Skip to main content
ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Citrus and Other Subtropical Products Research » Research » Publications at this Location » Publication #315233
Title: What's in a Number? Estimating numbers of viable Candidatus Liberibacter asiaticus cells in citrus and ACP

Author
item McCollum, Thomas
item Hilf, Mark

Submitted to: Citrograph
Publication Type: Trade Journal
Publication Acceptance Date: 3/2/2015
Publication Date: 4/17/2015
Citation: Mccollum, T.G., Hilf, M.E. 2015. What's in a Number? Estimating numbers of viable Candidatus Liberibacter asiaticus cells in citrus and ACP. Citrograph. 6(2):36-40.

Interpretive Summary: Huanglongbing (HLB) also known as citrus greening disease is having a devastating impact on the Florida citrus industry and is a serious threat to California citrus. Candidatus Liberibacter asiaticus (CLas) is a bacterial plant pathogen that is thought to be the causal agent of HLB. However, CLas is a challenging organism to study because it only lives in two places, 1) citrus phloem and 2) the Asian citrus psyllid (ACP), the insect that transmits the pathogen from plant to plant. To date, CLas has yet to be isolated and grown in culture, posing another problem for research aimed at understanding how CLas infection causes disease. Currently, confirmation of CLas infection by a DNA-based laboratory test known as Quantitative Polymerase Chain Reaction (qPCR). Unfortunately, although qPCR is a very sensitive method for detection of CLas DNA, it does not distinguish if the DNA came from live or dead CLas cells. Our results demonstrate that the use of DNA intercalating dyes can be used to estimate numbers of viable CLas cells. This research has significance for experiments aimed at understanding HLB development with the long term objective of developing solutions to overcome this devastating disease.

Technical Abstract: Knowledge of CLas viability is critical for answering questions about how the organism is proliferating within citrus and ACP, what is the response to therapeutic treatments, and for understanding disease development. Because CLas cannot be grown in culture, estimating cell viability has not been possible. In this project we investigated a novel method that allows for cell viability to be assayed by treatment of tissues with DNA intercalating dyes prior to DNA extraction and subsequent analysis by qPCR. When DNA intercalating dyes interact with DNA they prevent the DNA from being detected in qPCR. Because DNA intercalating dyes cannot pass through the membrane of living cells, their DNA is not affected by the dyes. On the other hand, DNA not contained within viable cells will interact with the dyes and is not detected by qPCR. By comparing the amount of DNA detected from tissues with and without exposure to DNA intercalating dyes, it is possible to estimate cell viability. In this research we evaluated two DNA intercalating dyes, ethidium monoazide and propidium monoazide for their utility in determining CLas viability in citrus and ACP. ACP had the highest total titer, but lowest proportion of viable cells among the various tissues that were evaluated. Roots had the lowest CLas titers, but greatest proportion of viable cells. In petioles, as total CLas titer increased, so did the proportion of viable cells. Using filtration and centrifugation we were able to produce fractions with enriched numbers of viable CLas cells from seed coat vascular bundles and ACP, this may have value in efforts to grow CLas in culture. Our results demonstrate that the use of DNA intercalating dyes can be used to estimate numbers of viable CLas cells. This research has significance for experiments aimed at understanding HLB development with the long term objective of developing solutions to overcome this devastating disease.