Quantitative assessment of phytopathogenic fungi in various substrates using a DNA macroarray.
Detection, identification and quantification of plant pathogens are the cornerstones of preventive plant disease management. To detect multiple pathogens in a single assay, DNA array technology currently is the most suitable technique. However, for sensitive detection, polymerase chain reaction (PCR) amplification before array hybridization is required. To evaluate whether DNA array technology can be used to simultaneously detect and quantify multiple pathogens, a DNA macroarray was designed and optimized for accurate quantification over at least three orders of magnitude of the economically important vascular wilt pathogens Verticillium albo-atrum and Verticillium dahliae. A strong correlation was observed between hybridization signals and pathogen concentrations for standard DNA added to DNA from different origins and for infested samples. While accounting for specific criteria like amount of immobilized detector oligonucleotide and controls for PCR kinetics, accurate quantification of pathogens was achieved in concentration ranges typically encountered in horticultural practice. Subsequently, quantitative assessment of other tomato pathogens (Fusarium oxysporum, Fusarium solani, Pythium ultimum and Rhizoctonia solani) in environmental samples was performed using DNA array technology and correlated to measurements obtained using real-time PCR. As both methods of quantification showed a very high degree of correlation, the reliability and robustness of the DNA array technology is shown.
Lievens
Bart
B
Centre of Microbial and Plant Genetics (CMPG), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee-Leuven, Belgium.
Brouwer
Margreet
M
Vanachter
Alfons C R C
AC
Lévesque
C André
CA
Cammue
Bruno P A
BP
Thomma
Bart P H J
BP
D000383:Agriculture / Q000379:methods; D001203:Ascomycota / Q000235:genetics* / Q000472:pathogenicity; D017931:DNA Primers; D005069:Evaluation Studies as Topic; D018551:Solanum lycopersicum / Q000382:microbiology*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012988:Soil Microbiology
D017931:DNA Primers