Abstract:
BACKGROUND: Huanglongbing (HLB) or citrus greening is a devastating disease of citrus. The gram-negative bacterium
Candidatus Liberibacter asiaticus (Las) belonging to the α-proteobacteria is responsible for HLB in North America as well
as in Asia. Currently, there is no cure for this disease. Early detection and quarantine of Las-infected trees are important
management strategies used to prevent HLB from invading HLB-free citrus producing regions. Quantitative real-time
PCR (qRT-PCR) based molecular diagnostic assays have been routinely used in the detection and diagnosis of Las. The
oligonucleotide primer pairs based on conserved genes or regions, which include 16S rDNA and the β-operon, have
been widely employed in the detection of Las by qRT-PCR. The availability of whole genome sequence of Las
now allows the design of primers beyond the conserved regions for the detection of Las explicitly.
RESULTS: We took a complimentary approach by systematically screening the genes in a genome-wide fashion,
to identify the unique signatures that are only present in Las by an exhaustive sequence based similarity search
against the nucleotide sequence database. Our search resulted in 34 probable unique signatures. Furthermore,
by designing the primer pair specific to the identified signatures, we showed that most of our primer sets are
able to detect Las from the infected plant and psyllid materials collected from the USA and China by qRT-PCR.
Overall, 18 primer pairs of the 34 are found to be highly specific to Las with no cross reactivity to the closely related
species Ca. L. americanus (Lam) and Ca. L. africanus (Laf).
CONCLUSIONS: We have designed qRT-PCR primers based on Las specific genes. Among them, 18 are suitable for the
detection of Las from Las-infected plant and psyllid samples. The repertoire of primers that we have developed and
characterized in this study enhanced the qRT-PCR based molecular diagnosis of HLB.
