Anatomical and histochemical alterations caused by powdery mildew on Hydrangea macrophylla (Hydrangeaceae) leaves
Introduction: There are few studies concerning the morpho-anatomical and histochemical alterations caused by powdery mildew in H. macrophylla leaves in the scientific literature. Objective: To describe and analyze anatomical and histochemical aspects of this pathosystem. Methods: More than 90 leaves of H. macrophylla (both healthy and infected leaves by powdery mildew) were collected in the nursery El Jardín del Eden, Rionegro, Antioquia, Colombia. To carry out the identification of the mycopathogen, sections were stained with Lactophenol Blue, and contrasted with specialized taxonomic keys. Transverse fragments 1 cm thick were fixed in a mixture of formalin, alcohol, and acetic acid. These were subsequently dehydrated using an ethanol series, clarified in Xylene, and finally embedded in Paraplast plus® to obtain 5 µm sections. Schiff's periodic acid reaction (PAS) was used to detect structural and reserve polysaccharides, Ruthenium Red for pectins, Ponseau S and Lacmoid for callose, ferric chloride for polyphenols, Sudan Black for lipids and Uvitex 2B-Hematoxylin for chitin. The sections were observed using a Nikon 80i eclipse® photon microscope, with Uvitex 2B-Hematoxylin-stained sections examined by epifluorescence using a UV-2A filter. For the observation and description of the samples by scanning electron microscopy, healthy and infected leaves were fixed and dehydrated in 100 % methanol, critical point dried, and coated with gold. Results: H. macrophylla leaves are isobilateral and homobaric, with adaxial and abaxial epidermis of a single cellular layer. The palisade parenchyma consists of a layer of short cells, while the spongy parenchyma forms 6 to 7 cellular layers. All vascular bundles in the leaf blade are closed collaterals. Abundant idioblasts with raphides may be observed in the mesophyll, and starch is the main reserve carbohydrate present in the tissues. The leaves are hypostomatic and exhibit a paracytic pattern of superficial stomata which possess large substomatal cavities. The morphological data observed indicate that the mycopathogen is related to the genus Erysiphe. The epidermal cells affected by the pathogen exhibit thickened walls, granular cytoplasm, and papillae or cell wall appositions in the outer periclinal walls. With the deterioration of the epidermis, the underlying tissues are affected and become necrotic. Histochemical test indicate that infected plants thicken and reinforce their epidermal cell walls with primary wall materials; primarily cutin, pectins, and callose. When stained with Sudan Black, the presence of dark-colored agglomerates in the cytoplasm of epidermal cells may be related to plant defense mechanisms; and those observed in mesophilic cells to the disorganization of membrane systems. Polyphenols accumulate in the cytoplasm of infected epidermal cells. The fungal material present in epidermal tissues was clearly differentiated when stained with fluorochrome to detect chitin. Conclusions: Species of the genus Erysiphe are causative agents of powdery mildew in H. macrophylla. Necrosis of the epidermal cells is observed in response to the mycopathogen, possibly due to hypersensitive response.