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Forest trees frequently interact with a diverse range of microorganisms including dark septate endophytes (DSEs) and fungal pathogens. Plant defense responses to either individual pathogens or endophytes have been widely studied, but very little is known on the effect of coinfection on host defenses. To study the impact of coinfection or tripartite interaction on plant growth and host defenses, Norway spruce (Picea abies (L.) Karst) seedlings were inoculated with a DSE Phialocephala sphaeroides or with a root pathogen Heterobasidion parviporum Niemela & Korhonen or coinfected with both fungi. The results showed that the DSE promoted the root growth of spruce seedlings. Control seedlings without any inoculum were subjected to sequencing and used as a baseline for identification of differentially expressed genes (DEGs). RNA-seq analysis of seedlings inoculated with P. sphaeroides, infected with H. parviporum or coinfected with both fungi resulted in a total of 5269 DEGs. The majority of DEGs were found in P. sphaeroides-inoculated seedlings. Lignin biosynthesis pathways were generally activated during fungal infections. The pattern was distinct with endophyte inoculation. The majority of the genes in the flavonoid biosynthesis pathway were generally suppressed during fungal infections. A specific transcriptional response to P. sphaeroides inoculation was the increased transcripts of genes involved in jasmonic acid biosynthesis, mitogen-activated protein kinases signaling pathway, plant hormone signal transduction and calcium-mediated signaling. This may have potentially contributed to promoting the root growth of seedlings. Although the coinfection suppressed the induction of numerous genes, no negative effect on the growth of the spruce seedlings occurred. We conclude that the subsequent H. parviporum infection triggered reprogramming of host metabolism. Conversely, the endophyte (P. sphaeroides), on the other hand, counteracted the negative effects of H. parviporum on the growth of the spruce seedlings.

The major threats to the sustainable supply of forest tree products are adverse climate, pests and diseases. Climate change, exemplified by increased drought, poses a unique threat to global forest health. This is attributed to the unpredictable behavior of forest pathosystems, which can favor fungal pathogens over the host under persistent drought stress conditions in the future. Currently, the effects of drought on tree resistance against pathogens are hypothetical, thus research is needed to identify these correlations. Norway spruce (Picea abies (L.) H. Karst.) is one of the most economically important tree species in Europe and is considered highly vulnerable to changes in climate. Dedicated experiments to investigate how disturbances will affect the Norway spruce—Heterobasidion sp. pathosystem are important, in order to develop different strategies to limit the spread of H. annosum s.l. under the predicted climate change. Here, we report a transcriptional study to compare Norway spruce gene expressions to evaluate the effects of water availability and the infection of Heterobasidion parviporum. We performed inoculation studies of three-year-old saplings in a greenhouse (purchased from a nursery). Norway spruce saplings were treated in either high (+) or low (−) water groups: high water group received double the water amount than the low water group. RNA was extracted and sequenced. Similarly, we quantified gene expression levels of candidate genes in biotic stress and jasmonic acid (JA) signaling pathways using qRT-PCR, through which we discovered a unique preferential defense response of H. parviporum-infected Norway spruce under drought stress at the molecular level. Disturbances related to water availability, especially low water conditions can have negative effects on the tree host and benefit the infection ability of the pathogens in the host. From our RNA-seq analysis, 114 differentially expressed gene regions were identified between high (+) and low (−) water groups under pathogen attack. None of these gene pathways were identified to be differentially expressed from both non-treated and mock-control treatments between high (+) and low (−) water groups. Finally, only four genes were found to be associated with drought in all treatments.

The major threats to the sustainable supply of forest tree products are adverse climate, pests and diseases. Climate change, exemplified by increased drought, poses a unique threat to global forest health. This is attributed to the unpredictable behavior of forest pathosystems, which can favor fungal pathogens over the host under persistent drought stress conditions in the future. Currently, the effects of drought on tree resistance against pathogens are hypothetical, thus research is needed to identify these correlations. Norway spruce (Picea abies) is one of the most economically important tree species in Europe, and is considered highly vulnerable to changes in climate. Dedicated experiments to investigate how disturbances will affect the Norway spruce - Heterobasidion sp. pathosystem are important, in order to develop different strategies to limit the spread of H. annosum s.l. under the predicted climate change. Here, we report a transcriptional study to compare Norway spruce gene expressions to evaluate the effects of water availability and the infection of Heterobasidion parviporum. We performed inoculation studies of three-year-old saplings in a greenhouse (purchased from a nursery). Norway spruce saplings were treated in either high (+) or low (-) water groups: high water group received double the water amount than the low water group. RNA was extracted and sequenced. Similarly, we quantified gene expression levels of candidate genes in biotic stress and jasmonic acid (JA) signaling pathways using qRT-PCR, through which we discovered a unique preferential defense response of H. parviporum-infected Norway spruce under drought stress at the molecular level. Disturbances related to water availability, especially low water conditions can have negative effects on the tree host and benefit the infection ability of the pathogens in the host. From our RNA-seq analysis, 114 differentially expressed gene regions were identified between high (+) and low (-) water groups under pathogen attack. None of these gene pathways were identified to be differentially expressed from both non-treated and mock-control treatments between high (+) and low (-) water groups. Finally, only four genes were found to be associated with drought in all treatments.

Heterobasidion spp. pathogenic fungi produce conspicuous fruitbodies on infected wood, which may represent a habitat for a range of organisms, including invertebrates. The aim was to: (i) assess and compare invertebrate diversity in Heterobasidion spp. fruitbodies, infected Picea abies wood and adjacent soil, (ii) test methods for collecting invertebrates from Heterobasidion spp. fruitbodies. A total of 69 Heterobasidion fruitbody samples, 46 wood samples and 19 soil samples were collected at two forest sites in Latvia. In total, 7311 invertebrate individuals were collected representing 62 different taxa, among which 55 were in fruitbodies, 47 in wood and 36 in soil. The dominant invertebrates in fruitbodies were Acari (82.4%), Collembola (6.2%), Coleoptera (4.7%), in wood were Acari (78.6%), Collembola (9.6%), Diplopoda (3.1%), in soil were Acari (90.6%), Collembola (3.4%) and Coleoptera (1.4%). Among all taxa, 17.7% were found exclusively in fruitbodies, 3.2% in wood, 4.8% in soil, while 48.4% were shared among different substrates. Although many invertebrate taxa were shared, their relative abundance differed substantially among different substrates. The sampling methods used provided valuable information on invertebrate communities. Further development of these methods may be needed to obtain more detailed and specific information about invertebrates inhabiting different habitats.

The literature review focuses on the effect of forest soil properties on infection of coniferous trees and stumps by Heterobasidion spores and further growth of mycelium from tree to tree. Spread of the fungus is greater in alkaline soil. Forest plantations on former agricultural lands have an increased risk of infection, due to lack of antagonistic soil microorganisms. In Latvia, severe infection of spruce stands by Heterobasidion root rot has been observed on peat soils.

Diversity of fungi was investigated in Scots pine and Norway spruce stumps showing symptoms of butt rot in managed coniferous forests located in the northernmost distribution area of Heterobasidion butt rot in Finland. During a survey of 101 recently clear-cut stands, a total of 479 stumps showing signs of decay were sampled. Fungi were isolated from wood chips taken at the transition zone between healthy and decayed wood. A subsample of 209 isolates from Norway spruce and nine from Scots pine was selected for sequencing ITS-DNA. In Norway spruce 59 species and in Scots pine six fungal species were detected based on ≥98% similarity and 62 genera based on ≥95% similarity with sequences in GenBank. Three sequences did not match to any identified sequence in GenBank. The majority of the isolates (74%) were Ascomycota while 18% were Basidiomycota and 6% Zygomycota. The dominance of Ascomycota over Basidiomycota was pronounced in spruce stumps of stands with a butt rot incidence above 20% and the decayed area was larger in sample discs from which only Ascomycota were isolated than in discs inhabited by Basidiomycota. The result suggests that there is a high site- and/or tree -specific variation of the mycoflora in the Norway spruce decaying butt. Based on these results no single fungal species can be suspected to act as causal agent of butt rot occurring in Norway spruce nor in Scots pine in Northern Ostrobothnia and Kainuu counties.

Stumps play a pivotal role in the epidemiology of the fungal forest pathogens Heterobasidion spp. because they are the main courts of primary airborne infections. The aims of this study were (i) to determine the susceptibility of seven tree species (i.e., Larix sibirica, Picea abies, Picea sitchensis, Pinus contorta, Pinus strobus, Pinus sylvestris and Pseudotsuga menziesii) to primary infection by H. annosum and H. parviporum through comparative inoculation experiments of conidia on wood discs in controlled conditions, (ii) to compare the susceptibility of wood discs of the same tree species to natural airborne infections in two Latvian Norway spruce forest stands infested either by H. annosum or H. parviporum, (iii) to explore the rates of infection of wood discs at increasing distances from spore sources in these two forests to make inferences on the range of spores dispersal. Results obtained by spraying wood discs with conidial suspensions in controlled conditions are in agreement with those obtained by exposing wood discs to the natural airborne inoculum in the forests, as clearly supported by the significant correlation (r = 0.79, p &lt, 0.05) between the two sets of data. Susceptibility was highest in Pinus species, followed by P. abies and P. sitchensis. Susceptibility was lowest for L. sibirica and P. menziesii. The area colonized by Heterobasidion spp. in the sapwood of wood discs was much greater than that colonized in the heartwood. A sharp decrease in the rate of infection of wood discs with distance from spore sources (i.e., fruiting bodies) was observed, further confirming the importance of local spore sources in the epidemiology of Heterobasidion spp. Taken together, these findings could help designing tactics to manage these fungal forest pathogens.

Significant hybridization between the invasive North American fungal plant pathogen Heterobasidion irregulare and its Eurasian sister species H. annosum is ongoing in Italy. Whole genomes of nine natural hybrids were sequenced, assembled and compared with those of three genotypes each of the two parental species. Genetic relationships among hybrids and their level of admixture were determined. A multi-approach pipeline was used to assign introgressed genomic blocks to each of the two species. Alleles that introgressed from H. irregulare to H. annosum were associated with pathways putatively related to saprobic processes, while alleles that introgressed from the native to the invasive species were mainly linked to gene regulation. There was no overlap of allele categories introgressed in the two directions. Phenotypic experiments documented a fitness increase in H. annosum genotypes characterized by introgression of alleles from the invasive species, supporting the hypothesis that hybridization results in putatively adaptive introgression. Conversely, introgression from the native into the exotic species appeared to be driven by selection on genes favoring genome stability. Since the introgression of specific alleles from the exotic H. irregulare into the native H. annosum increased the invasiveness of the latter species, we propose that two invasions may be co-occurring: the first one by genotypes of the exotic species, and the second one by alleles belonging to the exotic species. Given that H. irregulare represents a threat to European forests, monitoring programs need to track not only exotic genotypes in native forest stands, but also exotic alleles introgressed in native genotypes.

Patterns of forest disease are shifting worldwide. Pine woodlands in the Mediterranean Basin deliver important functions that may be disturbed by pathogen damage. In this chapter, I briefly review general concepts with examples on how and why forest pathogens are affecting pines. The main threat is the one posed by invasive pathogens that continually enter Europe via trade of living plants. While this aspect is capturing most of the attention, more efforts should be placed on understanding the role of native pathogens that may become more aggressive under global warming. For instance, the role of pathogens driving plant-soil feedbacks in pine woodlands, as well as their role in benefitting from abiotic perturbations such as drought, fire, hail or wind, remains largely unexplored. Another risk may come from future land-use changes. Nowadays, pines cover extensive areas with minimal management. However, increasing demand for timber products may put pressure on these stands and increase the prevalence of roots rots, particularly those caused by Heterobasidion annosum. Simulation data show that stump infection by Heterobasidion can be prevented by spraying thinning stumps with spores of the biocontrol fungus Phlebiopsis gigantea. Promoting mixed stands can be beneficial too. Diplodia sapinea is another pathogen to consider given its association with drought and hail. Concerning exotics, the pine wood nematode still poses the highest risk, however new invaders such as Fusarium circinatum, Dothistroma spp. or H. irregulare require attention. Greater involvement of society and stakeholders in preventing disease is needed. Several general directions for research and some general management recommendations are provided.

The Heterobasidion annosum s.l species complex comprises the most damaging forest pathogens to Norway spruce. We revisited previously identified Quantitative Trait Loci (QTLs) related to Heterobasidion-resistance in Norway spruce to identify candidate genes associated with these QTLs. We identified 329 candidate genes associated with the resistance QTLs using a gene-based composite map for Pinaceae. To evaluate the transcriptional responses of these candidate genes to H. parviporum, we inoculated Norway spruce plants and sequenced the transcriptome of the interaction at 3 and 7 days post inoculation. Out of 298 expressed candidate genes 124 were differentially expressed between inoculation and wounding control treatment. Interestingly, PaNAC04 and two of its paralogs in the subgroup III-3 of the NAC family transcription factors were found to be associated with one of the QTLs and was also highly induced in response to H. parviporum. These genes are possibly involved in the regulation of biosynthesis of flavonoid compounds. Furthermore, several of the differentially expressed candidate genes were associated with the phenylpropanoid pathway including a phenylalanine ammonia-lyase, a cinnamoyl-CoA reductase, a caffeoyl-CoA O-methyltransferase and a PgMYB11-like transcription factor gene. Combining transcriptome and genetic linkage analyses can help identifying candidate genes for functional studies and molecular breeding in non-model species.

Plant microbiome plays an important role in maintaining the host fitness. Despite a significant progress in our understanding of the plant microbiome achieved in the recent years, very little is known about the effect of plant pathogens on composition of microbial communities associated with trees. In this study, we analysed the mycobiome of different anatomic parts of asymptomatic and symptomatic Norway spruce trees naturally infected by Heterobasidion spp. We also investigated the primary impact of the disease on the fungal communities, which are associated with Norway spruce trees. Our results demonstrate that the structure of fungal communities residing in the wood differed significantly among symptomatic and asymptomatic Heterobasidion-infected trees. However, no significant differences were found in the other anatomic regions of the trees. The results also show that not only each of individual tree tissues (wood, bark, needles and roots) harbours a unique fungal community, but also that symptomatic trees were more susceptible to co-infection by other wood-degrading fungi compared to the asymptomatic ones.

We tested if root colonisation by ectomycorrhizal fungi (EMF) could alter the susceptibility of Norway spruce (Picea abies) seedlings to root rot infection or necrotic foliar pathogens. Firstly, spruce seedlings were inoculated by various EMF and challenged with Heterobasidion isolates in triaxenix tubes. The ascomycete EMF Meliniomyces bicolor, that had showed strong antagonistic properties towards root rot causing Heterobasidion in vitro, protected spruce seedlings effectively against root rot. Secondly, spruce seedlings, inoculated with M. bicolor or the forest humus, were subjected to necrotrophic foliar pathogens in conventional forest nursery conditions on peat substrates. Botrytis cinerea infection after winter was mild and the level of needle damage was independent of substrate and EMF colonisation. Needle damage severity caused by Gremminiella abietina was high in seedlings grown in substrates with high nutrient availability as well as in seedlings with well-established EMF communities. These results show that albeit M. bicolor is able to protect spruce seedlings against Heterobasidion root rot in axenic cultures it fails to induce systemic protection against foliar pathogens. We also point out that unsterile inoculum sources, such as the forest humus, should not be considered for use in greenhouse conditions as they might predispose seedlings to unintended needle damages.

Heterobasidion viruses have previously been shown to affect each other’s transmission and phenotypic effects on their hosts in a complex way. In this work, Heterobasidion parviporum strains hosting five coinfecting viruses simultaneously were constructed and used as donors in transmission experiments. They showed that viruses move more frequently between the mycelia of the same species than between the mycelia of H. parviporum and Heterobasidion annosum. One of the strains was used to show that coinfection of five viruses is relatively unstable in a natural environment and analyses of the growth rate and competitive ability of Heterobasidion strains hosting various virus combinations revealed that viral effects are not additive. The results also supported the view that the transmission of the promising virocontrol agent HetPV13-an1 may be enhanced by coinfecting viruses in the donor mycelium. However, its detrimental effects may be blocked by the presence of other viruses in the same mycelium. Repositories GenBank accession number MN058080.

Heterobasidion spp. cause economically important losses in conifer forests in the Northern Hemisphere, especially in Norway spruce stands. Freshly cut stumps are the main route for Heterobasidion spp. infection. Even small stumps of spruce seedlings are known to be susceptible to spore infection, however, very little is currently known about the susceptibility of small stumps of understory spruce to Heterobasidion spore infection. To determine the frequency of spore infections among stumps of understory trees, we analyzed 756 Norway spruce stumps in eight sample plots in eastern Latvia. Understory trees 35&ndash, 68 years of age, with a stump diameter of 2&ndash, 10 cm, were felled 33&ndash, 48 weeks before sampling. In total, 514 (68%) of the spruce stumps were infected by Heterobasidion spores. The infection frequency among the plots varied from 56% to 86%. Both infection frequency and stump surface area occupied by Heterobasidion positively correlated with stump diameter, but neither correlated with the time span elapsed between felling and sampling. Colonization of trees by a competitor fungus, Phlebiopsis gigantea (Fr.) J&uuml, lich, was observed in 30 (4%) of investigated stumps, but did not have any effect on the frequency of Heterobasidion infections. Our data show that Norway spruce stumps can be highly susceptible to Heterobasidion spore infection. In order to control airborne Heterobasidion infections, stump treatment should be considered during the sporulation period of Heterobasidion spp.

Main conclusion Two terpene compounds and four genes were identified as potential biomarkers for further evaluation for Scots pine susceptibility or tolerance against Heterobasidion annosum. Scots pine (Pinus sylvestris) is one of the main sources of timber in the boreal zone of Eurasia. Commercial pine plantations are vulnerable to root and butt rot disease caused by the fungus Heterobasidion annosum. The pathogen affects host growth rate, causes higher mortality and decreases in timber quality, resulting in considerable economic losses to forest owners. Genetic and biochemical factors contributing to Scots pine tolerance against H. annosum infection are not well understood. We assessed the predictive values of a set of potential genetic and chemical markers in a field experiment. We determined the expression levels of 25 genes and the concentrations of 36 terpenoid compounds in needles of 16 Scots pine trees randomly selected from a natural population prior to artificial infection. Stems of the same trees were artificially inoculated with H. annosum, and the length of necrotic lesions was documented 5 months post inoculation. Higher expression level of four genes included in our analysis and encoding predicted alpha-pinene synthase (two genes), geranyl diphosphate synthase (GPPS), and metacaspase 5 (MC5), could be associated with trees exhibiting increased levels of necrotic lesion formation in response to fungal inoculation. In contrast, concentrations of two terpenoid compounds, beta-caryophyllene and alpha-humulene, showed significant negative correlations with the lesion size. Further studies with larger sample size will help to elucidate new biomarkers or clarify the potential of the evaluated markers for use in Scots pine disease resistance breeding programs.

Global change potentially increases forest vulnerability. Different abiotic and biotic factors may interact to cause forest decline and accelerated tree mortality. We studied a mixed Mediterranean continental forest where Pinus pinaster Ait. (maritime pine) shows widespread decline to analyse the role of different abiotic and biotic factors on health status and growth dynamics both at the individual and plot levels. We also analysed stand composition and regeneration of tree species to check whether there is a change in species dominance. Fungal pathogens were seldom present and we detected no pervasive fungi or insect infestation and no presence of pathogens like Heterobasidion or Phytophthora. Infection of hemiparasite plants like Viscum album L. (mistletoe) can reduce leaf area and its abundance is generally considered an expression of host decline. Yet, the existence among declining trees of high defoliation levels without mistletoe, but not vice versa, suggests that defoliation in response to some abiotic stressor could be a predisposing factor preceding mistletoe infection. Compared to healthy trees, declining and dead trees exhibited higher defoliation rates, smaller needles and lower recent growth with steeper negative trends. Dead and declining trees showed similar negative growth trends since the early 1990s droughts, which we interpreted as early warning signals anticipating mortality of currently declining trees in the near future. Mortality of maritime pine extending across all size classes, the lower presence of this species in the smallest size classes and its lack of regeneration suggest it is potentially losing its current dominance and being replaced by other co-occurring, more drought-tolerant species. Our results unravelled that maritime pine decline seems to be mainly driven by a combination of predisposing and inciting abiotic factors (microenvironment and drought stress) and biotic factors (mistletoe). The absence of widespread fungal pathogens suggests that they may have a minor role on pine decline acting only eventually as contributing factors. Although there could be other interrelations among factors or other biotic agents at play, our results strongly suggest that water stress plays a major role in the decline process of the dominant species on an ecosystem with strong land-use legacies.

Because intensive forestry favors the spread of Heterobasidion spp., increasing amounts of decayed Norway spruce (Picea abies) wood in forest stands should be taken into account in the management of commercial forests. Spruce wood colonized by Heterobasidion spp. ensures fruit body formation, which increases the risk of new infections by basidiospores and, subsequently, the spread of Heterobasidion root rot. To date, very little is known about the prevalence of Heterobasidion fruit bodies on decayed spruce wood in different environments. To assess Heterobasidion fruit body production on various soil types and on different parts of trees, more than 36,000 fruit bodies on 425 decayed logs, 321 decayed trees, and 262 stumps of Norway spruce located on mineral and peat soils were surveyed. The most abundant fruit body formation occurred on infected spruce logs left on peat soil. Significantly more fruit bodies (average total fruit body area cm2 m−3) were found on logs on drained peat soil compared to drained mineral soil (4960 and 2674 cm2, respectively). On stems of windthrown, uprooted trees, approximately three times more actively sporulating fruit bodies were formed compared to roots. Further, approximately two times more Heterobasidion fruit bodies occurred on the roots of partly uplifted stumps than on stumps with roots remaining in the soil. H. parviporum was identified in 88% and H. annosum in 12% of analyzed spruce individuals. In cases where the tree was infected by H. annosum s.s., fruit bodies of this fungus were also commonly produced on spruce wood. Abundant development of fruit bodies was also observed in abandoned agricultural and pasture lands. Moreover, rich vegetation in fertile forest types favored fruit body development on both mineral and peat soils. On such sites, infected logs and windthrown trees, as well as partly uplifted spruce stumps, should be removed to limit the spread of Heterobasidion spp. by basidiospores.

Heterobasidion irregulare is one of the most problematic forest pathogens in the northern hemisphere, but has only been found relatively recently in the north central United States. Discovered in Wisconsin in 1993, but probably established sometime before that, it quickly spread throughout the state. In November 2014, it was found in southeastern Minnesota. Field surveys were then conducted throughout Minnesota with the focus in the southeast near the initial discovery. To find additional infection sites, surveys were conducted with accompanying aerial imagery of red pine (Pinus resinosa Aiton) stands that were previously thinned. Samples were collected from selected sites with dead and dying trees as well as samples from stumps in recently thinned pine stands. These samples were processed first with a nested polymerase chain reaction (PCR) protocol, which was replaced by a real-time PCR assay after its development. No samples tested positive for H. irregulare using these methods and no cultures from isolations were obtained outside the original infection area. Other indigenous fungi were also identified. The majority were wood decay fungi in the Basidiomycota. A spore collection study was also conducted after field surveys. Automated rotary arm spore collectors were used and assayed with an ITS TaqMan real-time PCR assay. Collectors were placed strategically in different areas of Minnesota. A positive control was used in an infected red pine plantation in Wisconsin and this location had the highest number of spores trapped, with 63,776 over a week period. Spores of H. irregulare were detected at several sites in Minnesota, with the highest spore total observed in traps at 413 over a week period. All other locations sampled also had some spores collected except Itasca State Park located in northwestern Minnesota. The weekly deposition of spores ranged from 0 to 1.26 m&minus, 2 h&minus, 1. Low spore levels occurring in Minnesota indicate that some spores are present, but they are currently being detected in amounts that may not be sufficient for colonization to be successful.

Phlebiopsis gigantea (Fr.) Jul. is one of the most common fungal species in coniferous forests and commonly used as a biological control agent to prevent aerial infections of conifers by Heterobasidion fungi. We used feeding experiments to examine whether Hylobius abietis L. (Coleoptera: Curculionidae) could serve as a vector for P. gigantea and associated viruses by disseminating the fungus in its faecal pellets. Two different P. gigantea strains were used in the experiments: the virus-free Rotsop biocontrol strain and P. gigantea 93073 infected with the virus strain PgLV-1. The Rotstop strain showed 100% viability during insect feeding, while the viability of the virus-infected strain 93073 was only 35%. Virus persistence was 100% during the passage of the host fungus through the alimentary tract. Based on growth experiments using virus-infected and virus-free strains obtained by hyphal tip or oidial isolation, the presence of PgLV-1 did not significantly affect the growth rate of the host fungus.

Although impacts of intensive forest harvesting practices such as removing logging residue and stumps for bioenergy on forest ecosystem has been extensively studied, no information is available about the long-term effects of stump harvesting on the health status of young Norway spruce stands. In the present study, we surveyed damage caused by the large pine weevil (Hylobius abietis L.) and other causal agents as well as the height growth of the Norway spruce seedlings for 12 years in stump-harvested and non-harvested experimental plots. The overall effect of stump harvesting combined with mounding on the health and survival of spruces was positive: 70% of spruce seedlings in stump-harvested plots were spared any damage, while 58% of seedlings remained undamaged in undisturbed control plots. The positive effect of stump removal and mounding on tree health was mainly due to reduced pine weevil damage. In all, 14.8 and 24.6% of spruce seedlings were gnawed by pine weevil in stump-harvested and control plots, respectively. In stump-harvested plots, pine weevil damage was slighter and lasted for a shorter time than in control plots. In addition, damage caused by Hylastes spp., black root beetles, was significantly less on stump-harvested plots. By contrast, vole damage was most frequent in stump-harvested plots during the population peak when the vole density was exceptionally high. Irrespective of whether stumps were harvested or not, stump roots provided a source of Heterobasidion inoculum in the next spruce regeneration. During this early stage of stand development only a few spruce seedlings were killed by Heterobasidion root rot and there were neither temporal nor quantitative treatment-related differences between the treatments. From the third year onwards the height growth of undamaged seedlings was more rapid in stump-harvested than non-harvested plots. Our results suggest that the overall risk of damage in young Norway spruce stands can be reduced by stump harvesting. Thus, stump harvesting and mounding seem to decrease the need for supplementary planting, which reduces regeneration costs that represent a considerable financial investment for a forest owner.

In boreal forests, stumps of Norway spruce are removed after clear cutting to gather wood for energy and, additionally, to reduce damage caused by the root rot fungus, Heterobasidion parviporum. In order to understand the behavior of H. parviporum at stump-harvested Norway spruce sites, the survival of Heterobasidion mycelia in buried root fragments and their ability to infect nearby spruce seedlings was investigated in southern Finland. The longevity of Heterobasidion mycelium in investigated root pieces exceeded the follow-up period of 83 months. After six years, 18% of the root fragments yielded living Heterobasidion mycelia. The survival of H. parviporum in root fragments was highest in sandy soils and increased with increasing volume of root fragment. The probability of occurrence of the fungus was highest when at least 60% of the root volume was originally colonized by the fungus, irrespective of the state of decay. It took 4.5 years until roots of the first seedling reached the buried root fragment and the seedling became infected by H. parviporum mycelia. After six years, 8% of the seedlings were found to be infected. In addition, the number of root residuals, i.e., the potential infection sources of the next tree generation, was determined at five stump-harvested Norway spruce sites. At these slightly infested spruce sites, on average 1500 potential disease sources ha−1 (in the form of decayed root fragments ⩾ 1.5 cm in diameter) were found in the upper soil layer after stump removal. Thus, our results indicate that stump harvesting procedure aimed at gathering wood for energy may not be an efficient way to remove infected wood material from forest soils. Furthermore, our survey on stored stumps next to clear-cut areas showed that the formation of Heterobasidion fruiting bodies may be abundant in decayed stumps in the lowest parts of the storage pile. In order to avoid increased risks of spore infection in nearby forests, decayed spruce stumps, including the lowest muddy stumps, should be transported within two years from forest storage to the end-user.

The biological control agent Rotstop® composed of a suspension of spores of Phlebiopsis gigantea (Fr.) Jul. is widely used for protecting conifer stumps from aerial infection by Heterobasidion species. The efficacy of Rotstop application on Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) stumps was determined in several locations and at different seasons in Latvia. Mean efficacy in controlling natural infection by Heterobasidion spp. in spruce stumps was 64%, calculated on the basis of number of infected stumps, and 89%, calculated on the basis of area of infected wood on sample discs cut from the stumps. Corresponding proportions for pine were 82% and 95%. The results show that Rotstop can be successfully used for stump treatment in Latvia, although improved efficacy is desirable, particularly in spruce. A Latvian isolate of P. gigantea, selected from numerous isolates in preliminary tests, was included in one experiment and was shown to be as effective as the Rotstop isolate. In un...

The southeastern United States is among the most productive forested areas in the world. Four endemic southern pine species – loblolly, longleaf, shortleaf, and slash – contribute significantly to the economic and ecological values in the region. A recently described phenomenon known as Southern Pine Decline (SPD) has been reported as having widespread impact in the southern pine region, particularly on loblolly pine. Root-feeding weevils and their associated fungi have been suggested as causal agents, even though literature and empirical research suggests that they are secondary insects colonizing weakened trees. Further, no published information exists about whether their associated fungi can cause mortality of mature trees in the southeastern U.S. Since there are significant management implications for pine health, we reviewed and critically examined the SPD phenomena on the southern landscape. Our regional analyses of USDA Forest Inventory and Analysis data show no discernable patterns related to pine growth or mortality, especially as related to topographic factors. There are no large-scale patterns related to pine mortality suggesting multiple interacting factors impacting tree health at stand-level. As such, the hypothesis that SPD is a regionally important decline syndrome and labeling declining southern pine stands as SPD is not supported. Instead, we discuss many abiotic (soil types, climate) and biotic (insects, pathogens, genetics) factors that may be interacting with each other and affecting southern pine health. Finally, we suggest management recommendations for landowners with pine health issues.

Effects of different pre-planting soil preparations and post-harvest wood debris applications in a clear-cut Scots pine plantation, on the abundance, diversity, and activity of culturable microorganisms were investigated. The investigation was done 9 years after the re-plantings had been done. This formed part of an investigation of silvicultural practices for conservation and the biological control ofArmillariaandHeterobasidionin northern temperate forests (Poland). The treatments being compared, were expected to have altered the soil’s physical and chemical properties, and consequently, its biological properties. Only soft-rot microfungi from the Ascomycota and Zygomycota were detected in the soil. Fungi, including those antagonistic toArmillariaandHeterobasidion, were more abundant after shallow ploughing than after deep ploughing or ridging, and where chipped rather than coarse wood debris was left on the soil surface or incorporated. Scots pine trees had the most biomass and the least mortality after ridging and leaving coarse wood debris on the surface (associated with only a relatively moderate abundance of fungi).

A Thesis for applying for the degree of Doctor of Philosophy in Forestry The aim of the doctoral thesis was to investigate the survival ability of pathogenic fungi Heterobasidion spp. and Diplodia sapinea and the saprotrophic fungus Phlebiopsis gigantea and viruses, after passing through the digestive tract of large pine weevils (Hylobius abietis L.). In laboratory experiments the growth of Heterobasidion spp. (infected with viruses HetPV2-pa1, HetPV4-pa1, HetRV6-pa17) and strains of P. gigantea (virus infected strain PgLV-1) and virus free strain (Rotstop®) were examined. Pine weevil adults that were potentially infected by D. sapinea were also investigated. The results of the study revealed that the large pine weevil is indeed able to carry viable spores of pathogenic and saprotrophic fungi, as well as viruses, through their digestive tract. The viruses may occur within hosts (fungi) without causing any consequences or they can also have a negative effect, which manifests through inhibited fungal growth or virulence to the host. The effect of viruses on Heterobasidion spp. can be different: exhibiting no symptoms or, under suitable conditions (i.e. new host), interfering with the fungal growth. After passing through the large pine weevil, the P. gigantea virus infected strain has lower viability and growth rate compared to the same species strain without viruses. While P. gigantea is used to produce the preparation Rotstop® for the treatment of conifer stumps against Heterobasidion spp., it is essential to ensure that a virus free strain of P. gigantea was utilized. When using a virus infected strain of P. gigantea, the effectiveness of protection may be lower. D. sapinea is a pathogen on Scots pine (Pinus sylvestris). D. sapinea will spread over short distances via spores, but the fungus can spread much more extensively and intensively by adult pine weevils. The significance of large pine weevils in managed forest can increase in the future, since the adult weevils are able to transport viable spores of different fungi through their digestive tract. Doktoritöös uuriti patogeensete seente juurepessu (Heterobasidion spp.) ja Diplodia sapinea ning levinud saprotroofse seene hiidkooriku (Phlebiopsis gigantea) ellujäämust hariliku männikärsaka valmikute seedetrakti läbimisel. Analüüsiti juurepessu viiruste (HetPV2-pa1, HetPV4-pa1, HetRV6-pa17) ja hiidkoorikuga seotud viiruse (PgLV-1) ellujäämisvõimet mardikate seedetraktis. Võrreldi okaspuupuistutes juurepessu tõrjel kasutatava biopreparaadi Rotstop® aluseks oleva hiidkooriku viirusevaba tüve kasvu viirusega nakatunud seenetüve arenguga. Lisaks analüüsiti hariliku männikärsaka potentsiaali kanda edasi universaalse patogeeni D. sapinea nakkust. Uuringutest selgus, et harilik männikärsakas suudab seedetraktis elujõulisena edasi kanda patogeensete ja saprotroofse seente eoseid, samuti juurepessu ja hiidkoorikuga seotud viiruseid. Viirused võivad eksisteerida koos peremeesorganismiga sellele mõju avaldamata, kuid neil võib esineda ka negatiivne toime, mis avaldub peremeesorganismi (seene) kasvu pärssimises ja tõvestusvõimelisuses. Juurepessul esinevate viiruste toime võib seenele olla erinev: viirused võivad esineda sümptomiteta või sobivates tingimustes (uus peremeesorganism) pärssida seene kasvu. Hiidkooriku kahe tüve võrdlemisel selgus, et viirusega nakatunud seenetüvi oli viirusevaba tüvega võrreldes peale putuka seedetrakti läbimist madalama ellujäämisvõime ja aeglasema kasvuga. Rakendades hiidkoorikul põhinevat preparaati praktikas tuleks jälgida, et kasutatakse viirusevabu seenetüvesid. Kasutades viirusega nakatunud hiidkooriku tüvesid, võib efektiivsus juurepessu tõrjel osutuda oluliselt madalamaks. D. sapinea on harilikul männil esinev patogeen. D. sapinea levib eostega lühemaid vahemaid, kuid hariliku männikärsaka kaasabil võib seene levik looduses osutuda laialdasemaks. Saadud tulemuste põhjal saab väita, et hariliku männikärsaka mõju metsale võib olla senisest veelgi olulisem, sest putukad on võimelised seedetraktis edasi kandma erinevate patogeensete seente eoseid. Publication of this dissertation is supported by the Estonian University of Life Sciences

The effects of plant symbionts on host defence responses against pathogens have been extensively documented, but little is known about the impact of pathogens on the symbiosis and if such an impact may differ for nonnative and native pathogens. Here, this issue was addressed in a study of the model system comprising Pinus pinea, its ectomycorrhizal symbiont Tuber borchii, and the nonnative and native pathogens Heterobasidion irregulare and Heterobasidion annosum, respectively. In a 6-month inoculation experiment and using both in planta and gene expression analyses, we tested the hypothesis that H. irregulare has greater effects on the symbiosis than H. annosum. Although the two pathogens induced the same morphological reaction in the plant-symbiont complex, with mycorrhizal density increasing exponentially with pathogen colonization of the host, the number of target genes regulated in T. borchii in plants inoculated with the native pathogen (i.e. 67% of tested genes) was more than twice that in plants inoculated with the nonnative pathogen (i.e. 27% of genes). Although the two fungal pathogens did not differentially affect the amount of ectomycorrhizas, the fungal symbiont perceived their presence differently. The results may suggest that the symbiont has the ability to recognize a self/native and a nonself/nonnative pathogen, probably through host plant-mediated signal transduction.

Interactions between three genotypes of a silver fir (Abies alba Mill.) embryogenic callus and Heterobasidion abietinum, H. parviporum, and H. annosum were examined in dual cultures. The aim of this study was to determine whether dual cultures can be used to evaluate the degree of fungal virulence at an embryogenic level, and whether different genotypes of a callus show different susceptibility. The dual cultures were performed on Schenk and Hildebrandt medium. Mycelial growth of H. parviporum and H. annosum was significantly stimulated in the presence of the callus but was not directional in nature. The embryogenic callus died between six and nine days after being colonized by H. parviporum suggesting that this was the most virulent species. By contrast, the callus remained healthy for up to 50 d after colonization with H. abietinum suggesting that this was the least virulent species. The callus of the A. alba genotype which originated in the mountain region of Poland remained healthy and alive for a significantly longer period than the other two genotypes in the dual cultures with all three Heterobasidion species even though overgrown by mycelium suggesting that the mountain genotype had the strongest defence response to Heterobasidion infection.

Summary We studied the effect of climate warming on Heterobasidion root rot in boreal forests by measuring respiration activity of pure cultures of Heterobasidion parviporum in Norway spruce (Picea abies) sawdust and by linking these data to temperature data obtained from three spruce forests located along a north-south transect stretching from northern Germany to northern Finland. The pure cultures applied in this investigation were homokaryotic, but in a separate investigation, we found no significant difference between the activity of homo- and heterokaryotic isolates. We also found that the temperature response curves of growth and respiration rates of this fungus were similar and propose that respiration reflects the general activity of H. parviporum. The respiration data were scaled up to annual cumulative respiration activity using daily temperature measurements from soil and air in the spruce forest sites. The annual respiration activity of H. parviporum showed a linear relationship with the average annual air temperature. An increase in the annual air temperature by 5°C would raise the annual activity of H. parviporum in spruce roots in northern Finland, southern Finland and northern Germany by 91%, 53% and 40%, respectively. This increase remains below the predicted increase in forest growth in northern Finland but exceeds considerably the predictions for southern Finland. According to the previous literature, a number of other decay fungi show a similar activity response to temperature as H. parviporum, suggesting that this result can be generalized to decay fungi with similar ecological habits.

Conifer trees, including Norway spruce, are threatened by fungi of the Heterobasidion annosum species complex, which severely affect timber quality and cause economic losses to forest owners. The timely detection of infected trees is complicated, as the pathogen resides within the heartwood and sapwood of infected trees. The presence of the disease and the extent of the wood decay often becomes evident only after tree felling. Fourier-transform infrared (FT-IR) spectroscopy is a potential method for non-destructive sample analysis that may be useful for identifying infected trees in this pathosystem. We performed FT-IR analysis of 18 phloem, 18 xylem, and 18 needle samples from asymptomatic and symptomatic Norway spruce trees. FT-IR spectra from 1066 – 912 cm−1 could be used to distinguish phloem, xylem, and needle tissue extracts. FT-IR spectra collected from xylem and needle extracts could also be used to discriminate between asymptomatic and symptomatic trees using spectral bands from 1657 – 994 cm−1 and 1104 – 994 cm−1, respectively. A partial least squares regression model predicted the concentration of condensed tannins, a defense-related compound, in phloem of asymptomatic and symptomatic trees. This work is the first to show that FT-IR spectroscopy can be used for the identification of Norway spruce trees naturally infected with Heterobasidion spp.

Healthy Scots pine (Pinus sylvestris L.) transplants had in rhizosphere a community of saprotrophic fungi which considerably suppressed the growth of severe root pathogens Heterobasidion annosum and Armillaria obscura. A community from transplants affected by needle cast (Lophodermium spp.) suppressed both pathogens to a much smaller extent.

Summary Heterobasidion annosum (Fr.) Bref. sensu lato is an important fungal parasite of coniferous trees throughout the temperate regions of the world. Approximately 15% of Heterobasidion isolates are infected by dsRNA viruses, which are considered as obligately intracellular and transmit vertically into both basidiospores and asexual conidia. Insects such as H. abietis and its larvae feeding on wood colonized by Heterobasidion fungi may carry Heterobasidion conidia and hyphae. In this study, we used H. abietis as a model species to reveal whether Heterobasidion viruses resist in their fungal host passing through the digestive tract of insects that may potentially serve as disseminators of fungal propagules. Pinus sylvestris branches were inoculated by three different strains of Heterobasidion parviporum, hosting taxonomically diverse virus species: Heterobasidion RNA virus 2, HetRV4 and HetRV6. Then, the inoculated branches were fed to H. abietis insects, and beetle excrements were investigated for Heterobasidion infections. All the inoculated fungal strains survived passage through the alimentary tract of the insects (survival rate 25–80%). The passage rates of the viruses inside their hosts varied considerably, ranging from 0 to 67%. Two different mycoviruses, HetRV2 and HetRV6, survived the intestinal passage of their fungal host, while the virus species HetRV4 was detected among none of the five H. parviporum isolates retrieved from insect faeces. The relative stability of fungi harbouring viruses suggests that if viruses are to be used for biological control against Heterobasidion species, it is likely that insect feeding does not considerably decrease the virus effect, but might instead enhance short-range dispersal of the viral biocontrol agent.