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

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.To grow to great heights, trees continually replace their water- and nutrient-conducting elements. Older elements, such as the heartwood that is formed in many trees, gradually become nonconductive. In contrast to the living sapwood, heartwood lacks active defense mechanisms against microbes. However, lignin, the polymer coating cell wall polysaccharides, is highly resistant to microbial degradation. In fact, white-rot fungi, besides having evolved the ability to tolerate or detoxify the secondary metabolites accumulating in heartwood, are the only organisms capable of efficiently degrading lignin. Following establishment in the heartwood of living trees, the colonies of pathogenic white-rot fungi expand and eventually also threaten the conductive sapwood.The white-rot fungus Heterobasidion annosum sensu lato, composed of three species with overlapping geographic distributions and host ranges in Europe (23), is the most important pathogen of Norway spruce (Picea abies L. Karst) in boreal forests. Primary infection of Norway spruce stands by H. annosum sensu lato takes place through fresh thinning stumps or wounds on roots and at the base of the stem. Basidiospores landing on these entrance points give rise to mycelia which colonize the root systems, and eventually the fungus spreads into the stem heartwood. At sites infested with Heterobasidion parviporum, a species primarily restricted to Norway spruce, roots of saplings can become infected by the fungus after around 10 years of growth (25). Stem colonization usually initiates only after the heartwood has started to develop, which in Norway spruce takes place in trees 25 to 40 years old (17). Due to relatively rapid axial spread within heartwood, the decay column caused by H. annosum sensu lato often is up to 10 m high in the stems of mature Norway spruce trees.In response to sapwood challenge by an expanding heartwood-based colony of H. annosum sensu lato, Norway spruce forms a so-called reaction zone (RZ) in the border area between healthy sapwood and colonized heartwood. This xylem defense is characterized by high pH due to increased carbonate content and enrichment of phenolic compounds, particularly lignans, some of which have shown antifungal properties in bioassays (14, 30, 31). Although several wood decay fungi are able to eventually penetrate the RZ regions formed in trees, the strategies employed by fungi to breach these unique defense barriers are poorly understood (24). The purpose of this study was to obtain information about the mechanisms operating in heartwood colonization and expansion of the decay column via penetration of the RZ. To do this, we examined spatial growth of H. parviporum and the associated substrate exploitation patterns within naturally colonized mature stems of Norway spruce.     

Spread of Heterobasidion annosum s.s. and Heterobasidion parviporum in Picea abies 15 years after stump inoculation

The tree pathogenic fungi Heterobasidion annosum s.s. and Heterobasidion parviporum cause root and butt rot in Norway spruce (Picea abies) and produce serious economic losses to the forest sector in Europe. We experimentally studied inter- and intraspecific differences between H. parviporum and H. annosum s.s. in the way they infect stumps and spread into neighbouring trees. Eleven H. parviporum and nine H. annosum s.s. isolates were artificially inoculated on stumps of two spruce stands after first thinning. After 15 years, the same isolates were reisolated from neighbouring trees. Heterobasidion parviporum spread more frequently from the inoculated stumps to the neighbouring trees than H. annosum s.s. The surroundings of H. annosum s.s. stumps that did not spread were often colonized by H. parviporum. Heterobasidion annosum s.s. spread was restricted mainly to the areas of the plot where no other Heterobasidion genotypes had been inoculated. In such cases, H. annosum s.s. tended to develop into bigger genets than H. parviporum. The probability of stump-to-tree spread of H. parviporum depended on the diameter of the stumps, suggesting that H. parviporum spread may relate to the presence of heartwood. Both H. parviporum and H. annosum s.s. proved to be strong pathogens on Norway spruce; however, when competing for the same trees, H. parviporum seemed capable of excluding H. annosum s.s. from the stand.     

Diversity and decay ability of basidiomycetes isolated from lodgepole pines killed by the mountain pine beetle

When lodgepole pines (Pinus contorta Douglas ex Louden var. latifolia Engelm. ex S. Watson) that are killed by the mountain pine beetle (Dendroctonus ponderosae) and its fungal associates are not harvested, fungal decay can affect wood and fibre properties. Ophiostomatoids stain sapwood but do not affect the structural properties of wood. In contrast, white or brown decay basidiomycetes degrade wood. We isolated both staining and decay fungi from 300 lodgepole pine trees killed by mountain pine beetle at green, red, and grey stages at 10 sites across British Columbia. We retained 224 basidiomycete isolates that we classified into 34 species using morphological and physiological characteristics and rDNA large subunit sequences. The number of basidiomycete species varied from 4 to 14 species per site. We assessed the ability of these fungi to degrade both pine sapwood and heartwood using the soil jar decay test. The highest wood mass losses for both sapwood and heartwood were measured for the brown rot species Fomitopsis pinicola and the white rot Metulodontia and Ganoderma species. The sap rot species Trichaptum abietinum was more damaging for sapwood than for heartwood. A number of species caused more than 50% wood mass losses after 12 weeks at room temperature, suggesting that beetle-killed trees can rapidly lose market value due to degradation of wood structural components.     

Effect of Spruce Root Constituents on Extracellular Enzymes of Fomes annosus

Fractions of acetone extracts from heartwood, sapwood and wood from the reaction zone in roots of Norway spruce, attacked by Fomes annosus, were tested concerning their effect on the activity of cellulase, polygalacturonase, aryl-β-glucosidase and laccase, obtained from culture filtrates of the same fungus. A specific inhibition of polygalacturonase was recorded with the resinous fractions. The hydrolysing enzymes were more inhibited by the lignans of the reaction zone in the presence of fungal laccase than in the absence of laccase. The pH dependence of the inhibiting effect of phenols varied among the enzymes. The four enzymes were inhibited by partly different lignan fractions, and synergistic effects between minor compounds were indicated. The phenolic fractions inhibited weakly the biosynthesis of the enzymes. It was obvious that results from enzyme inhibition studies in vitro do not reflect the complicated mutual interrelationships between production and activity of the extracellular fungal enzymes and the chemical constituents of the host in vivo.     

Spruce wood degradation by Pleurotus abieticola in comparison with Phlebiopsis gigantea and Heterobasidion parviporum: in vitro experiments with isolates

The objective of this study was to evaluate the in vitro mycelium growth of Pleurotus abieticola and its competitive ability to decompose sapwood and heartwood wood, as compared to the activity of Phlebiopsis gigantea and Heterobasidion parviporum. Over the last several decades, P. gigantea has routinely been used for biocontrol of the conifer pathogen Heterobasidion annosum s.l.; however, its protective effect on Norway spruce stands was recently demonstrated to be not satisfactory. P. abieticola was proposed instead, as a promising species that might successfully compete with H. parviporum. We investigated the growth of mycelium and the ability of P. abieticola isolates to decompose wood of Norway spruce, in the experiment with isolates of P. gigantea and H. parviporum. Heartwood was better decomposed than sapwood by the majority isolates used in the experiment. Linear growth of the investigated fungi showed a more rapid mycelium development for P. gigantea and H. parviporum, compared to that of P. abieticola. In dual cultures, H. parviporum was overgrown only by P. gigantea. All the tested isolates of P. abieticola showed weaker wood decomposition than those of P. gigantea and H. parviporum. Further study is required to better understand the role of P. abieticola for the protection of spruce stands.     

Superior wood for violins--wood decay fungi as a substitute for cold climate

Violins produced by Antonio Stradivari during the late 17th and early 18th centuries are reputed to have superior tonal qualities. Dendrochronological studies show that Stradivari used Norway spruce that had grown mostly during the Maunder Minimum, a period of reduced solar activity when relatively low temperatures caused trees to lay down wood with narrow annual rings, resulting in a high modulus of elasticity and low density. The main objective was to determine whether wood can be processed using selected decay fungi so that it becomes acoustically similar to the wood of trees that have grown in a cold climate (i.e. reduced density and unchanged modulus of elasticity). This was investigated by incubating resonance wood specimens of Norway spruce (Picea abies) and sycamore (Acer pseudoplatanus) with fungal species that can reduce wood density, but lack the ability to degrade the compound middle lamellae, at least in the earlier stages of decay. Microscopic assessment of the incubated specimens and measurement of five physical properties (density, modulus of elasticity, speed of sound, radiation ratio, and the damping factor) using resonance frequency revealed that in the wood of both species there was a reduction in density, accompanied by relatively little change in the speed of sound. Thus, radiation ratio was increased from 'poor' to 'good', on a par with 'superior' resonance wood grown in a cold climate.     

Wood decay under the microscope

Many aspects of the interactions between host wood structure and fungal activity can be revealed by high resolution light microscopy, and this technique has provided much of the information discussed here. A wide range of different types of decay can result from permutations of host species, fungal species and conditions within wood. Within this spectrum, three main types are commonly recognised: brown rot, white rot and soft rot. The present review explores parts of the range of variation that each of these encompasses and emphasizes that degradation modes appear to reflect a co-evolutionary adaptation of decay fungi to different wood species or the lignin composition within more primitive and advanced wood cell types. One objective of this review is to provide evidence that the terms brown rot, white rot and soft rot may not be obsolete, but rigid definitions for fungi that are placed into these categories may be less appropriate than thought previously. Detailed knowledge of decomposition processes does not only aid prognosis of decay development in living trees for hazard assessment but also allows the identification of wood decay fungi that can be used for biotechnology processes in the wood industry. In contrast to bacteria or commercial enzymes, hyphae can completely ramify through solid wood. In this review evidence is provided that wood decay fungi can effectively induce permeability changes in gymnospermous heartwood or can be applied to facilitate the identification of tree rings in diffuse porous wood of angiosperms. The specificity of their enzymes and the mild conditions under which degradation proceeds is partly detrimental for trees, but also make wood decay fungi potentially efficient biotechnological tools.     

Endophytes dominate fungal communities in six-year-old veteranisation wounds in living oak trunks

Old trees are rare in the landscape, as are many of their associated species. Veteranisation is a method by which attempts are made to create microhabitats, otherwise found only in old trees, in younger trees at an earlier stage than would occur naturally. Here, we analysed the early fungal succession in 6 y-old veteranisation wounds in ca. 100 y old living oak trunks by DNA-barcoding of the wood at eight sites in Sweden and Norway. We hypothesised basidiomycetes would be most abundant, and exposed sapwood and heartwood would select for different communities. We identified 686 fungal taxa, mainly ascomycetes, with a large overlap in species composition and surprisingly similar species richness, i.e. 325 vs. 308–360, between intact and different types of damaged wood, respectively. Endophytes continued to be present and common in damaged wood. The results demonstrate that damage to sapwood and heartwood partly select for different fungi and that 6 y is too early to evaluate if veteranisation can positively favour fungi of conservation interest.     

How does the richness of wood-decaying fungi relate to wood microclimate?

Microclimatic conditions in dead wood influence fungal growth and hence also species composition, but it remains unclear how they influence species richness in nature. We analysed fungal species richness based on the occurrence of fruit bodies on 2 m long segments of both standing and lying trunks of Norway spruce (Picea abies). The number of non-red-listed species was related positively to moisture, and negatively to both temperature extremes and fluctuations. The numbers of both red-listed and non-red-listed species were further differently influenced by trunk diameter and by trunk properties related to the progression in wood decay. These results indicate that the richness of fungal communities in dead wood is shaped by an interaction of wood decay, moisture and temperature fluctuations.     

Modelling the hyphal growth of the wood-decay fungus Physisporinus vitreus

The white-rot fungus, Physisporinus vitreus, degrades the membranes of bordered pits in tracheids and consequently increases the permeability of wood, which is a process that can be used by the wood industry to improve the uptake of wood preservatives and environmentally benign wood modification substances to enhance the use and sustainability of native conifer wood species. To understand and apply this process requires an understanding of how a complex system (fungus-wood) interacts under defined conditions. We present a three-dimensional fungal growth model (FGM) of the hyphal growth of P. vitreus in the heartwood of Norway spruce. The model considers hyphae and nutrients as discrete structures and links the microscopic interactions between fungus and wood (e.g. degradation rate and degree of opening of pits) with macroscopic system properties, such penetration depth of the fungus, biomass, and distribution of destroyed pits in early- and latewood. Simulations were compared with experimental data. The growth of P. vitreus is characterized by a stepwise capture of the substrate and the effect of this on wood according to different model parameters is discussed.     

Resistance of bioincised wood treated with wood preservatives to blue-stain and wood-decay fungi

Bioincising is a biotechnological process that aims at the improvement of wood preservative uptake in wood species with a low permeability, such as Norway spruce (Picea abies (L.) Karst). The process is based on a short-term pre-treatment with white-rot fungus Physisporinus vitreus. During incubation the membranes of bordered and half bordered pits are supposed to be degraded by fungal activity resulting in a better treatability of the wood structure for wood preservatives. In the present study, first of all the resistance of bioincised Norway spruce heartwood and untreated controls against blue-stain and wood-decay fungi (white- and brown-rot) was determined. Then, bioincised and untreated specimens were dipped or vacuum impregnated with six wood preservatives and substance uptake was assessed gravimetrically. Additionally, the penetration of 3-iodo-2-propynyl butylcarbamate (IPBC) into the wood was analyzed by high-pressure liquid chromatography (HPLC). Finally, wood resistance was assessed according to the European standards EN 152 and EN 113. Results showed no difference between bioincised wood without preservatives and the untreated wood against blue-stain discolouration. However, a significant (P < 0.05) increase in susceptibility against wood decay was recorded. In the bioincised wood samples a significantly higher uptake of all the different preservatives was determined and the HPLC-method revealed that IPBC penetrated deeper into bioincised wood than into control samples. The improved uptake of preservatives into bioincised wood resulted in a significantly higher resistance against white- and brown-rot fungi. However, only a slight protection against wood discolouration by blue-stain fungi was recorded. The results of this study show for the first time that the biotechnological process with P. vitreus can be used to improve wood durability by increasing the uptake and penetration of wood preservatives.     

Characterisation of some Phlebiopsis gigantea isolates with respect to enzymatic activity and decay of Norway spruce wood

The activity of cellulase, peroxidase, phosphatase and dehydrogenase enzymes, together with the content of protocatechuic and vanillic acids, in samples of Norway spruce wood inoculated with 17 different isolates of Phlebiopsis gigantea was measured. The same isolates were used to compare decay activity in samples of Norway spruce wood after incubation for 3 and 6 months. Significant differences in enzyme activity and phenol production were found between aerial mycelium overgrowing the wood sample and the underlying wood. These differences indicated that the nature of the fungal mycelium appears to change depending on whether it is in contact with wood. After 6 months, highly extensive decomposition of the wood was shown by two British isolates. The results confirm a large difference in P. gigantea inoculum among isolates in natural conditions and reinforce the need for constant evaluation of the most active isolates to use in preparations for biocontrol: a problem for both users and registration bodies.     

Impact of Norway spruce pre-degradation stages induced by Gloeophyllum trabeum on fungal and bacterial communities

In forest ecosystems, fungi are the key actors in wood decay. They have the capability to degrade lignified substrates and the woody biomass of coniferous forests, with brown rot fungi being common colonizers. Brown rots are typically involved in the earliest phase of lignocellulose breakdown, which therefore influences colonization by other microorganisms. However, few studies have focused on the impact of introducing decayed wood into forest environments to gauge successional colonization by natural bacterial and fungal communities following partial decay. This study aimed to address this issue by investigating the bacterial and fungal colonization of Norway spruce (Picea abies) wood, after intermediate and advanced laboratory-based, pre-decay, by the brown rot fungus Gloeophyllum trabeum. Using Illumina metabarcoding, the in situ colonization of the wood blocks was monitored 70 days after the blocks were placed on the forest floor and covered with litter. We observed significant changes in the bacterial and fungal communities associated with the pre-decayed stage. Further, the wood substrate condition acted as a gatekeeper by reducing richness for both microbial communities and diversity of fungal communities. Our data also suggest that the growth of some fungal and bacterial species was driven by similar environmental conditions.     

Forest microsite effects on community composition of ectomycorrhizal fungi on seedlings of Picea abies and Betula pendula

Niche differentiation in soil horizons, host species and natural nutrient gradients contribute to the high diversity of ectomycorrhizal fungi in boreal forests. This study aims at documenting the diversity and community composition of ectomycorrhizal fungi of Norway spruce ( Picea abies ) and silver birch ( Betula pendula ) seedlings in five most abundant microsites in three Estonian old-growth forests. Undisturbed forest floor, windthrow mounds and pits harboured more species than brown- and white-rotted wood. Several species of ectomycorrhizal fungi were differentially represented on either hosts, microsites and sites. Generally, the most frequent species in dead wood were also common in forest floor soil. Ordination analyses suggested that decay type determined the composition of EcM fungal community in dead wood. Root connections with in-growing mature tree roots from below affected the occurrence of certain fungal species on seedling roots systems in dead wood. This study demonstrates that ectomycorrhizal fungi differentially establish in certain forest microsites that is attributable to their dispersal and competitive abilities. Elevated microsites, especially decayed wood, act as seed beds for both ectomycorrhizal forest trees and fungi, thus affecting the succession of boreal forest ecosystems.