Authors: P Trivedi U S Sagaram J S Kim R H Brlansky M E Rogers L L Stelinski C Oswalt N Wang
Publish Date: 2009/02/26
Volume: 124, Issue: 4, Pages: 553-563
Abstract
Citrus Huanglongbing HLB is a devastating disease of citrus known to be associated with a fastidious phloemlimited Gramnegative 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 realtime PCR QPCR by using a plasmid template consisting of a 703 bp DNA fragment of rplKAJLrpoBC βoperon 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 EMAQPCR 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 EMAQPCR 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 managementWe 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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