7.10 Incidence and Population of 'Candidatus Liberibacter asiaticus' in Asian Citrus Psyllids (Diaphorina citri) on Citrus Plants Affected by Huanglongbing in Florida

Abstract

The Asian form citrus huanglongbing (HLB) (ex. citrus greening) is associated with ‘Candidatus Liberibacter asiaticus’ (Las) and vectored by the Asian citrus psyllid (ACP) (Diaphorina citri). Robust methods for DNA extraction, detection and quantification of the bacterium from ACP were developed and validated. The quality of DNA extraction from suspect psyllids was easily determined by the following highly efficient primer and/or probe sets of positive internal control (PIC) developed in the study: WGfpr for real-time PCR (qPCR) WGf: 5’-GCT CTC AAA GAT CGG TTT GAC GG -3’ WGp: 5’-TET/TTA CTG ACC ATC ACT CTG GAC GC/3BHQ-2 WGr: 5’-GCT GCC ACG AAC GTT ACC TTC-3’) ACPfr FOR conventional PCR (cPCR) ACPf: 5’-ACG AGG CCA GCA AGA GGT A-3’ ACPr: 5’-CAC GTC AGT CAT ATC AAC ATC ACT GTC G-3’). The PIC set for qPCR also was reliably used to evaluate PCR reaction cocktails for a higher and stable PCR amplification efficiency, and to normalize qPCR data for accurate quantification of the Las populations in its vector insects.