A multiplexed immunofluorescence method identifies Phakopsora pachyrhizi Urediniospores and determines their viability.

Soybean rust, caused by Phakopsora pachyrhizi, occurs concomitantly wherever soybean is grown in the tropical and subtropical regions of the world. After reports of its first occurrence in Brazil in 2001 and the continental United States in 2004, research on the disease and its pathogen has greatly increased. One area of research has focused on capturing urediniospores, primarily by rain collection or wind traps, and detecting them either by microscopic observations or by immunological or molecular techniques. This system of detection has been touted for use as a potential warning system to recommend early applications of fungicides. One shortcoming of the method has been an inability to determine whether the spores are viable. Our study developed a method to detect viable P. pachyrhizi urediniospores using an immunofluorescence assay combined with propidium iodide (PI) staining. Antibodies reacted to P. pachyrhizi and other Phakopsora spp. but did not react with other common soybean pathogens or most other rust fungi tested, based on an indirect immunofluorescence assay using fluorescein isothiocyanate-labeled secondary antibodies. Two vital staining techniques were used to assess viability of P. pachyrhizi urediniospores: one combined carboxy fluorescein diacetate (CFDA) and PI, and the other utilized (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide] (FUN 1). Using the CFDA-PI method, viable spores stained green with CFDA and nonviable spores counterstained red with PI. Using the FUN 1 method, cylindrical intravacuolar structures were induced to form within metabolically active urediniospores, causing them to fluoresce bright red to reddish-orange, whereas dead spores, with no metabolic activity, had an extremely diffused, faint fluorescence. An immunofluorescence technique in combination with PI counterstaining was developed to specifically detect viable P. pachyrhizi urediniospores. The method is rapid and reliable, with a potential for application in forecasting soybean rust based on the detection of viable urediniospores.