This protocol is significant as it make it possible to identify the plant-fungus interaction. In addition to coloring the pectin, it also demonstrates defects on the pathogen cell wall polymer and allows the identification of fungus structures. This simple and uncomplicated technique can be performed using light microscopy and it doesn't require any expensive equipment such as scanning electron microscope.
In addition, it is a fast technique that obtains an excellent distinction of plant structures for histopathological studies. The technique identifies the plant-fungus interaction. Hence, it collaborates to diagnose the diseases caused by biotrophic and necrotrophic fungi such as coffee rust and cercosporiosis.
To begin, harvest and approximately 10 square millimeter leaf sample at the middle region of the lesion using a scalpel and tweezer, and immerse it in 30 milliliters of Karnovsky fixative solution. Subject the leaf sample to a low vacuum of 500 to 600 millibar for 15 minutes using an oil pump, at least four times to increase the permeability of the fixative solution in the leaf tissue. After fixation, wash the leaf sample three times in 0.5 M cacodylate buffer diluted in distilled water for five minutes each, and then transfer it to a graded ethanolic series for 15 minutes at each ethanol concentration.
To gradually transfer the samples to glycol methacrylate, make Solution A by mixing the one gram of glycol methacrylate powder with 100 milliliters of basic resin under magnetic agitation. Then, immerse the samples in ratio one to two of Solution A and 100%ethanol for three hours. Immerse the samples in ratio one to one for Solution A:100%ethanol for three hours, then immersed in a pure basic resin for two to four days.
Subject the sample to a low vacuum at least four times a day for 15 minutes, followed by rotation. Mix 15 milliliters of Solution A with one milliliter of the hardener in a beaker with rotation for two minutes to produce the polymerization Solution B.Put 1.2 milliliters of this polymerization Solution B in plastic molds. Using a wooden pick, transfer the lesioned leaf samples from pure basic resin to Solution B and avoid using tweezers as they can cause tissue crushing.
Ensure to quickly orient leaf samples perpendicular to the plastic molds as Solution B quickly becomes viscous. When the perpendicular orientation of the leaf samples is achieved, wait for 30 minutes and then transfer the plastic mold to a plastic or glass chamber containing silica gel to prevent humidity. Once the resin and the leaf sample are polymerized after two to three hours, detach the resulting block from the plastic mold by sanding the block base with a sanding file.
Then, glue the block to a piece of wood. Cut the block into five micrometer thick sections using a rotating microtome equipped with eight centimeter steel blades. Place the sections onto glass slides covered with distilled water.
Then, transfer the slides to a hot plate to dry at 40 degrees Celsius and promote the adhesion of the sections to the glass slides. After drying, label the glass slides with the block reference name and the slide number. Cover the sections with two milliliters of 5%cotton blue in lactophenol and heat them on a hot plate at 45 degrees Celsius for five minutes.
Remove the excess dye by washing the slide three times in a beaker filled with distilled water. Stain it with two milliliters of 0.01%ruthenium red in water for one minute. Remove the excess dye by washing the slide three times in a beaker filled with distilled water.
Put a drop of distilled water over the sections and cover the sections with a cover slip for performing light microscopy analysis. In the double staining method, Hemileia vastatrix hyphae containing cell walls and the dense protoplasts content appeared dark blue in both spongy and palisade parenchyma. The haustorium mother cell and the haustoria also exhibited a dark blue color.
The counter staining with ruthenium red clearly defined fungal distribution in the intercellular spaces. During the interaction, Hemileia vastatrix haustorium mother cell broke the host cell wall and developed a haustorial neck surrounded by pectic compounds. The pectin-rich pink-red colored encapsulation of the haustorial neck cannot prevent haustorial formation.
In some cases, the pectin-rich encapsulation encased the haustorium incompletely. And in some cases, the haustorium is fully encapsulated. The pectin-rich cell wall maintain the integrity at the lesion border, where the fungus is not present.
The double staining method demonstrated the presence of intercellular hyphae. In interaction zones, pectin cell walls appeared to lose their integrity due to dissolution. Reproductive structures such as conidia were found in the adaxial epidermis.
Cercospora coffeicola hyphae were found in the substomatic chamber as curling structures, and the palisade parenchyma in the lesion region seems to undergo cell wall lysis. Avoid using tweezers as they can cause tissue crushing. Further histopathological studies on rusts, anthracnose, cercosporiosis, smuts and other biotrophic, hemibiotrophic, and necrotrophic plant-fungal interactions should be conducted to investigate the potential use of this technique in different pathosystems.
