Double-Staining Method: A Technique to Visualize Plant-Fungus Interactions Using Ruthenium Red and Lactophenol Cotton Blue

In coffee rust, spores of Hemileia vastatrix - a pathogenic fungus - land on the plant surface and grow a germination tube, thus initiating an infection via the hyphae.

These hyphae spread inside the tissue and invade the plant cells via specialized finger-like structures called haustoria, which absorb the plant nutrients. Haustoria also secrete effector molecules that are recognized by the plant’s resistance proteins, leading to pectin-rich encapsulation of the haustoria to restrict the infection.

To visualize this interaction, begin with an ultrathin, resin-embedded, infected coffee leaf section. Now, treat the sample with lactophenol cotton blue - a histological stain comprising phenol, lactic acid, glycerol, and cotton blue. Heat the slide briefly.

As the dye components penetrate the tissue, the phenol eliminates any live organisms, while lactic acid and glycerol help preserve the cellular architecture. Simultaneously, the cotton blue molecules bind to the chitin moieties in the fungal cell wall and stain it blue.

Wash the sample with distilled water. Then, treat the sample with ruthenium red dye.

The positively charged ruthenium molecules bind to the negatively charged polysaccharides in the pectin-rich plant cell wall and stain it red. Again, wash the slide and visualize under a microscope.

The pectin-rich plant cell wall appears red, encapsulating the blue haustoria extending from the blue fungal hyphae.