A new diagnostic real-time PCR method for huanglongbing detection in citrus root tissue

Jong-Won Park,John V Da Graça,Greg Mccollum,Madhurababu Kunta,W Evan Braswell,Eliezer S Louzada,John E Rascoe,Philip A Stansly

doi:10.1007/s10327-018-0793-4

Abstract

Citrus fibrous root tissue was evaluated as an alternative source material for huanglongbing (HLB) diagnosis by real-time PCR using primer-probe set TXCChlb, developed in the present study based on 16S rDNA of “Candidatus Liberibacter asiaticus” (CLas). Real-time PCR data obtained with DNA samples prepared from leaf and fibrous root tissue collected from mature (> 10 years old) and young (4–5 years old) citrus trees growing in Texas and Florida confirmed that root HLB test is more sensitive than the leaf HLB test in terms of the detection rate of HLB positive trees. In addition, the current study confirmed that HLB can be diagnosed at the pre-symptomatic stage using the root HLB test to facilitate efficient removal of HLB-positive, asymptomatic trees that could serve as a source for HLB. The new HLB diagnostic method using root tissue described in the current study can assist in deploying a more efficient disease management strategy to deter HLB spread, especially at the pre-symptomatic stage.

Highlights

Huanglongbing (HLB), known as citrus greening, is the most serious disease affecting citrus production in Asia, Africa, Americas and the Arabian Peninsula (Bové 2006; Gottwald 2010)
The current study developed a new set of primers and probe based on the nucleotide sequence of Candidatus Liberibacter asiaticus” (CLas) 16S rDNA and investigated its usefulness for HLB detection in root tissue of pre-symptomatic citrus trees
To examine any potential crossreactivity of the newly designed primer set, TXCChlb, with 16S rDNAs of other soil bacteria, we did a BLASTn search of the prokaryotic ribosomal RNA database after excluding the CLas 16S rDNA sequence (NCBI TaxID 34,102) and found that TXCChlb-f has ≤ 66% sequence coverage with 100% identity to the sequences on the prokaryotic 16S rDNA database, while TXCChlb-r has a maximum of 90% sequence coverage with 100% identity to the prokaryotic 16S rDNA database

Summary

Introduction

Huanglongbing (HLB), known as citrus greening, is the most serious disease affecting citrus production in Asia, Africa, Americas and the Arabian Peninsula (Bové 2006; Gottwald 2010). Α-Proteobacteria “Candidatus Liberibacter asiaticus” (CLas) (Jagoueix et al 1994), “Ca. L. africanus” (CLaf) (Planet et al 1995), and “Ca. L. americanus” (CLam) (Teixeira et al 2005) Among these three “Ca. Liberibacter” species, CLas is the most widespread and poses the greatest threat to the citrus industry around the world, while CLaf and CLam are geographically isolated, mostly in Africa and South America, respectively (da Graça and Korsten 2004; Gottwald 2010; Teixeira et al 2005). Because the pathogen is spread by psyllids (Capoor et al 1967; McClean and Oberholzer 1965; Wang and Trivedi 2013) and through propagation of infected material, a three-pronged disease management approach that includes inoculum reduction by the removal of trees with HLB disease, control of psyllid vector populations, and production of healthy trees for planting is recommended (da Graça and Korsten 2004).

Methods

Results

Conclusion