The 18S rDNA sequence of Synchytrium endobioticum and its utility in microarrays for the simultaneous detection of fungal and viral pathogens of potato

Abstract

Resting spores extracted from wart (Synchytrium endobioticum)-infected potato tubers were used for DNA extraction and amplification of 18S rDNA. Analysis of the cloned, sequenced fragment revealed high similarity to members of the Chytridiomycota. Using this information, specific oligonucleotide probes were designed and arrayed onto glass slides for detection of the pathogen. Viral sequence information available in the databank was retrieved, or new viral sequences were generated, and used to design probes for specific detection of important quarantine viruses of potato. To determine the sensitivity and specificity of the oligonucleotide probes, total RNA from infected plants was reverse transcribed, labelled with Cyanine 5, and hybridised with the microarray. A significant number of the oligonucleotide probes exhibited high specificity to S. endobioticum, Andean potato latent virus, Andean potato mottle virus, Potato black ringspot virus, and Potato spindle tuber viroid. Hybridisation signals of sub-arrays within slides were reproducible (r = 0.79) with a high correlation coefficient of hybridisation repetitions (0.73). Our results demonstrate the potential of microarray-based hybridisation for identification of multiple pathogen targets, which will find application in quarantine laboratories, where parallel testing for diverse pathogens is essential.

Macroarray Detection of Eleven Potato-Infecting Viruses and Potato spindle tuber viroid

“…(31) and for detecting bacterial pathogens of potato (12), vascular wilt pathogens of tomato (22), apple pathogens (28), and viruses (2). Glass-slide-based microarrays for pathogen detection have been employed in work with a nematode (13) and a chytrid (1), but the majority of the applications have been in detecting plant viruses (11,20), especially those of potato (1,4,6,7).…”

“…Hybridization assays, when properly designed and executed, can provide both excellent sensitivity and the robust detection of many (all) isolates though, historically, this has been dependent on the availability of purified viral nucleic acids or recombinant DNA clones thereof. An attractive and practical alternative has been to synthesize virus-specific (complementary) DNAs (1,7,11,17,18,23,33). When viral genomic sequences are available, the only limitations in this approach are the costs associated with oligonucleotide synthesis, arrays, and reagents.…”

Management of plant health risks associated with processing of plant-based wastes: A review

“…A conventional PCR method (Niepold and Stachewicz, 2004) and a microarray method (Abdullahi et al, 2005) have not been properly validated to date.…”

Improved real-time PCR assay for detection of the quarantine potato pathogen, Synchytrium endobioticum, in zonal centrifuge extracts from soil and in plants

“…The 18S region of rDNA has also been used to develop a detection method for S. endobioticum (Abdullahi et al 2005). These developments in soil extraction and PCR technology were both a motivation to develop and validate a more sensitive and specific detection method for winter spores.…”