Histological and histochemical detection of defence responses in pine embryos challenged in vitro with Heterobasidion annosum

Embryos of Pinus sylvestris, Pinus nigra var. maritima and Pinus contorta were inoculated in vitro with Heterobasidion annosum and the histology and histochemistry of the host response determined. Three days after challenge, intercellular penetration of hyphae through the epidermis and into the cortex of the host species had occurred. In cells close to the invading hyphae, cell walls became thickened in all three pine species. Lignification and suberization of cell walls in the cortex was detectable in P. nigra var. maritima and P. contorta, but not in P, sylvestris. Moreover, cells in the cortex of P. nigra var. maritima and P. contorta became occluded with dense deposits staining positively for phenolic and lipidic materials. Starch grains had accumulated in host cells close to sites of penetration by day 3, and levels appeared to increase by day 6, except for tissues where heavy invasion by H. annosum had occurred. Host responses intensified between 3 and 6 days after challenge. By day 6, however, the pathogen had penetrated as far as the stele and into the developing xy]em vessels. Nine days after challenge, host tissues contained abundant intra- and inter-cellular hyphae, causing severe disruption and disintegration of all tissues. The utility of such in vitro systems in studies on the resistance responses of conifers to challenge with fungal pathogens is discussed.