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Multilaboratory comparison of quantitative PCR assays for detection and quantification of Fusarium virguliforme from soybean roots and soil

Yuba R. Kandel, James S. Haudenshield, Ali Y. Srour, Kazi Tariqul Islam, Ahmad M. Fakhoury, Patricia Santos, Jie Wang, Martin I. Chilvers, Glen L. Hartman, Dean K. Malvick, Crystal M. Floyd, Dare3n S. Mueller, Leonor F. S. Leandro
Phytopathology 2015 v.105 no.12 pp. 1601-1611
Fusarium virguliforme, bioassays, death, detection limit, diagnostic techniques, disease control, disease diagnosis, fungi, genes, intergenic DNA, microbial detection, pathogens, quantitative polymerase chain reaction, reproducibility, ribosomal DNA, roots, soil, soybeans
The ability to accurately detect and quantify Fusarioum virguliforme, the cause of sudden death syndrome (SDS) in soybean, in samples such as plant root tissue and soil is extremely valuable for accurate disease diagnoses and to address research questions. Numerous quantitative real-time polymerase chain reaction (qPCR) assays have been developed for this pathogen but their sensitivity and specificity for F. virguliforme have not been compared. In this study, six qPCR assays were compared in five independent laboratories using the same set of DNA samples from fungi, plants, and soil. Multicopy gene-based assays targeting the ribosomal DNA intergenic spacer (IGS) or the mitochondrial small subunit (mtSSU) showed relatively high sensitivity (limit of detection [LOD] = 0.05 to 5 pg) compared with a single-copy gene (FvTox1)-based assay (LOD = 5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (100%) and two IGS assays being slightly less specific (95 to 96%). Another IGS assay targeting four SDS-causing fusaria showed lower specificity (70%), while the two mtSSU assays were lowest (41 and 47%). An IGS-based assay showed consistently highest sensitivity (LOD = 0.05 pg) and specificity and inclusivity above 94% and, thus, it suggested as the most useful qPCR assay for F. virguliforme diagnosis and quantification. However, specificity was also above 94% in two other assays and their selection for diagnostics and research will depend on objectives, samples, and materials used. These results will facilitate both fundamental and disease management research pertinent to SDS.