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Management of citrus huanglongbing (HLB) requires rapid detection of infected psyllids and trees in an orchard. Detection of HLB associated bacteria (LAS) can be done using psyllids since detection in infected trees is usually delayed (Manjunath et al., 2008). We have developed an easy to use, rapid and affordable detection kit for grower use for testing psyllids for LAS at a reasonable price for initial investment and an operating cost of about $2 per sample.. Eight psyllid samples can be simultaneously tested within 45 minutes. The psyllid DNA extraction and detection of LAS are conducted using a SmartDART™ unit which is operated by software installed on any android device for visualizing real time results. The test results can be e-mailed for both storage and analysis.  The DNA prepared can be stored refrigerated and sent to a laboratory for validation. No other equipment (even pipets) is required for the test. The detection system was validated using a large number LAS isolates from many citrus varieties, from different countries; the results were comparable to that of traditional real time PCR data. Development of methods for multiplex detection of the pathogen and the host DNA from both psyllids and plant host are in progress. We believe the detection system will be useful for growers in intra-orchard management, for extension workers, nurserymen, and in areas where the disease has become endemic as well as in those areas where the disease has been recently introduced.

Liberibacters have not yet been successfully cultured; their minimal genomes carry multiple copies of several genes. Sequences identical to phage genomes have been found in many Liberibacters. Available evidences suggest that the Liberibacter genomes are adapting rapidly in different hosts and environments. Characterization of genomes of rapidly changing unculturable organisms can be challenging. We have used a model system based on Candidatus Liberibacter psyllaurous associated with tomato “psyllid yellows” (Hansen et al., 2008) to develop methodologies using alternate techniques for sequencing metagenomes. We have constructed a BAC library from infected tomato psyllids (Bactericera cockerelli). The library consists of 57,600 clones arrayed in 150 plates each with 384 wells. DNA from individual clones were pooled for screening purposes. Initial identification of clones with Liberibacter sequences were conducted based on 16s ribosomal sequences, and contiguous clones were characterized by end sequencing and identified as containing Liberibacter genome fragments. Screening of additional clones from the library was based on probes developed on such sequences. A total of 245 clones with Liberibacter genome fragments have been identified. A total of 63 bar-coded BAC clones were sequenced by using Roche 454 technology. BAC clones from this library contain large inserts (average size 70 kb). Similarities and differences with other well characterized genomes of Liberibacters (Duan et al., 2009, Lin et al., 2011) will be presented.

Citrus huanglongbing (HLB) has become a major disease and limiting factor of production in citrus areas that have become infected. The destruction to the affected citrus industries has resulted in a tremendous increase to support research that in return has resulted in significant information on both applied and basic knowledge concerning this important disease to the global citrus industry. Recent research indicates the relationship between citrus and the causal agent of HLB is shaped by multiple elements, in which host defense responses may also play an important role. This review is intended to provide an overview of the importance of HLB to a wider audience of plant biologists. Recent advances on host-pathogen interactions, population genetics and vectoring of the causal agent are discussed. [ABSTRACT FROM AUTHOR]

Background: Genus Citrus (Rutaceae) comprises many important cultivated species that generally hybridize easily. Phylogenetic study of a group showing extensive hybridization is challenging. Since the genus Citrus has diverged recently (4–12 Ma), incomplete lineage sorting of ancestral polymorphisms is also likely to cause discrepancies among genes in phylogenetic inferences. Incongruence of gene trees is observed and it is essential to unravel the processes that cause inconsistencies in order to understand the phylogenetic relationships among the species. Methodology and Principal Findings: (1) We generated phylogenetic trees using haplotype sequences of six low copy nuclear genes. (2) Published simple sequence repeat data were re-analyzed to study population structure and the results were compared with the phylogenetic trees constructed using sequence data and coalescence simulations. (3) To distinguish between hybridization and incomplete lineage sorting, we developed and utilized a coalescence simulation approach. In other studies, species trees have been inferred despite the possibility of hybridization having occurred and used to generate null distributions of the effect of lineage sorting alone (by coalescent simulation). Since this is problematic, we instead generate these distributions directly from observed gene trees. Of the six trees generated, we used the most resolved three to detect hybrids. We found that 11 of 33 samples appear to be affected by historical hybridization. Analysis of the remaining three genes supported the conclusions from the hybrid detection test. Conclusions: We have identified or confirmed probable hybrid origins for several Citrus cultivars using three different approaches–gene phylogenies, population structure analysis and coalescence simulation. Hybridization and incomplete lineage sorting were identified primarily based on differences among gene phylogenies with reference to null expectations via coalescence simulations. We conclude that identifying hybridization as a frequent cause of incongruence among gene trees is critical to correctly infer the phylogeny among species of Citrus. [ABSTRACT FROM AUTHOR]

Citrus tristeza virus (CTV), a member of the aphid-transmitted closterovirus group, is the causal agent of the notorious tristeza disease in several citrus species worldwide. The codon usage patterns of viruses reflect the evolutionary changes for optimization of their survival and adaptation in their fitness to the external environment and the hosts. The codon usage adaptation of CTV to specific citrus hosts remains to be studied; thus, its role in CTV evolution is not clearly comprehended. Therefore, to better explain the host–virus interaction and evolutionary history of CTV, the codon usage patterns of the coat protein (CP) genes of 122 CTV isolates originating from three economically important citrus hosts (55 isolate from Citrus sinensis, 38 from C. reticulata, and 29 from C. aurantifolia) were studied using several codon usage indices and multivariate statistical methods. The present study shows that CTV displays low codon usage bias (CUB) and higher genomic stability. Neutrality plot and relative synonymous codon usage analyses revealed that the overall influence of natural selection was more profound than that of mutation pressure in shaping the CUB of CTV. The contribution of high-frequency codon analysis and codon adaptation index value show that CTV has host-specific codon usage patterns, resulting in higheradaptability of CTV isolates originating from C. reticulata (Cr-CTV), and low adaptability in the isolates originating from C. aurantifolia (Ca-CTV) and C. sinensis (Cs-CTV). The combination of codon analysis of CTV with citrus genealogy suggests that CTV evolved in C. reticulata or other Citrus progenitors. The outcome of the study enhances the understanding of the factors involved in viral adaptation, evolution, and fitness toward their hosts. This information will definitely help devise better management strategies of CTV. [ABSTRACT FROM AUTHOR]