A number of our trap design features are currently being tested in the field

We have used genome sequence data to identify epitopes on the surface of CaLas. Genes that encode these epitopes have been cloned in E. coli, and the proteins expressed, purified and used to immunize rabbits. The major outer membrane protein A has been used as the basis of a immune tissue print assay for CaLas. A problem for assays of CaLas isthe well known erratic distribution of the pathogen in infected citrus trees. DNA based detection methods require extraction and purification of the DNA prior to the assay. The immune tissue print assays are done directly on plant material and preserve and record the distribution of the pathogen with resolution of single infected phloem cells. Using this method, the pathogen has been readily detected in all tissues of infected sweet orange and lemon trees, a portion of the leaf midrib has been identified as ideal site for sampling. The tissue print assay also scales well to many samples. The rabbit polyclonal antibody has also been used to purify CaLas cells from plant extracts prior to PCR. Immune tissue prints and standard qPCR assays have been carried out on the same samples and are complementary. When both immune tissue prints and qPCR assays are used, the combined data show instances where each assay records a false negative, but together nearly all infected samples are detected.Huanglongbing is a devastating bacterial disease of citrus which causes yield loss, tree decline and reduces fruit and juice quality. HLB is caused by Candidatus Liberibacter asiaticus and transmitted by the Asian citrus psyllid.Many horticultural management practices have evolved to manage HLB infection such as pest management practices,nft growing system the establishment of Citrus Health Management Areas and addition of bactericides. There has been much investment by many funding agencies in many research topics to find solutions to HLB, including plant breeding and biotechnology.

Plant improvement to develop HLB resistant or tolerant root stocks and scions through conventional or biotechnology is the most likely to provide sustainable solutions to HLB. The goal of Citrus Research Development Foundation is to support research and to deliver HLB solutions to growers as quickly as possible. There are several plant improvement research programs in the US using conventional and biotech methods. In Florida, there are researchers at the University of Florida and USDA-ARS research departments comprised of several programs and many researchers. Due to the diversity of programs, program objectives, approaches to research and evaluation, it is difficult to understand the scope and progress of post HLB research and the current status of projects. CRDF is investing in efforts to work with researchers to develop a common development, evaluation, and deployment platform to accelerate commercial release of economically viable HLB resistant or tolerant root stocks and scions.The Asian citrus psyllid Diaphorina citri Kuwayama is the insect vector of the fastidious plant pathogen bacterium Candidatus Liberibacter asiaticus , the causal agent of citrus greening disease, also known as Huanglongbing which is the most significant and widespread threat to the citrus industry. To investigate gene expression profiles that associate with ACP- CLas interactions and identify genes in response to CLas infection, we constructed RNA-seq libraries from CLas-infected and CLas-free ACP samples of three different developmental stages . With 150 bp paired-end sequencing on the Illumina Hiseq2500, we generated 152 Gb of sequence data from 56 million reads per library/replicate, which was assembled into 34,122 contigs with 18,827 being annotated, which were then further analyzed for potential functional classification and potential roles in infection. The results suggested that gene expression in different developmental stages did not respond in the same manner to CLas infection. With more contigs being up or down- regulated, nymphal instars 4-5 showed a more sensitive response to CLas infection than nymphal instars 1-3 and adults. A comprehensive analysis of the transcriptomes revealed vector life stage differences and differential gene expression in response to CLas infection, and identified specific genes with roles in nutrition, development, immune response and transmission pathways.

In Florida approval was granted for field application to citrus trees of formulations of streptomycin and oxytetracycline to control infections by ‘Ca. Liberibacter asiaticus’ and the subsequent development of the associated disease, huanglongbing . We developed a procedure for infecting individual trees by grafting a single symptomatic leaf to the tree. We wanted to determine if this procedure could serve as a small scale assay system for testing conventional antibiotics and other compounds for antimicrobial activity effective against ‘Ca. L. asiaticus’. The working hypothesis behind the assay was that the application of the candidate compound to the inoculum leaf before or after grafting would impede or prevent bacteria in the inoculum leaf from infecting the receptor tree. Aqueous solutions of oxytetracycline or streptomycin, containing either glycerol or the commercial compound Pentra-Bark to aid penetration were applied under 20 psi of pressure to both the adaxial and abaxial surfaces of individual symptomatic leaves before or after grafting the leaf to a receptor tree. Receptor trees were inspected periodically for symptoms and after five to seven months leaf samples were evaluated for infection using real time PCR to detect pathogen DNA. The cumulative results from six trials found 44/60 , 46/75 and 53/80 of trees infected by grafted leaves treated with oxytetracycline, streptomycin or no treatment, respectively. The data suggest that under the conditions of this study, these two compounds failed to impede infection of the receptor trees relative to non-treated controls. The reasons for the lack of success in preventing infection are unknown but could be related to either the effectiveness of the compounds or the method of application, or both. The Asian citrus psyllid is the insect vector of the bacterium Candidatus Liberibacter asiaticus , the causal agent for the citrus greening or Huanglongbing disease which threatens citrus species worldwide. This vector is the primary target of approaches to stop the spread of the pathogen. Accurate structural and functional annotation of the psyllid’s gene models and understanding its interactions with the pathogenic bacterium, CLas, is required for precise targeting using molecular methods. The draft genome was annotated with automated pipelines.

Knowledge transfer from well-curated reference genomes like Drosophila to a newly sequenced insect is challenging due to the diversity and complexity among all insect genomes. We opted for manual curation of gene families that have key functional roles in D. citri biology and pathology. The community effort produced more than 450 manually curated gene models across developmental, RNAi regulatory, and immune-related pathways. Curators included undergraduate and graduate students from multiple institutions as well as experienced expert annotators from the i5k community. Here we report the official gene set for the Asian citrus psyllid genome that includes manually curated genes involved in pathways of experimental interest for vector control strategies. The Asian citrus psyllid, Diaphorina citri is the insect vector of ‘Candidatus Liberibacter asiaticus’ ,nft hydroponic system the causal agent of HLB. Proteomic characterization of D. citri identified hemocyanin as central to the insect’s response to CLas-infected trees. Hemocyanin expression is highly induced psyllids reared on CLas-infected Citrus medica Linn. and has been found to physically interact with a CLas vitamin A biosynthetic enzyme. Hemocyanins function as respiratory proteins of mollusks and arthropods with conserved histidine residues forming a coordination complex with copper ions binding oxygen for transport in the hemolymph. Hemocyanins have evolved from oxygen-binding tyrosinase enzymes, which participate in melanin biosynthesis and have documented roles in insect defense, including parasite encapsulation. This study evaluated the role that hemocyanin plays in the insect response to CLas infection. Specific dsRNA to hemocyanin in D. citri were designed and delivered via artificial diet feeding. Effects of hemocyanin suppression on CLas transmission were evaluated using detached leaf transmission assays. The levels of hemocyanin mRNA expression were highly correlated with the different D. citri color morphs and thus, we hypothesized that the blue abdominal color commonly observed in D. citri may be attributed to the oxygen coordinating copper associated with hemocyanin in the insect. Insight into the function of D. citri hemocyanin may reveal novel strategies to specifically disrupt CLas transmission by the insect vector. The commonly employed technique for monitoring the Asian citrus psyllid utilizes yellow cards covered with glue. These have several disadvantages: the sticky surface is messy and indiscriminate for other insects or debris trapped, it is difficult to remove intact specimens for complete identification, and insects are not well preserved for tissue analysis. An alternative design that can preferentially attract ACP while avoiding the sticky substance, while also preserving the specimens for complete analysis, is therefore desirable. This project enhances the mitigation of citrus greening through early detection of huanglongbing through improved trapping technologies.

Trapping of ACP, with “smart” traps that make it possible to accurately test for the presence and spread of the HLB pathogen, Candidatus liberibacter spp., facilitates the rapid site management response to enable delimitation and potential containment of citrus greening in regions where the disease is not widespread. The first attempt at psyllid trap design was a simple, color-attractive, yellow cylinder that eliminated the sticky glue, but the catch yield was consistently lower than that of the sticky trap, even when tested inside a psyllid rearing facility where the target population was high. Since making manual tweaks to our design quickly became both labor and time intensive, we decided to make the various physical modifications to our prototype by utilizing computer modeling software coupled to a 3D printer. This approach allows us to virtually model the design before testing, and then we can print viable traps within a single day, without the time needed to purchase and assemble the components derived from various sources; this removes a major impediment to rapid innovation. The modeling software can be used to make iterative tweaks to successive designs or to immediately create entirely new models, and the designs are limited only by the capabilities of the 3D printer. The printers deposit heated plastic into intricate designs, a method that is not possible using injection molding techniques, so the shapes of our traps can include projections and voids in strategic places to exploit the natural behaviors of citrus psyllids. Trap designs can easily be developed within a single day, allowing modifications and adjustments to be made in real time as new field data is acquired. Our improved “SmartTrap” designs can favor the capture of certain insects only, such that >50% of the insects collected would be psyllids. The trap base also contains a preservative liquid that allows those captured psyllids to be analyzed by standard molecular techniques. This enables researchers to better study the vector of citrus greening, therefore leading to improvements in control and prevention methods that may be more effective than the expensive, broad-spectrum pesticide sprays. The yellow trap color may be an adequate psyllid attractant for sticky traps, but our group is experimenting with slight color variations of different types of plastics and LED lights, incorporating the UV wavelengths that have been identified as psyllid-specific attractants. The concept of our latest design is to attract ACP to colored plastics and LEDs, where they will land on the primary cylindrical body of the trap. As they naturally crawl upward on rubberized plastic projections, designed to resemble the tender new growth of citrus stems, they will pass under the shade of an opaque overhang, where they will then crawl toward a view of sunlight that appears to be coming through the holes in the trap body, protected from the elements by a clear umbrella dome. Once inside the trap they will remain inside, preferring not to reenter the holes which appear dark from the interior perspective. They then fall down a slippery, fluon-lined funnel and into a preservative-filled test tube, which can be removed for inspection without dismantling the trap itself. Other researchers have identified potential chemical and auditory attractants for psyllids, and these features can easily be incorporated into a single trap design, made possible by 3D print technology.This presentation will show our latest designs and provide commentary on our observations.