Spatial Patterns in Hyphal Growth and Substrate Exploitation within Norway Spruce Stems Colonized by the Pathogenic White-Rot Fungus Heterobasidion parviporum

ABSTRACT In Norway spruce, a fungistatic reaction zone with a high pH and enrichment of phenolics is formed in the sapwood facing heartwood colonized by the white-rot fungus Heterobasidion parviporum. Fungal penetration of the reaction zone eventually results in expansion of this xylem defense. To obtain information about mechanisms operating upon heartwood and reaction zone colonization by the pathogen, hyphal growth and wood degradation were investigated using real-time PCR, microscopy, and comparative wood density analysis of naturally colonized trees with extensive stem decay. The hyphae associated with delignified wood at stump level were devoid of any extracellular matrix, whereas incipient decay at the top of decay columns was characterized by a carbohydrate-rich hyphal sheath attaching hyphae to tracheid walls. The amount of pathogen DNA peaked in aniline wood, a narrow darkened tissue at the colony border apparently representing a compromised region of the reaction zone. Vigorous production of pathogen conidiophores occurred in this region. Colonization of aniline wood was characterized by hyphal growth within polyphenolic lumen deposits in tracheids and rays, and the hyphae were fully encased in a carbohydrate-rich extracellular matrix. Together, these data indicate that the interaction of the fungus with the reaction zone involves a local concentration of fungal biomass that forms an efficient translocation channel for nutrients. Finally, the enhanced production of the hyphal sheath may be instrumental in lateral expansion of the decay column beyond the reaction zone boundary.

Calcium translocation, calcium oxalate accumulation, and hyphal sheath morphology in the white-rot fungus Resinicium bicolor

The white-rot fungus Resinicium bicolor was cultured on wood blocks in a modified soil block assay and was observed by environmental scanning electron microscopy and scanning electron microscopy. Resinicium bicolor was found to translocate calcium in mycelial cords in quantities greater than that found in the wood blocks and accumulated this calcium in the form of calcium oxalate. Calcium oxalate crystal clusters of mycelial cords were 3 × larger and far more numerous than the crystal clusters produced by the same fungus within the wood. Environmental scanning electron microscopy technology allowed for the examination of the hyphal sheath in a hydrated state. The hydrated hyphal sheath was found to be much thicker than the desiccated sheath observed after standard scanning electron microscope preparations. Calcium oxalate crystals were found to be embedded in the thick hyphal sheath, suggesting that previous observations of within-wall calcium oxalate precipitation may perhaps be better interpreted as artifacts generated during sample preparation. Key words: calcium oxalate, hyphal sheath, environmental scanning electron microscopy.

Environmental scanning electron microscopic observation of the hyphal sheath and mycofibrils in Postia placenta

Environmental scanning electron microscopic observations of Postia placenta grown on a defined medium and on red spruce wood allowed for the examination of the hydrated sheath of P. placenta. In the wood environment, mature hyphae that were not adhering to the substrate were observed to have a mycofibrillar morphology whereas hyphal tips and branch points had a smooth sheath morphology. A mycofibrillar adhesive matrix was observed on the hyphae growing on glass slides in the defined medium. These morphologies for hyphal sheaths in P. placenta are similar to those previously described by investigators from other laboratories who have used traditional electron microscopic preparative protocols that include dehydration steps. The potential future usefulness of environmental scanning electron microscopic technology in the study of the fine details of extracellular matrices is briefly discussed.Key words: mycofibrils, hyphal sheath, environmental scanning electron microscopy, extracellular matrix.

Myofibrillar cell wall extensions in the hyphal sheath of Postia placenta

Evidence is provided for the existence of linear extracellular fibrillar elements in the brown-rot fungus Postia placenta. These elements appear as structural components of the hyphal sheath and more closely resemble mycofibrils than fungal fimbriae. Mycofibrils are associated with and appear to originate from the hyphal surface when hyphae are grown on wood or inert substrates, such as glass cover slips and polycarbonate filters. These extracellular structures have a nominal diameter of 10–50 nm and are up to 25 μm in length. We conclude that mycofibrils are linear structural extensions of the hyphal cell wall. The precise function of mycofibrils in the brown-rot decay process of wood remains to be elucidated. Key words: Postia placenta, mycofibrils, fungal fimbriae, hyphal sheath, electron microscopy.

Immuno-scanning electron microscopic localization of extracellular wood-degrading enzymes within the fibrillar sheath of the brown-rot fungus Postia placenta

Extracellular wood-degrading enzymes of the brown-rot fungus Postia placenta were localized using colloidal gold labeled monoclonal antibodies to the β-1,4-xylanase (32 to 36 kDa) fraction of P. placenta. Postia placenta was grown from agar onto glass cover slips, immunolabeled with or without prior fixation, and examined by scanning electron microscopy. Enzymes were localized on the hyphal surface and on the clumped fibrillar elements mycofibrils of the hyphal sheath following fixation with glutaraldehyde. If fixation was omitted, labeling was diffuse and not localized on individual or clumped mycofibrils. We conclude that extracellular decay enzymes are weakly bound (noncovalently) to, but not identical with, the linear mycofibrillar elements of the hyphal sheath. The linear structural elements of the hyphal sheath may play an important role in transport and presentation of wood-degrading enzymes during the decay process. Key words: brown-rot fungi, enzymes, mycofibrils, hyphal sheath, immunolabeling, monoclonal antibodies, colloidal gold, scanning electron microscopy.

Identification of endophytic hyphae of Lophodermium piceae in tissues of green, symptomless Norway spruce needles by immunoelectron microscopy

Antiserum specific for Lophodermium piceae hyphae was obtained by absorbing a rabbit L. piceae antiserum with hyphal material of different fungal isolates. The specificity of this absorbed antiserum was tested with hyphae of endophytic fungi isolated from green, asymptomatic needles of Norway spruce (Picea abies Karst.) using the on-section immunogold labeling technique. With this specific, absorbed antiserum, a homogenous labeling was obtained with cultured hyphae of L. piceae, whereas all other spruce needle endophytes examined remained practically unlabeled. Insignificant cross-reactivity was also observed with needle tissues. The absorbed antiserum was then applied for the immunoelectron microscopical identification of L. piceae hyphae in situ in infected tissues of green, asymptomatic Norway spruce needles. The function of papillalike structures observed in infected needle mesophyll as well as the role of the hyphal sheath surrounding endophytic L. piceae hyphae are discussed in connection with the interaction between L. piceae and asymptomatic Norway spruce needles.