Two simplified fluorescent staining techniques to observe infection structures of the oomycete Plasmopara viticola in grapevine leaf tissues.
Plasmopara viticola, the causal agent of grapevine downy mildew, is an obligate biotrophic oomycete that grows in the intercellular spaces of host tissues and develops haustoria in the cells. Histological observations are the most effective methods to visualize and quantify the development of the infection structures. We chose two staining techniques leading to high resolution and contrast between parasite structures and host-plant tissues with a minimum of sample preparation: Blankophor and KOH-aniline blue fluorescent stainings. Blankophor (50 ppm in water or 15% KOH) staining was used to study the zoospore encystement on the leaf surface after release from sporangia. The aniline blue dye (0.05% in 0.067 M K(2)HPO(4), pH 9-9.5, after hot KOH whitening) was used to observe the invasive structures inside host tissues that lead to the production of sporangiophores and infectious sporangia. We tested modifications of some parameters of the procedures to determine the most appropriate for high throughput analyses adapted to our pathosystem and equipment facilities.
Díez-Navajas
Ana María
AM
Unité Mixte de Recherche 1131, Institut National de la Recherche Agronomique-Université Louis Pasteur de Strasbourg, 28 rue de Herrlisheim, 68021 Colmar Cedex, France.
Greif
Charles
C
Poutaraud
Anne
A
Merdinoglu
Didier
D
D000814:Aniline Compounds; D005456:Fluorescent Dyes / Q000378:metabolism; D006878:Hydroxides; D008856:Microscopy, Fluorescence / Q000379:methods*; D009868:Oomycetes / Q000472:pathogenicity* / Q000502:physiology / Q000648:ultrastructure*; D010935:Plant Diseases / Q000382:microbiology; D018515:Plant Leaves / Q000382:microbiology / Q000648:ultrastructure; D017680:Potassium Compounds; D013172:Spores, Fungal / Q000648:ultrastructure; D013194:Staining and Labeling / Q000379:methods; D027843:Vitis / Q000382:microbiology / Q000648:ultrastructure
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D006878:Hydroxides; D017680:Potassium Compounds; C017006:aniline blue; C029943:potassium hydroxide