Repertoire of novel sequence signatures for the detection of Candidatus Liberibacter asiaticus by quantitative real-time PCR

Abstract

BackgroundHuanglongbing (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.ResultsWe 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).ConclusionsWe 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.

Genomes of ‘Candidatus Liberibacter solanacearum’ Haplotype A from New Zealand and the United States Suggest Significant Genome Plasticity in the Species

“…L. solanacearum' ZC1 were also predicted to be specific to this genome and annotated by RAST as hypothetical proteins (again, most were less than 70 amino acid residues in length). The lack of clarity surrounding the predicted function of many of these haplotype-or species-specific orthologs is consistent with Kogenaru et al (2014), who identified 18 coding sequences in 'Ca. L. asiaticus' that were not present in 'Ca.…”

“…Genome sequences have been used to develop tools for investigating genetic variability and improved detection of 'Ca. L. asiaticus' (Katoh et al 2011, Kogenaru et al 2014. Such tools are important for characterizing the strains used in biological experiments.…”

Quantification and ecological study of ‘Candidatus Liberibacter asiaticus’ in citrus hosts, rootstocks and the Asian citrus psyllid

“…It is proposed that two important strategies for prevention are sensitive detection methods and a healthy seedling system. In recent years, quantitative detection methods have been developed and improved for more sensitive and specific purposes (Li et al ., , ; Morgan et al ., ; Ananthakrishnan et al ., ; Hu et al ., ; Bertolini et al ., ; Kogenaru et al ., ; Feng et al ., ). Quantitative methods have been used to analyse the distribution of Las, showing uneven patterns in different infected tissues of various citrus cultivars (Tatineni et al ., ; Li et al ., ).…”

A review of techniques for detecting Huanglongbing (greening) in citrus

“…Fourier transform infrared spectroscopy has also been used for the diagnosis of diseased citrus plants, and mid-infrared spectroscopy has been used to study citrus greening infection; however, except for the LIBS method, these studies did not provide early diagnosis. Early detection and quarantine of Las-infected trees are important management strategies used to prevent HLB from invading HLB-free citrus-producing regions (Kogenaru et al 2014). In this review, we describe the current diagnostic techniques for HLB disease performed worldwide, because an early diagnosis and differentiation of Ca.…”

“…HLB symptoms can be confused with diseases like Citrus Tristeza Closterovirus, Phytophthora infection, citrus blight, and certain nutrient deficiencies (Shokrollah et al 2011). Methods such as electron microscopy, serology, DNA probes, enzymatic assay, enzyme-linked immunosorbent assays (ELISA), conventional polymerase chain reaction (PCR) (Yamamoto et al 2006), and quantitative PCR (qPCR) are used for the diagnosis and confirmation of HLB (Kogenaru et al 2014). X-ray fluorescence and laser-induced breakdown spectroscopy (LIBS) combined with chemometric strategies are used to successfully predict the condition of orchard plants infected with Ca.…”