Convenient detection of the citrus greening (huanglongbing) bacterium 'Candidatus Liberibacter asiaticus' by direct PCR from the midrib extract

PLoS One. 2013;8(2):e57011. doi: 10.1371/journal.pone.0057011. Epub 2013 Feb 20.

Abstract

A phloem-limited bacterium, 'Candidatus Liberibacter asiaticus' (Las) is a major pathogen of citrus greening (huanglongbing), one of the most destructive citrus diseases worldwide. The rapid identification and culling of infected trees and budwoods in quarantine are the most important control measures. DNA amplification including conventional polymerase chain reaction (PCR) has commonly been used for rapid detection and identification. However, long and laborious procedures for DNA extraction have greatly reduced the applicability of this method. In this study, we found that the Las bacterial cells in the midribs of infected leaves were extracted rapidly and easily by pulverization and centrifugation with mini homogenization tubes. We also found that the Las bacterial cells in the midrib extract were suitable for highly sensitive direct PCR. The performance of direct PCR using this extraction method was not inferior to that of conventional PCR. Thus, the direct PCR method described herein is characterized by its simplicity, sensitivity, and robustness, and is applicable to quarantine testing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Citrus / microbiology*
  • DNA, Bacterial / genetics
  • In Situ Hybridization, Fluorescence
  • Plant Diseases / microbiology*
  • Plant Leaves / chemistry
  • Plant Leaves / microbiology
  • Polymerase Chain Reaction
  • Rhizobiaceae / genetics*
  • Sensitivity and Specificity

Substances

  • DNA, Bacterial

Grants and funding

This study was supported in part by a Grant-in-Aid for Research and Development Projects for Application in Promoting New Policy of Agriculture, Forestry and Fisheries from the Ministry of Agriculture, Forestry and Fisheries of Japan. However, the funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study