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Open Access Research article

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

Sunitha Kogenaru12, Qing Yan1, Nadia Riera1, M Caroline Roper3, Xiaoling Deng4, Timothy A Ebert5, Michael Rogers5, Michael E Irey6, Gerhard Pietersen7, Charles M Rush8 and Nian Wang1*

Author Affiliations

1 Citrus Research and Education Center, Department of Microbiology and Cell Science, IFAS, University of Florida, Lake Alfred 33850, USA

2 Present address: Division of Nephrology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109-0676, USA

3 Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA

4 Department of Plant Pathology, South China Agricultural University, Guangzhou, Guangdong, P. R. China

5 Department of Entomology and Nematology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred 33850, USA

6 US Sugar Corporation, Clewiston, FL 33440, USA

7 Department of Microbiology & Plant Pathology, ARC-Plant Protection Research Institute, University of Pretoria, Pretoria, South Africa

8 Texas A&M AgriLife Research and Extension Center, Texas A&M University, Amarillo, USA

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BMC Microbiology 2014, 14:39  doi:10.1186/1471-2180-14-39

Published: 17 February 2014

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.

Keywords:
Detection system; Diagnostic; Candidatus Liberibacter asiaticus; Greening; Huanglongbing; Bacteria; Psyllid; Citrus