European Journal of Plant Pathology

, Volume 124, Issue 4, pp 553–563 | Cite as

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

  • P. Trivedi
  • U. S. Sagaram
  • J. -S. Kim
  • R. H. Brlansky
  • M. E. Rogers
  • L. L. Stelinski
  • C. Oswalt
  • N. Wang


Citrus Huanglongbing (HLB) is a devastating disease of citrus known to be associated with a fastidious, phloem-limited Gram-negative, yet to be cultured bacterium in the genus Candidatus Liberibacter. In the present study we have developed a method to quantify viable Candidatus Liberibacter asiaticus (Las) with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells. First, calibration curves were developed with the aid of quantitative real-time PCR (QPCR) by using a plasmid template consisting of a 703 bp DNA fragment of rplKAJL-rpoBC (β-operon) region. Standard equations were then developed to quantify Las genome equivalents in citrus, periwinkle, and Asian citrus psyllid, Diaphorina citri. To overcome the limitation of quantitative PCR in discriminating between live and dead bacterial cells, EMA was used to inhibit the amplification of DNA from the dead cells of Las in plant samples. By using the standard equations and EMA-QPCR methods developed in this study, we found that the proportion of viable cells in citrus and periwinkle ranged from 17–31% and 16–28%, respectively. It was determined that a minimum bacterial concentration is required for HLB symptom development by quantifying the population of Las in symptomatic and asymptomatic leaves. The EMA-QPCR methodology developed in the present study should provide an accurate assessment of viable HLB pathogen, providing a tool to investigate disease epidemiology and thus act as a crucial component for disease assessment and management.


Citrus greening Candidatus Liberibacter asiaticus Ethidium monoazide Quantitative PCR 



We thank Dr. Madhulika Sagaram for her assistance with statistics. We thank Dr. Satyanarayana Tatineni and Dr. Chunxian Chen for critical review of this manuscript. This work has been supported by Florida Citrus Production Research Advisory Council (FCPRAC).


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Copyright information

© KNPV 2009

Authors and Affiliations

  • P. Trivedi
    • 1
  • U. S. Sagaram
    • 1
  • J. -S. Kim
    • 1
  • R. H. Brlansky
    • 1
  • M. E. Rogers
    • 1
  • L. L. Stelinski
    • 1
  • C. Oswalt
    • 2
  • N. Wang
    • 1
  1. 1.Citrus Research and Education CenterUniversity of FloridaLake AlfredUSA
  2. 2.Polk County Cooperative Extension ServiceUniversity of FloridaBartowUSA

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