Department of Crop Sciences, University of Illinois, Urbana 61801, USA.
Phytopathology. 2012 Dec;102(12):1143-52. doi: 10.1094/PHYTO-02-12-0040-R.
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.
大豆锈病由 Phakopsora pachyrhizi 引起，在世界热带和亚热带地区大豆种植的地方都会同时发生。该病害于 2001 年在巴西首次报道，于 2004 年在美国大陆首次报道，自此之后，对该病害及其病原体的研究大大增加。研究领域之一集中在采集夏孢子，主要通过雨水收集或风阱收集，并通过显微镜观察或免疫或分子技术进行检测。这种检测系统被吹捧为用作推荐早期应用杀菌剂的潜在预警系统。该方法的一个缺点是无法确定孢子是否具有活力。我们的研究开发了一种使用免疫荧光测定法结合碘化丙啶（PI）染色来检测活的 P. pachyrhizi 夏孢子的方法。基于使用异硫氰酸荧光素标记的二级抗体的间接免疫荧光测定，抗体与 P. pachyrhizi 和其他 Phakopsora spp. 反应，但与其他常见的大豆病原体或大多数其他锈菌测试物不反应。两种重要的染色技术用于评估 P. pachyrhizi 夏孢子的活力：一种是结合羧基荧光素二乙酸酯（CFDA）和 PI，另一种是利用[2-氯-4-[2,3-二氢-3-甲基-(苯并-1,3-噻唑-2-基)-亚甲基]-1-苯基喹啉鎓碘化物]（FUN 1）。使用 CFDA-PI 方法，活孢子用 CFDA 染色呈绿色，而无活力的孢子用 PI 复染呈红色。使用 FUN 1 方法，在代谢活跃的夏孢子内诱导形成圆柱形的细胞内腔结构，使它们发出亮红色到红橙色荧光，而无代谢活性的死孢子则发出极度扩散、微弱的荧光。开发了一种结合 PI 复染的免疫荧光技术来特异性检测活的 P. pachyrhizi 夏孢子。该方法快速可靠，具有基于检测活夏孢子预测大豆锈病的潜力。
