However, the concentration of Liberibacter DNA has to be high enough to allow for confirmation by conventional PCR followed by cloning and sequencing. By the time titers are high enough in plants for this, the infection has already spread to adjacent trees—especially if Asian Citrus Psyllid are present. This scenario is completely inadequate if the California citrus industry is to keep HLB out of groves. Several early detection techniques are being evaluated by the California Citrus Research Board. These include both direct measurement of elicitor proteins produced by the bacterium and the detection of small interfering and messenger RNAs, proteins, metabolites, and volatile organic compounds produced by the citrus tree in response to infection. Many of these approaches measure systemic responses. This holds a huge advantage over any PCR technique which is significantly limited by sampling since Liberibacter are not uniformly distributed in an infected tree. Nevertheless, a PCR protocol is urgently needed to validate these early detection methods that would provide acceptable evidence of their efficacy to federal and state regulatory agencies. Droplet digital polymerase chain reaction amplification is such a protocol. The technique utilizes the same qPCR primers and probes and amplified products can be isolated, cloned, and sequenced from ddPCR as with conventional PCR. We have evaluated 87 transect/risk-based survey site samples from around the Hacienda Heights positive with ddPCR that have been evaluated by several of the early detection protocols. Correlation of ddPCR results with those of these methodologies will determine which protocol show the most promise for detecting HLB early enough in the infection process to make tree removal, and therefore elimination of inoculum, feasible.

Huanglongbing in Florida is caused by Candidatus Liberibacter asiaticus , a phloem-limited fastidious α-proteobacterium,10 liter drainage pot which is transmitted by Asian citrus psyllids . HLB is causing an unprecedented crisis for citrus industry in Florida and poses a severe threat to citrus production in California, Texas, and Arizona. Currently, no effective HLB management is available. We aim to control HLB by targeting critical traits of Las to break down its infection cycle. Interestingly, Las contains the General Secretory Pathway , which is important for the viability and secretion of putative Sec dependent effectors of Las. SecA, an ATPase, is vital for the function of the Sec pathway and a good target to develop antimicrobials. We have identified multiple SecA inhibitors with high antibacterial activity to Liberibacters and their relatives. We will represent our recent progress in controlling HLB using SecA inhibitors and other antimicrobial compounds. In addition, our study indicates Las contains a functional salicylic acid hydroxylase which breaks down SA and its derivatives. SA and its derivatives play a central role in plant defenses, e.g., systemic acquired resistance , and are exogenously applied on plants as SAR inducers to control plant diseases. Here, we will present our recent progress in controlling HLB by nullifying SA hydroxylase of Las. Finally, breaking down the interactions of virulence factors and their targets in planta has been suggested to be one strategy to generate disease resistant plants. We will present our recent progress in identifying SDEs and their putative targets. We aim to generate HLB resistant or tolerant plants by disrupting the interaction between SDEs and their targets in citrus. Conventional PCR and real-time PCR methods had been developed and systematically validated for detection of citrus Huanglongbing before the disease was detected and confirmed in Miami, Florida, in 2005. Since then, the two rapid and sensitive multiplex qPCR assays have been used to screen field samples for HLB as part of an on-going survey conducted by state, industry, and/or USDA-APHIS regional laboratories. The two screening qPCR assays can detect all three known species of HLB bacteria: ‘Ca. L. asiaticus’ , ‘Ca. L. africanus’, and ‘Ca. L. americanus’. Since 2005, over 200,000 samples of HLB host plants and Asian citrus psyllids collected mainly in Florida, Texas, South Carolina, Louisiana, Georgia, and California have been tested using the screening qPCR assays. Confirmatory tests which include three multiplex qPCR , three cPCR assays , and sequence analysis of the cPCR amplicons are done exclusively at USDA CPHST Beltsville laboratory on samples that test suspect positive in the screening qPCR assays.

The first regulatory detections of HLB-Las by the states using the screening assays were confirmed by the federal confirmatory assays in new quarantine areas. We combined the two screening qPCR assays and validated them as a single assay . The performance of the new combined assay remained unchanged; however, the cost was reduced by half. We have supplemented our confirmatory assays by developing and validating two additional qPCR assays that target two different conserved genes of Las. Although the current qPCR assay is at least 100-fold more sensitive than cPCR, which in 1996 proved useful for pre-symptomatic detection of the disease, in the future we will evaluate several emerging technologies and/or platforms such as digital PCR and digital sequencing for advanced HLB diagnosis. Huanglongbing , associated with Candidatus Liberibacter asiaticus , is the most devastating disease of citrus and threating the citrus industry in Florida. Early root infection has been suggested to play a central role in HLB disease development and of significance to tree health. Therefore, introduction of beneficial bacteria to roots to promote root health might be an alternative approach to management of HLB. Our recent investigations showed that three beneficial Bacillus and closely relative isolates were able to promote citrus plant development with stronger root systems and delay the development of HLB symptoms and Las populations in greenhouse assays. Here, we characterized the rhizosphere competence of these three isolates in both greenhouse and natural environment. Using culture-dependent and – independent approaches, bacterial populations of these isolates on roots of citrus and in rhizospheric soil were determined following soil inoculation. The bacterial populations on the roots of citrus and in soil one month after inoculation were approximately 5.0 × 104 CFU/g and 5.0 × 103 CFU/g , respectively, for the three isolates. The assays revealed a similarity in rhizosphere competence with survival rates ranging from approximately 0.05% to 0.5% for the three isolates. Further analysis revealed that the rhizosphere competence of these isolates may be associated with various phenotypic traits, including substrate utilization, nitrogen dissimilation, siderophore mediated iron acquisition, stress tolerance, copper resistance, and the production of antimicrobial substances.Globalization has increased long-distance human-mediated pathways for invasive disease introduction. Detection of initial introductions of exotic pathogens/pests is challenging because they occur in very low incidence. Optimal probability of eradication/mitigation depends on early detection prior to spread. The earlier the detection, the more likely the pathogen can be eliminated or the epidemic slowed,plant pot with drainage lessening impact over multiple years. To find point introductions across a broad geographic landscape of mixed agricultural/residential areas requires substantial manpower and fiscal resources. Point introductions often go undetected for prolonged periods until incidence exceeds the lower threshold of sampling sensitivity. The Census/Travel model utilizes probable pathways, parses regions into smaller areas , and predicts the most likely locations in a given geographic area for introduction. The model’s geospatial method uses US census and international travel data combined with a pathosystem’s epidemiological characteristics, i.e., latency; detection sensitivity, reliability of confirmation, reproductive rate, environmental suitability, dispersal rate, ease of control, etc. Combining existing foreign population habitat and international pathway data, the model generates a risk index map to identify locations with the highest introduction potential. The risk map is linked to a survey optimizer that calculates the number of samples to be taken in a given area based on risk, and estimates manpower and fiscal requirements. It also ranks foreign countries by their relative contribution to risk of disease introduction. Risk maps were generated for HLB and other pathogens/pests. For prior disease introductions in Florida with identified distributions, the model performed well and validated known points of introduction. The census/travel model is being integrated into existing risk-based model platforms to optimize early detection surveys in California, Texas, Arizona, in Florida to optimize disease intervention/control.

The model is independent of pathosystem, and can be extend to all States to predict introductions of human, animal, or plant diseases/pests. Citrus Health Management Areas facilitate the coordinated control of ACP populations, the clean-up of abandoned groves, and the removal of infected trees. Growers cooperating within a CHMA have been effective in suppressing ACP populations and slowing the spread of HLB in FL, indicating CHMAs are a viable management strategy. It has been confirmed that ACP populations are decreasing where coordinated spray efforts have been implemented in commercial groves in FL. However, the boundaries of CHMAs in FL are not optimized in size, but constructed primarily on arbitrary boundaries with an attempt to combine resources and garner neighbor participation. There are other various relevant factors, such as urban population size, abandoned groves’ acreage, ratio between commercial citrus and residential area, will have a significant contribution to CHMA performance. Improving from CHMA design deficiency in FL, we intend to construct CHMA boundaries for Central Valley based on estimated HLB/ACP risk level under 1-mile2 grid resolution. Where mixed landscapes exist, an optimal mix of residential and commercial landscape is considered so that regional disease management decisions can be implemented more effectively. Through cluster analysis and spatial statistics, we have developed maps that organize plantings based on the spatial pattern and dynamics of ACP populations and HLB risk. K-means clustering method is preferred to construct CHMA as homogenous as possible for risk. The CHMA size and locations are also optimized for cost-effective management. Through thousands of simulations and optimization, 28 CHMAs are currently proposed for California Central Valley, which explains more than 55% of the total risk variance. For super large CHMAs, sub-CHMAs can be further constructed with approximate balanced between acreage and ownership. A similar methodology can be applied for CHMA construction in other citrus producing areas or states .Citrus Huanglongbing , spread by the psyllid vector, is a devastating disease threatening nearly every citrus producing area worldwide with the exception of Australia and Mediterranean countries. The recent finds of ACP in California central valley emphasize the urgency for regulatory intervention and disease control, as this would pose a major threat to the viability of the citrus industry. Increasing ACP incidence and risk are considered inevitable for the Central Valley. A spatial explicit ACP simulation model is developed with attempt to understand the dynamic of ACP population changes, in particular, how ACP spread in a mixture of residential and commercial citrus landscapes in the Central Valley. A 16 mile2 area of Porterville, CA is used as the baseline for ACP spread simulation. The mathematical model considers the following parameters for ACP progression: psyllid life span and mortality, net reproduction rate in natural environment, psyllid dispersal distance, citrus host type and density, new flush production, and effect of different spray schemes.We then modeled factors influencing ACP population variation and interaction with new flush availability in relation to temperature for a period of 3 years using a daily time step. ACP spread occurs more frequently and faster within commercial citrus clusters, but comparatively slower for low density or well separated residential areas. This study also evaluates the behavior and distribution of ACP by subjected to different spray strategy scenarios. A comparison between simulation outputs confirms that the synchronize rate for coordinated spray plays an important role in slowing ACP epidemic development. Besides justifying the benefit of large-scale disease management, the outcome of this simulation model can also quantify the influence of input epidemiological parameters on ACP development, and can assist management decision-making by running specified scenario-based analyses. Powerful computational tools have enabled us to learn/extract patterns of residential host plant distribution. To a large extent, residential citrus biodiversity/choice is influenced by both the physical environment and preferences of household for specific host types. An understanding of social preferences for dooryard citrus tree is critical to residential citrus host density mapping and modeling. Dooryard citrus preferences are heterogeneous and far from random, where certain factors may be able to explain part of the variation in preference. In addition to local climatic and environmental factors, we postulate that a range of demographic and socioeconomic characteristics can also affect the residential preferences for citrus types.