As a response to infection by CLas, citrus may increase the production of its AHL mimic, which would bind to LuxR and possibly limit CLas bacterial growth by triggering cell aggregation and consequently limit bacterial movement in planta. The insect extract has a structurally related AHL, which may be produced by endosymbiontic bacteria and bind to CLas LuxR. As a result of this binding, CLas may form biofilm on the surface of the gut of Asian citrus psyllids , Diaphorina citri. Currently, we are investigating the effect of many compounds known to be signals for bacterial LuxR, especially those found in citrus phloem sap, on the activity of CLas LuxR. These compounds include, but are not restricted, to Indole-3-acetic acid , indole, γ-amino butyric acid , salicylic acid , Riboflavin and Lumichrome. We expressed CLas LuxR in citrus using the CTV-based vector system. LuxR expressing citrus showed equally distributed severe symptoms when infected with CLas. Rearing infected ACP on uninfected LuxR plants resulted in a diminishing CLas population. Interfering with CLas cell-to-cell signaling may lead to a new avenue of control strategies. Citrus Huanglongbing is the most destructive disease in citrus. HLB has caused a significant economic impact on citrus production in Florida, the second largest orange juice producer in the world. An available option to citrus growers is to uproot infected trees and/or to use enhanced nutritional supplements that mask symptoms and postpone the underlying problem. The latter strategy does not eradicate the bacterium, Candidatus Liberibacter asiaticus ,10 liter drainage collection pot which causes HLB. Judicious use of the pesticides against Asian citrus psyllid combined with use of biotechnological approaches, including RNAi, may be useful to control psyllid populations.

Towards this end, we modified citrus plants to become more attractive to psyllids. These plants may have application as perimeter trap plants along grove edges or interspersed with other plants within a grove. Additionally, these citrus plants should be kept regularly pruned so as to not allow flowering to prevent possible gene flow to the commercial population of trees. We observed that psyllids are attracted to yellow and mixed yellow areas and thus to the chlorotic and semi-chlorotic regions in plants. We have developed citrus plants with silenced endogenous genes, Phytoene desaturase that interferes with the carotenoid biosynthetic pathway and δ amino levulinic acid dehydratase that interferes with the chlorophyll biosynthetic pathway using silencing potential of the citrus tristeza vector. Citrus trees that have silenced PDS and δ-ALAd exhibited generalized chloritic phenotypes because of interference with their pigments’ biosynthesis and attracted more psyllids than normal plants. Developing citrus plants as trap plants that attract psyllids may contribute to protecting and increasing sustainability of new citrus plantings by employing the trap crop technique. While significant efforts are spent on remedies against Citrus Greening, its exact molecular mechanisms remain poorly understood. In order to successfully eradicate this plague in the long-term, we need to gain knowledge about these mechanisms, learn which molecules of the pathogen are causing the infection, which molecules of the host are the targets, and devise strategies to specifically interfere at molecular level. Comparative analysis of multiple genomes offers a bird’s eye view of a pathogenicity landscape and will generate hypotheses about molecular players that are most likely to be involved. We performed comparative analysis of available genomes of Liberibacter, citrus and psyllid with the emphasis on the differences between pathogenic and non-pathogenic Liberibacter species. The results on protein structure and function prediction are presented as a comprehensive web-site with web-pages dedicated to each protein. Based on our analysis, we formulated hypotheses about proteins that are more likely to be causing HLB and vulnerabilities in citrus targeted by these virulence factors.

Some of the important players involve the Liberibacter prophage, plant-like secreted factors, and steroid biosynthesis enzymes.Liberibacter crescens BT-1 is a gram-negative, rod-shaped, α- proteobacterium isolated from mountain papaya. It is a close relative of the causal citrus HLB agents Liberibacter asiaticus, L. africanus, and L. americanus. Citrus greening is one of the devastating diseases with high economical costs to the worldwide citrus industry. The inability to culture any of the HLB causal agents limits our understanding of the mechanism of infection and delays the development of proper treatments for citrus greening. L. crescens BT-1 is the first cultured member of the Liberibacter genus and can serve as a model to study methods to kill Liberibacter pathogens. Here, mutagenesis is used to identify essential genes in L. crescens BT-1 that can serve as antimicrobial targets for the closely related pathogens as has been done in other systems. Analysis of the essential genes, protein-coding sequences necessary for the survival of L. crescens BT-1, has not been previously attempted due to the limitations of available genetic tools for the organism. In order to gain insight on the virulence, metabolism, and culturability of the genus Liberibacter, a mini-Tn5 transposon derivative system consisting of a gene specifying resistance to kanamycin, flanked by a 19-base-pair terminal repeat sequence of Tn5, was used for the genome-wide mutagenesis of L. crescens BT-1 and created an insertion mutant library. By analyzing the location of insertions using massively-parallel sequencing technology , the genes essential for L. crescens BT-1 survival under in vitro conditions are determined. The information obtained may reveal unique aspects of the physiology and metabolism of L. crescens BT-1, aiding in the discovery and the development of therapies against the disease. We used electrophysiological techniques to record antennal signals and feeding behavior of the Asian citrus psyllid, Diaphorina citri, to gain insight into insect-plant interactions that may be of value in the fight to control Candidatus Liberibacter asiaticus, the bacterium responsible for Huanglongbing disease of citrus. We were unable to demonstrate antennal responses to most of the volatile organic compounds produced by citrus leaves using either head space volatiles or purchased pure compounds.

However,drainage planter pot large and consistent antennal responses to degradation products of common citrus volatiles were obtained under laboratory conditions. Glass cartridges loaded with β-ocimene or citral became stimulatory only after 3 to 9 days at room temperature. Gas chromatograph-coupled mass spectrometry demonstrated that both compounds degraded completely over 3 to 9 days to acetaldehyde, acetone, acetic acid, formic acid, and other compounds. GC-coupled electroantennographic detection identified two peaks that elicited consistent and large antennal responses: acetic and formic acids. Both acids were highly stimulatory to D. citri antennae and responses were correlated with log dose. Probing behavior of D. citri was studied by incorporating blends of citrus volatile compounds in varying proportions and amounts into an emulsified wax substrate . More probes were observed on SPLAT containing blends of acetic and formic acids compared with either acid separately or other compounds. Our study suggests that D. citri may orient to formic and acetic acid present in the citrus tree canopy. These observations of antennally active compounds, both constitutive and arising as degradation products from constitutive plant volatiles, may contribute to development of attractants and/or repellants for this important species. In Brazil, Huanglongbing was reported only in São Paulo, Paraná, and Minas Gerais, but the vector is widespread in the country. It is of the utmost importance to develop and improve sampling procedures in order to maximize the probability of early detection in regions where the disease is still unreported. Our objectives were: to find an efficient spatial sampling scheme to detect the disease as soon as possible, to know how many times one should scout a given orchard in order to find 100% of symptomatic plants, and to optimize the sampling frequency and intensity in order to maximize the detection probability before the disease reaches a given incidence. For the first two objectives, an excess of 650 observed field maps were used as a baseline. Objective was accomplished by simulating 21 intra-orchard sampling schemes, including patterns in X, W, Double W, Diagonals, in clusters, per planting rows and edge focused patterns. In each simulation, the plants were selected according to the sampling pattern and ‘evaluated’ considering the reported efficiency in Brazil . In all cases, a monomolecular function was fitted describing the relationship between HLB incidence and probability of detection. The best sampling procedures were always those which included the evaluation of plants on the orchard’s edges or systematically assessing a fraction of the planting rows. As the results consider only the detection probability, issues such as efficiency per assessed plant, sampling costs per plant and total sampling time should be further evaluated. Objective was accomplished by simulated scouting of 100% of the plants in an orchard, assuming a given detection efficiency. As the detection efficiency increases, a steep decrease in required re-inspections occurs, stabilizing after 50% of detection efficiency. Considering the reported detection efficiency in Brazil, an average of three re-inspections are needed to find 100% of the symptomatic plants. Objective was accomplished by developing a spatially explicit compartmental model; simulating HLB epidemics in a Brazilian citrus landscape; and simulating different sampling schemes, frequencies, and intensities. Results expressed as the probability of detecting the disease before the regional incidence reaches a given value allowed the development of scenarios to be used by policy makers.

Based on recent experimental results indicating that newly infected young flush can become infectious to nymphs and adult psyllids within 10-15 days, we incorporated this short time to infectiousness into an agent-based microsimulation model of HLB transmission. A major implication of our results is that waiting to implement control measures until trees are symptomatic is a strategic decision that can lead to unnecessary rouging. A high priority needs to be given to monitoring and ongoing surveillance of groves so that invasion by psyllids is detected when, or soon after, this process is initiated. Then intensive control of the psyllid population during the asymptomatic phase of transmission can profoundly enhance the useful fruit producing life of citrus trees.One potential control mechanism is RNAi constructs, which currently is being investigated. Citrus trees are infected with a genetically modified strain of CTV that produces extra double stranded RNA sequences specific to psyllid genes. When these RNAs are ingested by psyllids, they inhibit production of proteins vital to psyllid development. An RNAi construct may act by reducing the survival or reproduction of the psyllids, as well as inhibiting the acquisition of Ca. Las by the psyllids. We have extended our original model to incorporate the effects of various RNAi constructs. Our model predicts that RNAi constructs can significantly reduce the psyllid population and slow the spread of Ca. Las infection. Increasing the effectiveness of D. citri monitoring is of great importance to improve the control of this insect. Thus, this study aimed to evaluate the effect of periodic training of inspectors in identifying D. citri on yellow sticky cards and to compare the effectiveness and costs of reading the YSC in the field versus in the office. The effect of periodic training was determined by comparing the performance of nine inspectors who had been trained a year before and immediately after a new training session. Each inspector assessed 20 YSC that had been exposed in the field for 7 days. For comparing YSC reading performance in the field and in the office, eight inspectors assessed the presence of D. citri on 90 YSC. In both experiments, one psyllid was placed on some YSC at a marked position. The assessment was first conducted in the field and a week later in the office using the same YSC. For this study, the eight inspectors were analyzed as a single group and as two groups of four inspectors based on the highest and lowest performances in the field. Periodic training of inspectors improved the efficiency of D. citri detection on YSC by 28%. Overall, reading the YSC in the office increased the detection of psyllids by 15.8% compared to the field. The efficiency in detecting the insect on YSC in the office increased by 18.4 and 10.2% for inspectors with lowest and highest performances in the field, respectively. Furthermore, the assessment in the office reduced the difference of effectiveness in detecting D. citri between the worst and best inspectors from 12.5 to 3.8 percentage points. The assessment of YSC in the office reduced inspection costs at 60%.The official regulatory techniques for Huanglongbing diagnosis are quantitative polymerase chain amplification or conventional PCR analysis of DNA from the HLB causing Liberibacter.