Articles
DEVELOPMENT OF A QPCR TECHNIQUE TO SCREEN FOR RESISTANCE TO ASIATIC CITRUS CANKER
Article number
892_21
Pages
173 – 181
Language
English
Abstract
Asiatic citrus canker (ACC) (causal organism Xanthomonas citri subsp. citri (Xcc)) threatens sustainability of the Florida citrus industry.
Resistant cultivars, whether developed through conventional breeding or genetic transformation, will be the best solution for managing ACC. In Florida, prior to 2006, regulatory constraints made experiments with Xcc very difficult to conduct.
We initiated experiments to develop a method for screening large numbers of plants within the constraints imposed by the Citrus Canker Eradication Program (CCEP) in Florida.
We have used quantitative real time polymerase chain reaction (qPCR) to quantify Xcc in plant tissue and found results to be highly correlated with plate count data. qPCR is much more efficient than plate counting for enumeration of bacteria.
This is particularly advantageous when restrictions imposed by quarantine make dilution plating impractical.
In addition, the high specificity of qPCR overcomes problems associated with plating plant extracts which often contain a variety of microbes. qPCR evaluations have revealed several aspects of the citrus/Xcc pathosystem.
For example, Xcc growth is more rapid in leaves of grapefruit than in kumquat, suggesting that kumquat resistance to Xcc may occur prior to hypersensitivity.
Results with detached leaves were less clear than with attached leaves, most likely due to disruption of the vascular tissue in detached leaves.
One disadvantage of the qPCR method is that dead cells cannot be distinguished from live cells.
Cessation of the CCEP in Florida has resulted in relaxed restrictions on work with Xcc, allowing us to conduct some limited screening utilizing whole plant spray inoculation of citrus hybrids and transgenics developed in our citrus improvement program.
Resistant cultivars, whether developed through conventional breeding or genetic transformation, will be the best solution for managing ACC. In Florida, prior to 2006, regulatory constraints made experiments with Xcc very difficult to conduct.
We initiated experiments to develop a method for screening large numbers of plants within the constraints imposed by the Citrus Canker Eradication Program (CCEP) in Florida.
We have used quantitative real time polymerase chain reaction (qPCR) to quantify Xcc in plant tissue and found results to be highly correlated with plate count data. qPCR is much more efficient than plate counting for enumeration of bacteria.
This is particularly advantageous when restrictions imposed by quarantine make dilution plating impractical.
In addition, the high specificity of qPCR overcomes problems associated with plating plant extracts which often contain a variety of microbes. qPCR evaluations have revealed several aspects of the citrus/Xcc pathosystem.
For example, Xcc growth is more rapid in leaves of grapefruit than in kumquat, suggesting that kumquat resistance to Xcc may occur prior to hypersensitivity.
Results with detached leaves were less clear than with attached leaves, most likely due to disruption of the vascular tissue in detached leaves.
One disadvantage of the qPCR method is that dead cells cannot be distinguished from live cells.
Cessation of the CCEP in Florida has resulted in relaxed restrictions on work with Xcc, allowing us to conduct some limited screening utilizing whole plant spray inoculation of citrus hybrids and transgenics developed in our citrus improvement program.
Authors
G. McCollum, R. Stange, U. Albrecht, K. Bowman, R. Niedz, E. Stover
Keywords
Xanthomonas citri subsp. citri, canker, quarantine, pthA, quantitative real-time polymerase chain reaction, qPCR
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