyrrhocoricin, and other), and plants (defensins, and others). The underlined peptides are examples of those that have already been evaluated in citrus against the liberibacters. In 2009, field trials for transgenic citrus were established in Florida. Currently under evaluation for resistance to citrus canker and HLB are (i) a Carrizo citrange that carries a gene that encodes for a plant-based anti-microbial peptide, being tested by Southern Gardens Nursery LLC and Texas A&M University; and (ii) Carrizo citrange and grapefruit that carry a proprietary gene called ‘Disease Block”(i.e. a phage-based protein that disrupts the outer membrane of Gram negative bacteria but does not lyse the cells), being tested by Southern Gardens and Integrated

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CITRUS GREENING RESEARCH AND DEVELOPMENT AND INDUSTRY PREPAREDNESS 83 Plant Genetics. No field resistance data have been generated as of December 2009 but preliminary laboratory data for the transgenic citrus indicate efficacy against citrus greening (M. Irey, United States Sugar Corporation, Clewiston, FL, personal communication). Genes Involved in Systemic Acquired Resistance (SAR) SAR is an important defense mechanism in plants which, when activated by a specific microorganism or chemical, can provide protection against a variety of challenging pathogens. The Arabidopsis NPR1 gene is induced in SAR and in turn NPR1 induces the expression of a battery of downstream pathogenesis-related (PR) genes. Over-expression of NPR1 in Arabidopsis, as well as in rice and tomato, induces broad spectrum disease resistance. Constitutive expression or over-expression of NPR1 and other SAR genes in transgenic sweet orange is now being evaluated for their ability to confer resistance to CLas and other bacteria, with the possibility of producing broad-spectrum resistance. One concern about SAR proteins is they may adversely affect yield. Since the SAR transgenes are of plant origin, should they be found to be effective, there should be less regulatory concern than would be found for transgenes from other organisms. RNA Interference to Limit Psyllids Recently, plant-mediated RNA interference (RNAi) has been shown to effectively control plant insect pests, at least in the laboratory settings. Transgenic corn plants expressing corn rootworm double-stranded RNAs (dsRNAs) exhibited a significant reduction in rootworm damage, and they induced mortality of the insects (Baum et al., 2007). These dsRNAs, after being ingested by the insect, were processed in the insect into small RNAs. The natural gene silencing mechanism of the insect directs destruction of sequences corresponding to those in the dsRNAs in the target gene, leading to death of the insect. Similarly, transgenic cotton plants expressing the dsRNA of a cotton bollworm p450 mono-oxygenase gene effectively inhibited cotton bollworm. Therefore, there is a potential for using RNAi as a control measure for psyllids, and projects taking this approach are in progress. Essential genes for psyllid development or viability could be chosen for RNAi in citrus trees. As the various anti-CLas or anti-ACP genes are created, there will be a need to test them in transgenic citrus. Many of the programs that are investigating such genes do not have capabilities for producing transgenic plants. There will be an increased need for the production of transgenic material and the facilities presently available may not be able to meet those needs. The Core Citrus Transformation Facility (CCTF) at University of Florida Citrus Research and Education Center (UF-CREC) is a service laboratory open to qualified investigators. With additional funding for employees and equipment, the CCTF could increase its production significantly to meet the needs for transgenic citrus.

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84 STRATEGIC PLANNING FOR THE FLORIDA CITRUS INDUSTRY TRANSGENIC, VIRAL, AND BACTERIAL DNA VECTORS FOR MEDIATING GENE EXPRESSION IN CITRUS There is a general consensus that the most viable long-term approach to HLB mitigation is to deploy trees that are resistant to CLas, or ACP, or both . Intensive research efforts in Florida are devoted to this long-term objective, which will likely require years of effort. Resistant plants can be produced by (i) classical genetics and plant breeding, (ii) genetic transformation to produce transgenic plants, and (iii) introduction of a bacteria- or virus-derived DNA vector bearing a gene of interest. In an example of the latter case, Agrobacterium tumefaciens Ti plasmids are commonly used in plant research to express genes in pressure-infiltrated leaves, for example, without the need for time-consuming genetic transformation. Plant viruses are another source of DNA vectors for tests of gene expression without transformation. Usually, use of bacterium- or virus-mediated gene expression is termed “transient expression” because the longer term, multi-generational gene expression characteristic of genetic engineering is not observed. It is the good fortune of citrus research, and possibly the citrus industry, that W.O. Dawson of UF-CREC has developed CTV, itself a significant disease agent under specific circumstances in Florida, into a viral gene expression vector. This CTV vector was found, very surprisingly, to maintain gene expression for years. Citrus Tristeza Virus-Mediated Systemic Expression of Foreign Genes in Citrus The CTV vector is likely to find application because of the urgent need to have HLB mitigation measures in place in less time than the 10–15 years, after a functional anti-CLas gene has been identified. The latter would be required even for the most facile transformation of mature citrus tissue. The CTV-based vector is now being developed as an interim measure to protect citrus trees against HLB without the (as yet unavailable) long-term transformation procedures. That is, it appears that CTV vectors may be used not only to test transgene constructions for later genetic transformation into citrus but also possibly for “transient” expression of transgene constructions over a period of several seasons in the orchard. The CTV vector is applied without transformation, without regeneration, and without the use of a selectable marker or reporter gene. The CTV-based vector allows insertion of one, two or three genes into the viral genome, and these inserted genes are expressed together with virus genes as the virus vector replicates in the host plant. The vector can express high levels of the products from the inserted genes, systemically in both shoots and roots. Most interestingly, after having been amplified within an initial citrus tree, the CTV vector can be transmitted by graft- inoculation to other citrus trees of any size or variety. The longevity of CTV-mediated gene expression is truly surprising. So far, the vector has continually expressed foreign genes in citrus for six years. Twenty-seven of 30 citrus trees, which have been infected with the vector for 4–6 years, still contain the intact foreign gene. Based on this observation, it is very likely that a high percentage of the trees will retain the foreign peptide gene for ten years or more. CTV is endemic in many countries of Asia, Africa, America, Australia, as well as in some Mediterranean countries, as well as Florida, and citrus trees grown in these regions are tolerant to the virus. Problems arise from CTV primarily where citrus is grown on sour orange rootstock,

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CITRUS GREENING RESEARCH AND DEVELOPMENT AND INDUSTRY PREPAREDNESS 85 which rootstock is now avoided in Florida citrus orchards. Thus, using a CTV-based gene vector to infect citrus trees should not harm the trees. In summary, the advantages of the CTV-vector approach, in comparison to the transformation procedures for the production of transgenic trees from juvenile or mature tissues, are:  A CTV-vector would allow CLas-resistant trees to be available in much less time than would be required for production of transgenic trees from juvenile or mature citrus tissues.  If one or a few trees that are supporting the vector containing the beneficial gene for HLB control become available, the vector construction can be transferred to other citrus trees using vector-infected budwood or rootstock in the nursery or orchard.  The problems associated with transformation of juvenile tissue are avoided because juvenile trees infected with the vector can be a source of budwood to transfer the vector to mature trees.  Similarly, budwood from a CTV-vector-infected tree can be transferred to almost any citrus variety, whereas with genetic transformation the time-consuming and expensive transformation protocol would need to be repeated with each new citrus variety.  The CTV vector bearing an anti-CLas construction might be sufficiently potent to be applied not only to healthy trees as a prophylactic but also therapeutically to already infected trees by graft inoculation.  The CTV vector allows expression of more than one gene, so a properly designed construction could be effective against more than one disease agent.  Vector constructions that are compatible have been developed, which should allow re-inoculation of the same tree should a first construction fail or become obsolete.  The CTV-vector infected trees are not transgenic, so there is virtually no possibility of transgene transfer through pollen. However, CTV is aphid- transmitted, so aphids could spread the genes inserted into CTV if virus particles are formed. It should be possible to construct CTV vectors that will not result in gene transfer by aphids.  CTV is phloem-limited as is Clas and thus, anti-bacterial peptides or other genes would be expressed in the tissue where the pathogen is located. Screening of Anti-Bacterial Peptides using CTV-based Vectors The CTV-vector system is suited to the testing of candidate genes intended to act against any citrus-infecting disease agent (Dawson et al., 2005), but it is particularly well-suited to screen for the effectiveness of anti-CLas gene products. No long-term culture of CLas has been demonstrated, so conventional in vitro antimicrobial screening methods are not applicable. The CTV vector and CLas occupy the same site in the host plant, the phloem sieve tubes, and thus the

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86 STRATEGIC PLANNING FOR THE FLORIDA CITRUS INDUSTRY CTV-vector-encoded anti-bacterial product is produced in, and targeted at, the site inhabited by CLas where the liberibacters live and multiply. In practice, a citrus budwood line expressing an anti-bacterial peptide from the infecting CTV vector is propagated in various citrus cultivars including sweet orange. Infection by the CTV vector is confirmed by an enzyme linked immunosorbent assay or ELISA. The plants are then graft-inoculated with a citrus source of CLas and subsequently observed for symptom development and tested for CLas accumulation. In this way, over 100 genes for ABPs are being screened. Promising candidate peptides already have been identified by the CTV work. CITRUS CULTURAL PRACTICES MODIFIED TO ACCOMMODATE ENDEMIC HUANGLONGBING Advanced Citrus Production System The Advanced Citrus Production System (ACPS) is now being evaluated in Florida citrus groves for potential in maintaining sustainable and profitable citrus production in the presence of HLB and citrus canker. The aim of the ACPS is to shorten and enhance the citrus production cycle. Economic fruit production is expected to occur in as little as 3 years from planting, thus achieving an early return on investment. The intended early high yields are anticipated based primarily on high density planting (conventional grove: 116 trees/acre; high density grove: 363 trees/acre) and optimum nutrition-water relations using intensively managed computerized daily fertigation (fertilizer included in irrigation water), commonly referred to as “Open Hydroponics System” (OHS). The OHS will have to be adapted for Florida summer rainy season and sandy soil conditions. Several field experiments using newly planted and mature trees are in progress to evaluate the ACPS for field plot layouts, spacing, automated fertigation system design, equipment selection, irrigation scheduling, monitoring, remote control, fertilizer formulations, root density distribution, and girdling to enhance cropping and control tree vigor. Because ACPS involves higher tree densities, automated irrigation, and intensive nutrient management, annual cultural costs will increase. Estimates are available of the changes in these costs, as well as a determination of the required yield increase that a grove with an ACPS must achieve to be profitable. Induction of Systemic Acquired Resistance and Supply of Micronutrients In Florida, imidacloprid and aldicarb are the only two available systemic insecticides which provide effective control of psyllids on young non-bearing trees. Based on reports that imidacloprid can induce systemic acquired resistance (SAR, described above) to citrus canker and because of its intensive use for psyllid control in newly planted citrus trees, there is interest to determine whether imidacloprid is able to induce SAR against HLB. Experiments are currently underway to evaluate the effect of imidacloprid, as well as non-insecticide SAR inducers, on HLB symptom expression and CLas titers in citrus tissues. In other research, chemical inducers of SAR, such as isonicotinic acid (INA) and acibenzolar-s-methyl (ASM, Actigard®), are being investigated for efficacy against HLB. Initial experiments in which a SAR inducer was applied as a drench did not result in protection against budwood-inoculated CLas. Further experiments are aimed at testing SAR inducers under the

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CITRUS GREENING RESEARCH AND DEVELOPMENT AND INDUSTRY PREPAREDNESS 87 presumably less intense inoculation method that a citrus orchard would experience when exposed to psyllids. Curative levels of micronutrients have also been credited with eliminating mineral deficiency symptoms associated with HLB. Commercial-scale, replicated experiments are underway to test the effect of a combination of micronutrients and SAR inducers, with and without insecticidal psyllid control, on the spread and impact of HLB. Growers hope that the putative effect of SAR inducers and micronutrients will make removal of HLB-infected trees unnecessary. However, even if there is such an effect, the trees will not be cured and may continue to serve as sources of inoculum. MODEL SYSTEMS AND CHEMICAL SCREENING Two projects underway are using three model system approaches to understanding and combating HLB. In the first approach, compounds capable of suppressing CLas accumulation and their effects are being discovered by mass screening of plantlet cultures initiated from CLas- infected citrus and periwinkle. The source of compounds is a chemical library similar to the type used in the pharmaceutical industry for drug screening. Each plantlet is exposed to a different compound and tested for reduction in CLas titer and possible phytotoxic affects. Two approaches in another project rely on results from the model plant Arabidopsis. The Arabidopsis regulatory protein MAP kinase kinase 7 (MKK7) enhances basal resistance to pathogens and induces SAR in Arabidopsis. Both the Arabidopsis MKK7 gene and the corresponding gene from citrus are being over-expressed in citrus, which will be tested for resistance to CLas (and the canker organism). In the second approach, citrus treated with a mutagen is to be screened for mutants that show elevated activity of the oxidative pentose phosphate pathway (oxPPP), a condition that, in Arabidopsis, results in enhanced disease resistance. SUMMARIES OF EXPERIENCES WITH OTHER MAJOR PLANT DISEASES There are many plant diseases which, like HLB, present a serious threat to, and could even eliminate production of, a specific crop. Appendixes L and M summarize the disease and the actions taken in attempting or accomplishing mitigation for two such examples. AN OVERVIEW OF RESOURCES FOR CITRUS PEST MANAGEMENT PROJECTS Research that is focused on Florida’s pest problems is performed by state-funded personnel using facilities at Florida’s Land Grant University (University of Florida) and by staff of the US Department of Agriculture’s (USDA) Agricultural Research Service (ARS), using in-house funding at its facilities located in Florida. ARS’ in-house funds can be used to foster research partnerhips via specific cooperative agreements with outside entities such as universities and industry (http://www.ars.usda.gov/is/np/ SpecCoopAgreements/ SpecCoopAgreementsintro.htm). The Animal and Plant Health Inspection Service (APHIS), another unit of USDA, also provides funding citrus research infrastructure and personnel. These include the Center for Plant Health Science and Technology (CPHST) and the Citrus Health Response Program (CHRP). These resources provide the long-term infrastructure necessary to deal with pest management problems. There are also citrus/citrus pest research

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88 STRATEGIC PLANNING FOR THE FLORIDA CITRUS INDUSTRY programs in Parlier, California; Frederick and Beltsville, Maryland; and Weslaco and College Station, Texas and these can have an impact on citrus in Florida (www.aphis.usda.gov). Citrus pest management research is supported by several important national, regional, state and industrial entities. As with many agricultural systems, citrus researchers have found support from USDA in its various programs which include:Agriculture and Food Research Initiative (AFRI) competitive grants program, which replaced the long standing National Research Initiative (NRI) program and the much shorter-lived Initiative for Future Agriculture and Food Systems (IFAFS). These programs have provided major funding for citrus research. AFRI funds programs that advance “fundamental sciences in support of agriculture.” (http://www.csrees.usda.gov/fo/agriculturalandfoodresearchinitiativeafri.cfm). The Pest Management Alternatives Program provides support for and encourages “the development and implementation of integrated pest management (IPM) practices, tactics, and systems for specific pest problems….” (http://www.csrees.usda.gov/fo/pestmanagementalternativessrgp.cfm). Integrated Pest Management funds projects that “develop and implement new ways to address complex pest management issues”. (http://www.csrees.usda.gov/integratedpestmanagement.cfm). The Interregional Project Number 4 (IR-4) “provides expert assistance with product development and registration for minor crops” such as citrus (http://www.csrees.usda.gov/fo/ir4minorcroppestmanagement.cfm). In addition to these longer-standing USDA programs, new programs have been more recently established, including the large Specialty Crops Research Initiative (SCRI). SCRI was established to “solve critical industry issues through research and extension activities and gives priority to projects that are multistate, multi- institutional, or trans-disciplinary; and include explicit mechanisms to communicate results to producers and the public”. (http://www.csrees.usda.gov/fo/specialtycropresearchinitiative.cfm). In addition to these USDA programs that cover the broad landscape of agriculture, there were, in the past, special research grants programs specific to citrus, such as the Citrus tristeza virus (CTV) research program (http://www.csrees.usda.gov/fo/fundview.cfm?fonum=1079). An important third level of funding for pest management research in Florida is that supplied by the Florida citrus industry. For example, Southern Gardens Citrus has planted the first research field trials of potential canker and greening disease-resistant citrus trees in its Hendry County citrus groves. The trees, noted to be resistant to canker and greening in the laboratory, were planted in small plots to determine if the trees are disease resistant under commercial grove conditions. (http://www.growingproduce.com/news/flg/?storyid=2047). Florida, California and Arizona also have also funded research. Prior to the detection of HLB in Florida, funding from citrus producing states was directed towards research that pertained to their own industry’s needs. With HLB now present in Florida and with the possibility of HLB reaching other citrus-producing states, industry representatives from Florida and California have begun to discuss combining their financial and laboratory resources in order to arrive at HLB management strategies as soon as possible. In 2008, representatives from Florida traveled to California to learn how the California research funding programs are structured. Most recently, industry representatives met in Dallas, Texas in December 2009 to identify citrus research needs on a national level and to discuss how federal funding could be best allocated. In addition, growers in both Florida and California will vote on the potential establishment of a national citrus research program. All citrus growers will be assessed on a per box of harvested fruit basis to fund this program. Management of this program will be by a board composed of representatives from all citrus-producing states. This vote is planned to take place in 2010. Over $4 million of the California Citrus Research Board (CRB) annual budget is directed towards HLB and ACP. Approximately $2 million is allocated to research and an additional $2 million funds an operational program that monitors and traps ACP in production citrus groves and supports a high-throughput diagnostic laboratory.

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CITRUS GREENING RESEARCH AND DEVELOPMENT AND INDUSTRY PREPAREDNESS 89 It is important to note that these USDA funding agencies do not have a long-term strategic plan for pest management specific to citrus in Florida. Furthermore, it appears that there are no coordinated long-term plans across agencies for citrus research and extension. RECENT HISTORY OF INDUSTRY FUNDING IN FLORIDA The Florida Citrus Production Research and Advisory Council (FCPRAC) has operated since 1991 under the Florida Citrus Production Research Marketing Order (FCPRMO). The order allows growers to tax themselves up to 1 cent per box of citrus and direct those funds to research through a competitive grants program with the Board of FCPRAC making the funding decisions. Historically, this tax raised about $1.5 million annually and, in 2007/08, nearly all of these funds were spent on HLB and canker. In 2007/08, the state provided a grant of $3.5 million and this was combined with $1.5 million from FCPRAC and $2 million from the Florida Department of Citrus (FDOC), the latter being a state agency that is funded by an excise tax placed on each box of citrus that moves through commercial channels. The $7 million of funding in 2007/08 funded 100 projects that were reviewed by FCPRAC, with about 50 percent of the projects going to IFAS and 40 percent to ARS. In the fall of 2007, industry started an effort to convince the Florida Citrus Commission (FCC), a 12-member board appointed by the governor to oversee the FDOC, that a significant research investment (e.g. $20 million) from the Citrus Advertising Trust Fund (CATF) and other sources was needed to deal with HLB. On January 16, 2008, the FCC passed a resolution that ‘… expresses willingness to provide support … with expectation of accountability and participation’ and on January 28, the Florida Citrus Industry Research Coordinating Council’s (FCIRCC) Greening Research Task Force formed the Greening Research Oversight Committee to start development of a comprehensive Research Management Plan. In early January 2008, representatives of the Florida citrus industry formed an ad-hoc committee to determine a strategic research plan for dealing with HLB in Florida. They examined the Pierce’s Disease Program in California to determine how it operated for the coordination and management of a large strategic research and extension program. The group then hired Tom Turpen from the Technology Innovation Group, Inc. (TIG), a consulting group that “promotes the diffusion of innovation through commercialization”. In February 2009, they met with the National Research Council (NRC) to suggest forming an expert panel that will develop a list of HLB research priorities and with ARS to form a SWAT-Team for the future of the Florida Citrus Industry. These meetings and interactions resulted in creation of a Greening and Canker Research Management Organization. The conceptual model and the various members and their relationships are diagrammed in Figure 3-1 The Greening and Canker Program was focused on: 1) Urgently finding solutions to production problems, with an emphasis on Greening and Canker but with the realization that other problems will arise in the future. 2) Providing accountability and responsibility for projects and maintaining grower control of the program. 3) Communicating and coordinating activities to stakeholders.

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90 STRATEGIC PLANNING FOR THE FLORIDA CITRUS INDUSTRY The National Academies Real Time Web Reporting Management System Turpen - TIG Department of Citrus FCPRAC Research Community o USDA o Universities Internal o Others External Funding Funding Regional Growers FCIRCC Association Florida Citrus Manual FIGURE 3-1 Conceptual model of agency relationships. Diagram courtesy of R. Norberg, Florida Department of Citrus, Lakeland, Florida. FLORIDA CITRUS ADVANCED TECHNOLOGY PROGRAM A major jump in funding citrus research for Florida was achieved when FCPRAC launched the Florida Citrus Technology Program (FCATP) in 2008, which was the core of what was envisioned in the diagram above. The stated objective of the program is “to apply new technologies to improve the profitability of the Florida citrus industry”. The NRC was commissioned to handle the review of the proposals that were submitted to the FCATP Grants Program in 2008. A total of 205 proposals were received by the FCATP and an initial 83 projects were selected for funding. The total amount of funding awarded was $16,298,243. The target for research funding in 2009 is $34,700,000. A key element to the FCATP Grants Program should be accountability. In the 2008 RFP, the reporting requirements include a 1-page quarterly report with a more complete annual report stating “progress towards stated milestones…and next steps toward field application”. These are considered to be minimal reporting requirements, intended to cut down on paperwork and PI time use. Consideration also should be given to more direct interactions between researchers and growers or grower representatives. One method of doing this is described in the set of recommendations below.

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CITRUS GREENING RESEARCH AND DEVELOPMENT AND INDUSTRY PREPAREDNESS 91 RESEARCH COMMUNICATION AND INFORMATION EXCHANGE The greater the number and size of research projects that are supported in the effort to control HLB, the more complex becomes the task of sharing results between investigators and fully monitoring advances. An effective HLB control strategy may arise out of the combination of results from two or more otherwise unconnected projects, whether the combination is synergistic or merely additive. Presumably the probability of finding such a happy combination should increase with the number and size of research projects, as should the number of new ideas on control strategies, providing the merits of the new combination can be recognized. Appendix M presents a case study on the benefits of improving communications between researchers working on various aspects of the same general problem. The great benefit of co- location of researchers, as well as good communications with a wide array of researchers at other sites, may be seen from the experience with the investigation of maize stunting disease in the southern edges of the corn belt. As reported in Chapter 2, citrus research in Florida includes efforts at two major centers, the University of Florida’s Citrus Research and Education Center (CREC) at Lake Alfred and the USDA-ARS US Horticultural Research Laboratory (USHRL) at Forrt Pierce, as well as efforts of smaller numbers of researchers at several other locations. The recent, marked expansion of support for research on HLB resulted in the involvement of more laboratories, on a national scale. That is, the scale of the effort that HLB is receiving and should receive is greatly in excess of possible co-location of researchers. The experience with research projects on Pierce’s disease of grapevine, and associated organizations providing various services, may be informative here. The introduction and establishment of a new insect vector, the glassy winged sharpshooter (GWSS), into southern California vineyards resulted during the 1990s in a greatly increased spread of the Pierce’s disease causative agent, the Gram-negative bacterium Xylella fastidiosa. The resulting losses of vines and entire vineyards in southern California, and the specter of GWSS invasion of the more valuable and extensive vineyards of northern California, stimulated the initiation of several competitive research grant programs. Two of these remain, the wine-grower check-off-supported Pierce’s Disease Control Program of the California Department of Food and Agriculture (CDFA) and the USDA-University of California (UC) Pierce’s Disease Research Grants Program. The grant review process for these two programs is entirely integrated, with the CDFA managing the solicitation and collection of ad hoc reviews, the USDA-UC program managing the panel reviews, and a joint meeting of the advisory committees for the respective groups making the final recommendations for new and continued funding of projects by the two programs. Ad hoc and panel reviewers are recruited on a national basis, as is the solicitation of proposals. A valuable activity of the CDFA and USDA-UC grant programs is the Pierce’s Disease Symposium (http://www.cdfa.ca.gov/pdcp/Research_Symposium_Index.html), held in early December or late November each year. Participation by the Principal Investigator (PI), and preferably other research group members as well, in the Symposium is a condition of grant support from the programs. Verbal presentations are solicited, or accepted on a volunteer basis, from a subset of the PIs. The Symposium also attracts researchers who are not supported by either of the grants programs, as well as grower members of the Pierce’s Disease Control Board and members of the CDFA’s Pierce’s Disease Research Scientific Advisory Panel. All Symposium participants are encouraged to make one or more poster presentations. Panel discussions bring together PIs who are taking similar research directions so that status of related approaches may be compared directly. Roundtable discussions are structured to encourage

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92 STRATEGIC PLANNING FOR THE FLORIDA CITRUS INDUSTRY “brainstorming” of new approaches. Discussions at the Symposium have resulted in PIs taking new directions in their research. Research and development communication on Pierce’s disease research is also greatly facilitated through a contract from the CDFA with the University of California’s Public Intellectual Property Resource for Agriculture (PIPRA). Two of the important services provided by PIPRA take the form of databases (http://www.piercesdisease.org). One of these is a nearly constantly updated repository of all references to research and other publications on Pierce’s disease. The other is a compendium of progress reports submitted by research grant-supported Pierce’s disease projects. The California Pierce’s Disease Control Board has taken the responsibility of public education about Pierce’s disease and has employed a public relations firm to prepare announcements and other documents for growers and the general public. An example is “Pierce’s Disease. A Decade of Progress,” which is available at http://www.cdfa.ca.gov/pdcp/.