Huanglongbing (HLB) or citrus greening is the most devastating disease of citrus, threatening the citrus industry worldwide, and leading to massive reduction in fruit production as well as death of infected trees . The causal agents of HLB are three closely related gram-negative, phloem-limited α-proteobacteria Candidatus Liberibacter species [2, 3]. The heat tolerant strain Ca. L. asiaticus (Las) is the most widespread in Asia as well as in the USA whereas Ca. L. americanus (Lam) is mostly limited to South America [2–4]. Ca. L. africanus (Laf) is heat sensitive and localized to the African continent. All the three Liberibacter species are currently uncultured and are known to reside in the sieve tubes of the plant phloem  or in the gut of the phloem-feeding psyllids . Psyllids are the natural vectors in transmitting the bacteria between plants [1, 6]. The Asian psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae) is responsible for transmitting Las and Lam in Asia and America, while the African citrus psyllid, Trioza erytreae Del Guercio (Homoptera: Psyllidae), is the natural vector of Laf in Africa . The characteristic symptoms of the infected plants include the yellow shoots, foliar blotchy mottles, along with poor flowering and stunting . HLB also results in poorly colored, unpleasant tasting, reduced size fruit that shows staining of vascular columella and seed abortion . Generally the fruit may remain partially green, for this reason HLB is also called citrus greening . Chronically infected trees are sparsely foliated and display extensive twig or limb die-back and eventually die within three to five years . Moreover, the disorders induced in diseased plants vary with cultivar, tree maturity, time of infection, stages of disease and other abiotic or biotic agents that affect the tree . HLB symptoms also share certain similarities to nutrient deficiency , citrus stubborn disease caused by Spiroplasma citri and a HLB-like disease caused by a phytoplasma [9, 10]. Early diagnosis and differentiation of Las infections from those defects and agents mentioned above, is thus critical to reducing the spread and devastation of this disease locally and via international trade, as well as minimizing the economic impact of potential false positive diagnoses.
Importantly, HLB and the Asian citrus psyllid (D. citri) are expanding to new citrus production areas. Currently, Asian citrus psyllid has been found in Florida, Texas, California, Arizona, Hawaii, Louisiana, Georgia, and Alabama in the USA, as well as in parts of South and Central America, Mexico, and the Caribbean. Meanwhile, HLB has not only been identified in Florida, Louisiana, South Carolina, Louisiana, Georgia, Texas and California of the USA; it has also been discovered in Cuba, Belize, Jamaica, Mexico, and other countries in the Caribbean . While HLB and D. citri have been found in different producing areas, the number of infected trees and the psyllid vector population vary dramatically among different regions. Thus, different strategies of management of HLB are recommended for different regions, according to the corresponding severity of HLB and occurrence of psyllid vectors.
Currently, no efficient management strategy is available to control HLB. For the recently Las-infected citrus producing areas such as California, prevention and eradication of HLB are the most efficient and cost-effective approaches. Additionally, Las infected trees are most often found to be asymptomatic during the early stage of infection. Thus, accurate early detection of Las in citrus plants and psyllids is critical for enacting containment measures in non-endemic citrus producing areas. For the citrus producing areas without HLB, such as the Mediterranean region, accurate detection is critical for the success of quarantine measures against Ca. Liberibacter.
Methods such as biological indexing using graft, dodder transmission , isothermal loop amplification (LAMP) , electron microscopy , DNA probes , enzyme-linked immunosorbent assays (ELISA) , conventional PCR [16–22] and quantitative real-time PCR (qRT-PCR) [22–26] are used for the diagnosis and confirmation of HLB. Although diagnostic tools like conventional PCR and LAMP showed good sensitivity, they were not consistent in detection of Las bacterium from infected plant and psyllid materials [6, 13, 25]. The current HLB diagnostic detection mainly employs qRT-PCR based methods due to their sensitive and quantitative nature. The initial qRT-PCR oligonucleotide primer sets for the detection of Las, targeted rplKAJL-rpoBC operon (β-operon: CQULA04f/r) , 16S ribosomal RNA gene (rDNA) (HLBasf/r) , EUB338f/EUB518r , ALF518f/ EUB518r  or species specific variable regions. EUB338f/EUB518r primers are universal to Eubacteria , while ALF518f/EUB518r primers identify α-proteobacteria universally  including Las, therefore not specific. Furthermore, the primers based on the conserved 16S and β-operon regions are popular but nevertheless have been shown to pose a potential specificity issue, as both false negatives and false positives have been reported . Therefore, efforts have been directed towards developing effective qRT-PCR primers that target other non-conserved sequences. Recent studies made use of intragenic repeat regions of the prophage sequence for the detection of Las by qRT-PCR . However, the intragenic repeat regions of the prophage sequence were also identified in Lam. Therefore, these primer pairs, hyvi/hyvii did not distinguish between Las and Lam, posing a specificity issue . Consequently, primer pairs that specifically detect Las and make clear distinction among other phylogenetically closely related bacteria are essential.
Here we took a complimentary approach to identify the genes that are unique to Las by a bioinformatic analysis with the goal of expanding the arsenal of tools for Las detection. The advancement in the genome sequencing of Las  provides an opportunity to design primers based on species specific sequences for the detection of Las. We designed the oligonucleotide primer pairs specific to the identified unique genic signatures. We further validated their specificities and selectivity against closely related strains that demonstrated the application to Las-infected tissues and insect vectors by a qRT-PCR.