Multi-laboratory comparison of quantitative PCR assays for detection and quantification of Fusarium virguliforme from soybean roots and soil

Authors: KANDAL, Y - Iowa State University; Haudenshield, James; SROUR, ALI - Southern Illinois School Of Medicine; ISLAM, KAZI - Southern Illinois University; FAKHOURY, AHMAD - Southern Illinois University; SANTOS, PATRICIA - University Of Nevada; WANG, JIE - Michigan State University; CHILVERS, MARTIN - Michigan State University; Hartman, Glen; MALVICK, DEAN - University Of Minnesota; FLOYD, CRYSTAL - University Of Minnesota; MUELLER, DAREN - Iowa State University; LEANDRO, LEONOR - Iowa State University

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/15/2015
Publication Date: 12/1/2015
Publication URL: http://handle.nal.usda.gov/10113/61873
Citation: Kandal, Y.R., Haudenshield, J.S., Srour, A.Y., Islam, K.T., Fakhoury, A.M., Santos, P., Wang, J., Chilvers, M.I., Hartman, G.L., Malvick, D.K., Floyd, C.M., Mueller, D.S., Leandro, L. 2015. Multi-laboratory comparison of quantitative PCR assays for detection and quantification of Fusarium virguliforme from soybean roots and soil. Phytopathology. 105(12):1601-1611.

Interpretive Summary: Accurate identification and quantification of Fusarium virguliforme, the cause of sudden death syndrome in soybean, within root tissue and soil are needed to understand and optimize disease management stratgies. The presence of several Fusarium spp. and other closely related fungi in soybean fields makes quantification of F. virguliforme challenging. Several quantitative molecular assays (qPCR) have been developed but not compared. In this study, six independent moecular assays were compared in five independent laboratories using a set of 78 DNA samples. The study identified strengths and weaknesses of six qPCR assays in root and soil samples. For example, one assay that is based on the amplification of multi-copy genes showed a relatively high sensitivity (the ability to amplify a small amount of DNA) compared to a single copy gene assay. However, the highest specificity (the ability to exclude from detection any non-F. virguliforme species DNA) among the assays was the single copy gene assay. The choice of assay for diagnosis or research will depend on objectives, samples, and materials used. These results will help promote both fundamental and disease management research pertinent to SDS. This research is important to soybean pathologists, mycologists, and other scientists interested in soybean pathogen detection methodologies using molecular assays.

Technical Abstract: Accurate identification and quantification of Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, within root tissue and soil are important tasks. Several quantitative PCR (qPCR) assays have been developed but there are no reports comparing their use in sensitive and specific detection of F. virguliforme. In this study, six independent qPCR assays were compared in five independent laboratories using a set of 78 DNA samples. Multi-copy gene based assays targeting the intergenic spacer (IGS) or the mitochondrial small subunit (MtSSU) showed a relatively high sensitivity (LOD= 0.05 to 5 pg) compared to single copy gene FvTox1-based assay (LOD =0.5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (0.97) and two IGS assays being slightly less specific (0.92 to 0.93). Another IGS assay targeting four SDS-causing fusaria showed even lower specificity (0.68), while MtSSU assays showed the lowest (0.40 and 0.46). This study has identified strengths and weaknesses of six qPCR assays used to detect and quantify F. virguliforme in root and soil samples. The choice of assay for diagnosis or research will depend on objectives, samples and materials used. These results will help promote both fundamental and disease management research pertinent to SDS.