A multiplexed immunofluorescence method identifies Phakopsora pachyrhizi urediniospores and determines their viability

Authors: VITTAL, RAMYA - University Of Illinois; Haudenshield, James; Hartman, Glen

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/14/2012
Publication Date: 12/10/2012
Citation: Vittal, R., Haudenshield, J.S., Hartman, G.L. 2012. A multiplexed immunofluorescence method identifies Phakopsora pachyrhizi urediniospores and determines their viability. Phytopathology. 102:1143-1152.

Interpretive Summary: Soybean rust, caused by the fungus Phakopsora pachyrhizi, has been reported in most countries in the tropical and subtropical regions of the world that grow soybean. After reports of its first occurrence in Brazil in 2001 and the continental United States of America in 2004, research on the disease and its pathogen has greatly increased. One area of research has focused on capture of 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 methods used to detect urediniospores has been to determine whether the spores are viable. Using antibodies to fluorescently tag P. pachyrhizi urediniospores and a dye that stains viable spores green and a second dye that stained non-viable spores red, we developed methods 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. This information will be useful to microbiologists, plant pathologists and epidemiologists interested in knowing more about techniqeus to measure vaibility of fungal spores.

Technical Abstract: Soybean rust, caused by Phakopsora pachyrhizi, has been reported in most tropical and subtropical countries of the world that grow soybean. After reports of its first occurrence in Brazil in 2001 and the continental United States of America in 2004, research on the disease and its pathogen has greatly increased. One area of research has focused on capture of 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 methods used to detect urediniospores has been 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 did not react with other common soybean pathogens 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 non-viable 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.