|Luster, Douglas - Doug|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/6/2008
Publication Date: 10/1/2008
Citation: Baysal, F., Dorrance, A., Ivey, M.L., Luster, D.G., Frederick, R.D., Czarnecki, J., Boehm, M., Miller, S. 2009. An Immunofluorescence Assay to Detect Urediniospores of the Soybean Rust Pathogen, Phakopsora pachyrhizi. Plant Disease. 92:1387-1393.
Interpretive Summary: A diagnostic assay was developed to detect spores in microscopic samples of soybean rust collected from passive air samplers in soybean fields. The assay was tested comparing spores trapped in double stick tape or petroleum jelly on microscope slides retrieved from the air samplers. The assay was carried out in solution in test tubes and on standard glass microscope slides. Double-stick tape affixed to slides was superior to petroleum jelly-coated slides both in retaining spores and in spore detection efficiency. The assay was used to confirm the identity of soybean rust spores captured on glass slides in passive air samplers from Georgia, Kentucky and Ohio during 2006.
Technical Abstract: An indirect immunofluorescence spore assay (IFSA) was developed to detect urediniospores of Phakopsora pachyrhizi, utilizing rabbit polyclonal antisera produced in response to intact non-germinated (SBR1A) or germinated (SBR2) urediniospores of P. pachyrhizi. Both antisera were specific to Phakopsora spp. and did not react with other common soybean pathogens or healthy soybean leaf tissue in enzyme-linked immunosorbent assays. SBR1A and SBR2 bound to P. pachyrhizi spores was detected with goat anti-rabbit Alexa Fluor 488-tagged antiserum using a Leica DM IRB epifluorescent microscope with an I3 blue filter, (excitation 450-490 nm, emission 515 nm). The assay was carried out in solution in test tubes and on standard glass microscope slides. Double-stick tape affixed to slides was superior to petroleum jelly-coated slides both in retaining spores and in immunofluorescence due to non-specific binding of the Alexa Fluor 488-tagged antisera to the latter. The IFSA was used to confirm the identity of P. pachyrhizi urediniospores captured on glass slides in passive air samplers from Georgia, Kentucky and Ohio during 2006.