Unequivocal evidence on the antagonistic effects of endophytic fungi associated with woody plants against insect herbivores has been documented in only a few cases so far. Experimental evidence of the significance of needle endophytes to coniferous trees has remained scant because it is difficult to obtain trees with needles free of endophytes that could be used as comparable controls for trees infected with endophytes. Previously we reported a new methodology to get Norway spruce (Picea abies (L.) Karst.) saplings without needle endophytes and to inoculate them with a needle endophyte Lophodermium piceae (Fuckel) Höhn. Here we describe the first trial where spruce saplings with and without needle endophytes were provided as substrate for insect larvae. We transferred larvae of two sawfly species, Neodiprion sertifer Geoffroy and Gilpina pallida Klug, to the seedlings. Even though the main host of these sawfly species is not Norway spruce, but Scots pine (Pinus sylvestris L.), they are also known to occasionally feed on spruce. In this experiment the larvae did not develop to pupae with the provided spruce substrate but consumed measurable amounts of needles. No significant difference was found between the extent of needles consumed by either of the two sawfly species in inoculated and uninoculated saplings.

Lophodermium piceae is the most common endophyte of Norway spruce ( Picea abies ) needles, and it probably occurs in the total distribution area of Norway spruce. Its significance to the host tree is still largely unknown mainly because no methodology is known for generating endophyte-free saplings nor for inoculation of intact needles with this fungus. We report here how a daily heat treatment at 40 °C for 8 h eradicates endophytes of Norway spruce needles within a few days without causing visible harm to the plants. We compared various methods for inoculation of endophyte-free saplings. In order to obtain effective inocula, we investigated what factors activate dormant L. piceae infections in the needle tissue resulting in ascomata formation. Best inoculation success was achieved by suspending naturally shed, highly infected needles above the saplings. Infection frequencies of up to 70% of needles were achieved with repeated inoculations in the course of 3 years. Once established in needles, individual infections by L. piceae can persist for at least 5 years. Individual infections remain small in young needles, but at a needle age of 6 years, they start to grow and spread resulting in presence all over the needle from base to tip at a needle age of 7 years, but still without causing visible symptoms on the needles. The presented methodology for generating both endophyte-free and endophyte-infected saplings of Norway spruce provides a new approach for testing the significance of infections by L. piceae to insect herbivory and diseases of Norway spruce needles.

Dothistroma septosporum, the primary causal agent of Dothistroma needle blight, is one of the most significant foliar pathogens of pine worldwide. Its wide host and environmental ranges have led to its global success as a pathogen and severe economic damage to pine forests in many regions. This comprehensive global population study elucidated the historical migration pathways of the pathogen to reveal the Eurasian origin of the fungus. When over 3800 isolates were examined, three major population clusters were revealed: North America, Western Europe, and Eastern Europe, with distinct subclusters in the highly diverse Eastern European cluster. Modeling of historical scenarios using approximate Bayesian computation revealed the North American cluster was derived from an ancestral population in Eurasia. The Northeastern European subcluster was shown to be ancestral to all other European clusters and subclusters. The Turkish subcluster diverged first, followed by the Central European subcluster, then the Western European cluster, which has subsequently spread to much of the Southern Hemisphere. All clusters and subclusters contained both mating-types of the fungus, indicating the potential for sexual reproduction, although asexual reproduction remained the primary mode of reproduction. The study strongly suggests the native range of D. septosporum to be in Eastern Europe (i.e., the Baltic and Western Russia) and Western Asia.

Diplodia sapinea is a cosmopolitan endophyte and opportunistic pathogen having occurred on several conifer species in Europe for at least 200 years. In Europe, disease outbreaks have increased on several Pinus spp. in the last few decades. In this study, the genetic structure of the European and western Asian D. sapinea population were investigated using 13 microsatellite markers. In total, 425 isolates from 15 countries were analysed. A high clonal fraction and low genetic distance between most subpopulations was found. One single haplotype dominates the European population, being represented by 45.3% of all isolates and found in nearly all investigated countries. Three genetically distinct subpopulations were found: Central/North European, Italian and Georgian. The recently detected subpopulations of D. sapinea in northern Europe (Estonia) share several haplotypes with the German subpopulation. The northern European subpopulations (Latvia, Estonia and Finland) show relatively high genetic diversity compared to those in central Europe suggesting either that the fungus has existed in the North in an asymptomatic/endophytic mode for a long time or that it has spread recently by multiple introductions. Considerable genetic diversity was found even among isolates of a single tree as 16 isolates from a single tree resulted in lower clonal fraction index than most subpopulations in Europe, which might reflect cryptic sexual proliferation. According to currently published allelic patterns, D. sapinea most likely originates from North America or from some unsampled population in Asia or central America. In order to enable the detection of endophytic or latent infections of planting stock by D. sapinea, new species-specific PCR primers (DiSapi-F and Diplo-R) were designed. During the search for Diplodia isolates across the world for species specific primer development, we identified D. africana in California, USA, and in the Canary Islands, which are the first records of this species in North America and in Spain.

Diplodia sapinea is an important pathogen of Pinus spp. in many parts of the world's temperate and sub-tropical zones. In Central Europe, this fungus has been known since its first description in the 19th century. But it was only in 2007 that the fungus was identified using DNA-markers in northern Europe (in Estonia). Viljo Kujala reported observing D. sapinea on logging debris of Norway spruce in Finland in 1945, but we have not been able to verify the identifications from his herbarium samples. In 2004, we collected cones under asymptomatic Scots pine (Pinus sylvestris) trees at one location near the southern coast of Finland but failed to isolate D. sapinea from the samples. In 2015 and 2016, we collected fallen Scots pine cones in 15 Scots pine stands in various parts of Finland and the fungus was found at five locations in southwestern Finland including the location investigated in 2004. Frequency of cones with pycnidia of D. sapinea varied from 1% to 12% in the infested stands. No symptoms of Diplodia tip blight or resinous cankers were detected in trees in the stands where the cones were collected. The results suggest a recent introduction after 2004 of the D. sapinea in Finland.

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.

Lophodermium piceae represents the most common Norway spruce needle endophyte. The aim of this study was to find out whether subpopulations of L. piceae in climatically different environments (in which Norway spruce occurs natively) are adapted to local thermal conditions. L. piceae’s ability for thermal adaptation was investigated by determining growth rates of 163 isolates in vitro at four different temperatures: 2, 6, 20 and 25 °C. Isolates were obtained between 1995 and 2010 from apparently healthy needles sampled in Finland, Poland, Switzerland, Italy and southeastern Siberia. The sampling sites represent seven climatically distinct locations. Results were evaluated in relation to the age and geographic origin of the isolate, in addition to the highest and lowest average monthly temperature of the sampling location. We found a significant correlation between the growth rate and the age of the isolate at 25 °C. Variation in growth rates between subpopulations was low compared to within subpopulations. Only at 2 °C did statistically significant differences between the average growth rates of subpopulations emerge. These results suggest that L. piceae covers the whole distribution area of Norway spruce but that generally the thermal reaction norm of its subpopulations has not changed according to local temperature ranges, despite high contrast in thermal conditions across this vast area. Therefore, it would appear that the thermal environment is not a crucial factor in assessing the fitness of this fungal species within the native range of Norway spruce.

Heterobasidion parviporum and H. annoswn are the most damaging root and butt rot pathogens of conifers in southern Fennoscandia where their distribution and frequency are reasonably well described in literature. However, in the most northern boreal forests of Europe the occurrence and significance of these pathogens is poorly known. In the future global warming may increase root and butt rot damages in the North. In this research the distribution of Heterobasidion spp. in northern Finland was compiled from literature and various available databases and from a field study in two counties of northern Finland, Northern Ostrobothnia and Kainuu. Conifer stumps from 101 regeneration cutting sites were inspected and sample discs from stumps with signs of decay were investigated for the presence of Heterobasidion spp. In all, 11,846 stumps were investigated. Decay was found in 429 Norway spruce stumps and 50 Scots pine stumps. H. parviporum was isolated from five spruce discs, each from a different site. H. annoswn was not detected. According to previous records H. annostan has been identified only twice in northern Finland and these locations are over 600 km south of the northernmost Scots pine stands in Fennoscandia. Instead, H. parviporum occurs sporadically in the whole distribution area of Norway spruce in northern Finland. Most probably H. parviporum can be found in overmature dense stands at fertile sites but even there only at low frequencies. In northern Finland butt rot of conifers is caused mainly by other decay fungi than Heterobasidion. Low pH of the organic soil layer is probably a significant factor hampering H. parviporum from spreading in the North.

Heterobasidion butt rot causes considerable losses to forestry in coniferous forests in many parts of Europe including southern Fennoscandia. In northern Fennoscandia, including central and northern Finland, both Heterobasidion annoswn and H. parviporum are rare. This study aimed to determine the incidence of butt rot in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) north of the area commonly infested by Heterobasidion spp. in Finland. The incidence of butt rot in these conifers was investigated in Northern Ostrobothnia and Kainuu using two data sets: stump inspections in 101 regeneration cutting sites and the Finnish National Forest Inventory (NFI) data. According to stump inspections butt rot occurred in 1% of Scots pine trees and 10% of Norway spruce trees. In the most recent NFI data from 2009 to 2011 the incidence of trees with rot in mature managed stands of these two counties was 1% and 5% for Scots pine and Norway spruce, respectively. According to NFI, these values are nearly equal to the average incidence of butt rot in mature pines and spruces of the whole of Finland. Consequently, the low incidence of Heterobasidion butt rot in these parts of Finland does not mean lower occurrence of butt rot. Butt rot incidence varied greatly between sites: 0-24% of Norway spruce stumps in spruce dominated sites and 0-13% of Scots pine stumps in pine dominated sites were infected. High butt rot incidence in Norway spruce was associated with high site fertility, stand age, low organic matter content of soil and low temperature sum of the site. In Scots pine butt rot appeared after stand age exceeded 120 years while decay occurred already in 40-80 years old Norway spruces. High butt rot incidence in Scots pine was associated also with high flow accumulation of the site. Precipitation, plot slope, plot aspect, ditches, stand density and elevation were insignificant variables in models explaining decay incidence. During 1991-2013 butt rot incidence in Scots pine or Norway spruce has not changed in the study area. Similarly, to the more southern areas with high Heterobasidion risk, butt rot control in the North should focus especially on fertile mineral soil sites.

Dothistroma septosporum, a notorious pine needle pathogen with an unknown historical geographic origin and poorly known distribution pathways, is nowadays found almost in all areas inhabited by pines (Pinus spp.). The main aim of this study was to determine the relationship between North European and East Asian populations. In total, 238 Eurasian D. septosporum isolates from 11 countries, including 211 isolates from northern Europe, 16 isolates from Russian Far East and 11 isolates from Bhutan were analysed using 11 species-specific microsatellite and mating type markers. The most diverse populations were found in northern Europe, including the Baltic countries, Finland and European Russia. Notably, D. septosporum has not caused heavy damage to P. sylvestris in northern Europe, which may suggest a long co-existence of the host and the pathogen. No indication was obtained that the Russian Far East or Bhutan could be the indigenous area of D. septosporum, as the genetic diversity of the fungus there was low and evidence suggests gene flow from northern Europe to Russian Far East. On the western coast of Norway, a unique genetic pattern was observed, which differed from haplotypes dominating other Fennoscandian populations. As an agent of dothistroma needle blight, only D. septosporum was documented in northern Europe and Asia, while D. pini was found in Ukraine and Serbia.

We investigated the causative agent of a disease outbreak affecting small-leaved limes (Tilia cordata Mill.) and resulting in darkening of the leaf petioles and excessive defoliation during summer 2016 in southern Finland. The fungal species composition of the symptomatic petioles was examined by culture isolation and molecular identification using ITS rDNA sequences, which revealed the most prevalent fungal species present in the petioles as Apiognomonia errabunda (Roberge) Hhn. Based on reviewing curated herbarium specimens deposited at the Universities of Helsinki and Turku, A. errabunda is native and widely distributed in small-leaved limes in Finland, and occasionally infects also other broadleaved trees, including Quercus robur L. and ornamental species of Tilia L. and Fagus L. The ITS sequence analysis conducted during this study revealed minor within-species polymorphisms similar to those observed earlier in the Central European and Russian populations of A. errabunda, and reports the first nucleotide sequences of this species from the Nordic countries.

Alien invasive pathogens have caused numerous disastrous epidemics around the globe during the last two centuries. The frequency of these catastrophes has increased in parallel with the increase of international plant trade. Effective control of the risks requires understanding of factors governing vulnerability of indigenous plants. We tested whether the threat caused by alien pathogens of Asian origin is random among various tree species in Europe or whether it relates to their distribution range. A database including distribution ranges of 75 European tree species and literature-derived information on their susceptibility to invasive forest pathogens (IFPs) of Asian origin was compiled. Analysis on this database indicated that the susceptibility to Asian pathogens is significantly more common among tree species that occur only within Europe than among species with distributional ranges from Europe to Siberia (disease susceptibility percentage, DSP, 52 and 19 %, respectively). Notably, all severely attacked tree species are strictly European while tree species with distribution ranges extending from Europe to Siberia show at most only mild or moderate symptoms of Asian IFPs. Furthermore, the proportion of European broadleaf tree species susceptible to Asian IFPs is significantly higher than that of conifer species. Our results suggest that in Europe, Asian pathogens cause a higher risk to temperate and Mediterranean forests, largely composed of broadleaved species with distributional ranges restricted to Europe, than to boreal forests dominated by conifers distributed to Siberia.

The significance of endophytic needle infections to Norway spruce is still largely unsolved. Lophodermium piceae is the most common species representing typically over 90 % of the cultivable isolates obtained from surface sterilized needles. Generally, more than 50 % of the needles are infected with this species and single healthy looking needles can accommodate several, even more than 30 individual infections, while still looking green and healthy (Müller et al. 2001). The foliage of Norway spruce (Picea abies) is noteworthy resistant against herbivores and diseases in North Europe as compared to needles of Scots pine (Pinus sylvestris). Interestingly, Scots pine needles are not colonized with any single dominating endophyte species similarly as needles of Norway spruce. Therefore, it appears intriguing to find out if resistance of Norway spruce needles against herbivores and diseases is mediated by L. piceae.

Most plant species harbour a diverse community of endophytic, but their role is still unknown in most cases, including ecologically and economically important tree species. This study describes the culturable fungal endophytic community of Pinus sylvestris L. twigs in northern Spain and its relationship with diametric growth of the host. In all, 360 twig samples were collected from 30 Scots pines in fifteen stands. Isolates were obtained from all twig samples and 43 fungal taxa were identified by morphogrouping and subsequent ITS rDNA sequencing. All isolates were Ascomycetes, being Dothideomycetes and Sordariomycetes the most abundant classes. Half of the species were host generalists while the others were conifer or pine specialists. We found three new endophytic species for the Pinaceae: Biscogniauxia mediterranea, Phaeomoniella effusa and Plectania milleri, and additional six new species for P. sylvestris: Daldinia fissa, Hypocrea viridescens, Nigrospora oryzae, Ophiostoma nigrocarpum, Penicillium melinii and Penicillium polonicum. The endophytic community of fast and slow growing trees showed differences in species composition, abundance and evenness, but not in diversity. Phoma herbarum was associated to fast growing trees and Hypocrea lixii to those growing slow. Our results support the hypothesis that some endophytic species may affect growth of P. sylvestris.

A silica-based propagation medium was developed for large-scale production of ectomycorrhizal (ECM) fungal inoculum by solid state fermentation. Development of the medium was started by screening for an optimal growth medium among six different semisynthetic agar media traditionally used in cultivation of ECM fungi. The majority (65 %) of the twenty tested ECM fungal strains that typically colonize Norway spruce (Picea abies) seedlings grew best on modified Melin-Norkrans (MMN) medium with reduced sugar content (½MMN). In order to develop a nutritionally similar medium for large-scale cultivation of the ECM fungi, we chose silica to form a solid matrix and light brewery malt extract to provide nutrients. The medium was supplemented with a commercial humic acid product that was shown to boost fungal growth. The optimal concentration of the constituents was screened for in two assays by determining the growth rates of seven potential inoculant ECM fungal strains (Amphinema sp., Cenococcum geophilum, Hebeloma sp., Meliniomyces bicolor, Paxillus involutus, Piloderma byssinum, and Tylospora asterophora). As a result, we composed a silica-based mass propagation medium (pH 5.8) containing 2.5 % brewery malt extract and 0.5 g/l humic acid product Lignohumate AM. This medium is easily produced and supported good growth of even the slowly growing and rarely studied Athelioid ECM strains. Furthermore, root systems of Norway spruce nursery seedlings were colonized by the tested ECM fungi by using solid inoculum formulated from the silica medium.

SummaryTrees are known to have adapted to local climatic conditions, but the adaptation of their pathogenic associates is poorly understood. Heterobasidion parviporum causes root and butt rot in spruce. In this work, the respiration of H. parviporum subpopulations from climatically diverse environments was examined at various temperatures. Isolates were obtained from three areas in Europe (southern Finland, Denmark and northern Italy) and from two locations in Central Siberia (Krasnoyarsk and Irkutsk). Respiration rates were measured in gas tight vials at eight temperatures from 0 to 33°C, using spruce saw dust as the sole substrate. Strains from Siberian locations with cold winters had higher activity at low temperatures (2–15°C) than strains from European locations with mild winters. Respiration rates of Siberian subpopulations increased more than those of European strains when the temperature rose from 0 to 6°C, but the increase was greater with the European subpopulations when the temperature increased further from 6 to 20°C. Only small differences were found among European as well as Siberian subpopulations. Variation in respiration rates between subpopulations was low compared to variation within subpopulations. Using strains isolated 2–18 years ago and thereafter stored at 5°C, we found lower respiration rates at 20°C in older isolates, independent of geographical origin, suggesting phenotypic plasticity of H. parviporum in regard to responses to temperature. Based on these findings, we propose that subpopulations of H. parviporum have the potential to adapt to global warming.

The type species of the genus Tiarosporella, T. paludosa, is epitypified and confirmed as a member of the Botryosphaeriaceae. Based on morphology and DNA sequence data of the large subunit nuclear ribosomal RNA gene (LSU, 28S) and the internal transcribed spacers (ITS) and 5.8S rRNA gene of the nrDNA operon, the genus Tiarosporella is shown to be poly- and paraphyletic. A group of isolates morphologically similar to T. paludosa cluster to the Phacidiaceae (Phacidiales, Leotiomycetes) and we accommodated them in Darkera, a genus associated with needle diseases of conifers, with D. picea introduced as a novel taxon. This new taxon includes isolates occurring on needles of Picea spp. in Europe (Finland, Norway and Switzerland) and differs from D. parca according to a five-locus alignment consisting of ITS, LSU, partial 18S nuclear ribosomal RNA, translation elongation factor 1-alpha and beta-tubulin genes. Four novel genera are introduced for tiarosporella-like fungi, namely Eutiarosporella based on E. tritici on Triticum aestivum from South Africa, Marasasiomyces based on M. karoo on Eriocephalus sp. from South Africa, Mucoharknessia based on M. cortaderiae on Cortaderia selloana from Argentina, and Sakireeta based on S. madreeya on Aristida setacea from India. Together with the genus Botryobambusa, these genera represent a subclade in the Botryosphaeriaceae that is ecologically diverse, occurring on Poaceae, as well as woody hosts, including endophytes, saprobes, and plant pathogens.

The trade of plants and plant products is globally the most important pathway for alien pests and pathogens causing considerable damage to plant health. The introductions have increased exponentially in Europe, and continued even in Australia, where extreme quarantine efforts are implemented. The commercial actors in international plant trade lack a strong motive to reduce pest and pathogen risks as the true costs of the practiced trade are not internalized in the pricing of the products but footed to taxpayers and land owners. Therefore a shared responsibility of international plant traders is suggested to complement the current legislative restrictions in controlling exotic pests and pathogens. (C) 2013 Elsevier Ltd. All rights reserved.

Summary We studied the effect of climate warming on H eterobasidion root rot in boreal forests by measuring respiration activity of pure cultures of H eterobasidion parviporum in Norway spruce ( P icea abies ) sawdust and by linking these data to temperature data obtained from three spruce forests located along a north‐south transect stretching from northern G ermany to northern F inland. 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.

Fungal viruses (mycoviruses) with RNA genomes are believed to lack extracellular infective particles. These viruses are transmitted laterally among fungal strains through mycelial anastomoses or vertically via their infected spores, but little is known regarding their prevalence and patterns of dispersal under natural conditions. Here, we examined, in detail, the spatial and temporal changes in a mycovirus community and its host fungus Heterobasidion parviporum, the most devastating fungal pathogen of conifers in the Boreal forest region. During the 7-year sampling period, viruses accumulated in clonal host individuals as a result of indigenous viruses spreading within and between clones as well as novel strains arriving via airborne spores. Viral community changes produced pockets of heterogeneity within large H. parviporum clones. The appearance of novel viral infections in aging clones indicated that transient cell-to-cell contacts between Heterobasidion strains are likely to occur more frequently than what was inferred from genotypic analyses. Intraspecific variation was low among the three partitivirus species at the study site, whereas the unassigned viral species HetRV6 was highly polymorphic. The accumulation of point mutations during persistent infections resulted in viral diversification, that is, the presence of nearly identical viral sequence variants within single clones. Our results also suggest that co-infections by distantly related viral species are more stable than those between conspecific strains, and mutual exclusion may play a role in determining mycoviral communities.