Developing a sensitive, high-throughput tool for rapid detection of agronomically important seed-borne pathogens of tomato

[摘要] The limited specificity, sensitivity and multiplex capacity of detection techniques currently availablefor important seed-borne pathogens of tomato is a significant risk for the global tomato trade andproduction industry. These pathogens can be associated with seed at low concentrations but, due totheir highly virulent nature, these low levels can be sufficient to infect germinating seedlings andspread to neighbouring plants and fields, potentially causing epidemics and economic losses. In thisstudy, detection techniques currently available for phytodiagnostics were evaluated for the capacityto accurately detect and identify five agronomically important seed-borne pathogens of tomato:Pepino mosaic virus (PepMV), Tomato mosaic virus (ToMV), Clavibacter michiganensis subsp.michiganensis (Cmm), Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv.tomato. A prototype diagnostic microarray was also designed in an attempt to develop a tool thatcould simultaneously detect these five seed-borne pathogens from a single sample. Viral detectionbased on serological techniques was rapid, accurate and reliable but only detected a single pathogenper assay and required supplementary bioassays to indicate the viability of detected viral pathogens.Selective media plating for bacterial detection demonstrated unreliable recovery of targetedbacteria from infected seed and leaf samples and required supplementary tests to validate theidentity of presumptive positives. Assays were lengthy, laborious and sometimes too ambiguous foraccurate diagnosis of bacterial pathogens. Nucleic acid-based technologies demonstrated improvedsensitivity and specificity for detection of targets from pure culture, leaf and seed extracts,compared to conventional and serological methods, yet also required supplementary bioassays ormedia assays to validate the viability of detected pathogens. Amplification efficiency however, wasaffected by the presence of PCR inhibitors and despite positive detection, variable banding intensityin electrophoretic analysis of amplified products necessitated the use of reference cultures tovalidate diagnosis. The developed microarray incorporated 152 pathogen-specific and control probesto facilitate diagnosis and taxonomic classification of detected pathogens. The array was challengedwith pure culture extracts of the five target pathogens, selected related and non-target, unrelatedpathogens of tomato. Positive detection of each of the pathogens was demonstrated but theproduction of hybridisation signals was highly variable and extremely sensitive to minor technicaldifferences. Each of the five pathogens were successfully detected in combination proving thatdifferent classes of seed-borne pathogens could be detected from a single sample using thedeveloped microarray. This prototype microarray has good potential for phytodiagnostic screeningof the five targeted pathogens, and further validation, optimisation and extension for testing tomatoseed samples may facilitate incorporation of this array into standard diagnostic protocols.

[发布机构] University of the Witwatersrand