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Four species of the destructive forest pathogen Heterobasidion annosum sensu lato (s.l.) are present in Europe: H. annosum sensu stricto (s.s.), H. abietinum and H. parviporum are native species, while H. irregulare is a non‐native invasive species currently reported only in Italy, yet recommended for regulation throughout Europe. In this study, real‐time PCR detection tests were developed for each of the four species, which can be used simultaneously or individually thanks to probes labelled with species‐specific fluorescent dyes. The different performance criteria of each assay were evaluated, and it was determined that they were theoretically capable of detecting amounts of DNA corresponding to 311, 29 and 29 cell nuclei in H. annosum s.s., H. irregulare and H. parviporum, respectively. The specificity of each assay was assessed with a wide set of strains. Real‐time PCR tests successfully detected Heterobasidion species from 36 fruiting bodies taken from the forest, as well as from artificially inoculated or naturally infected wood samples. The multiplex real‐time PCR assays developed in this study could have practical applications both in forest management and in phytosanitary monitoring. [ABSTRACT FROM AUTHOR]

Elucidation of the role of infectious agents putatively involved in gill disease is commonly hampered by the lack of culture systems for these organisms. In this study, a farmed population of Atlantic salmon pre-smolts, displaying proliferative gill disease with associated Candidatus Branchiomonas cysticola, Ca. Piscichlamydia salmonis and Atlantic salmon gill pox virus ( SGPV) infections, was identified. A subpopulation of the diseased fish was used as a source of waterborne infection towards a population of naïve Atlantic salmon pre-smolts. Ca. B. cysticola infection became established in exposed naïve fish at high prevalence within the first month of exposure and the bacterial load increased over the study period. Ca. P. salmonis and SGPV infections were identified only at low prevalence in exposed fish during the trial. Although clinically healthy, at termination of the trial the exposed, naïve fish displayed histologically visible pathological changes typified by epithelial hyperplasia and subepithelial inflammation with associated bacterial inclusions, confirmed by fluorescent in situ hybridization to contain Ca. B. cysticola. The results strongly suggest that Ca. B. cysticola infections transmit directly from fish to fish and that the bacterium is directly associated with the pathological changes observed in the exposed, previously naïve fish. [ABSTRACT FROM AUTHOR]

Dieback of European ash was first observed in Europe in the early 1990s. The disease is caused by the invasive ascomycete Hymenoscyphus fraxineus, proposed to originate from Far East Asia, where it has been considered a harmless saprotroph. This study investigates the occurrence of H. fraxineus in tissues of local ash species in the Russian Far East, and assesses its population-specific genetic variation by ITS sequencing. Shoot dieback symptoms, characteristic of H. fraxineus infection on European ash, were common, but not abundant, on Fraxinus mandshurica and Fraxinus rhynchophylla trees in Far East Russia. High levels of pathogen DNA were associated with necrotic leaf tissues of these ash species, indicating that the local H. fraxineus population is pathogenic to their leaves. However, the low levels of H. fraxineus DNA detected in shoots with symptoms, the failure to isolate this fungus from such tissues, and the presence of other fungi with pathogenic potential in shoots with symptoms indicate that local H. fraxineus strains may not be responsible (or their role is negligible) for the observed ash shoot dieback symptoms in the region. Conspicuous differences in ITS rDNA sequences detected between H. fraxineus isolates from Russian Far East and European populations suggest that the current ash dieback epidemic in Europe might not directly originate from the Russian Far East. Revision of the herbarium material shows that the earliest specimen of H. fraxineus was collected in 1962 from the Russian Far East and the oldest H. fraxineus specimen of China was collected in 2004. [ABSTRACT FROM AUTHOR]

Ten saplings of European ash ( Fraxinus excelsior L.) naturally infected by the invasive ash dieback pathogen Hymenoscyphus fraxineus were collected in Ukraine and Norway and examined for bark necrosis and extension of discoloration in sapwood and pith in a stem region. Tissue-specific colonization profiles were determined by spatial analyses of symptomatic and visually healthy stem tissues using a H. fraxineus-specific qPCR assay and light microscopy. Our data suggest that hyphal growth in the starch-rich perimedullary pith is of particular importance for both axial and radial spread of H. fraxineus, but that most of its biomass accumulates in sapwood parenchyma. The study confirms the results from earlier work and presents new information that refines the current stem invasion model. [ABSTRACT FROM AUTHOR]

The white-rot fungus Heterobasidion parviporum Niemelä & Korhonen establishes a necrotrophic interaction with Norway spruce (Picea abies (L.) H.Karst.) causing root and butt rot and growth losses in living trees. The interaction occurs first with the bark and the outer sapwood, as the pathogen enters the tree via wounds or root-to-root contacts. Later, when the fungus reaches the heartwood, it spreads therein creating a decay column, and the interaction mainly occurs in the inner sapwood where the tree creates a reaction zone. While bark and outer sapwood interactions are well studied, little is known about the nature of the transcriptional responses leading to the creation of a reaction zone. In this study, we sampled bark and sapwood both proximal and distal to the reaction zone in artificially inoculated and naturally infected trees. We quantified gene expression levels of candidate genes in secondary metabolite, hormone biosynthesis and signalling pathways using quantitative polymerase chain reaction. An up-regulation of mainly the phenylpropanoid pathway and jasmonic acid biosynthesis was found at the inoculation site, when inoculations were compared with wounding. We found that transcriptional responses in inner sapwood were similar to those reported upon infection through the bark. Our data suggest that the defence mechanism is induced due to direct fungal contact irrespective of the tissue type. Understanding the nature of these interactions is important when considering tree breeding-based resistance strategies to reduce the spread of the pathogen between and within trees. [ABSTRACT FROM AUTHOR]

Armillaria species have a global distribution and play variable ecological roles, including causing root disease of diverse forest, ornamental and horticultural trees. Accurate identification of Armillaria species is critical to understand their distribution and ecological roles. This work focused on characterizing an unidentified Armillaria isolate from a Serbian forest using pairing, sequencing of the partial large subunit and intergenic spacer-1 regions of r DNA ( LSU- IGS1) and the translation elongation factor-1 alpha gene ( tef-1α) genes, and phylogenetic analyses. Despite previously obtained LSU- IGS1 RFLP patterns that matched the newly described North American Armillaria altimontana, pairing tests and phylogenetic analyses of LSU- IGS1 and tef-1α sequences clearly demonstrate that the unidentified isolate is not A. altimontana. Based on LSU-IGS1, Armillaria gallica isolates were polyphyletic, and the Serbian isolate clustered with a subset of European A. gallica isolates within a well-supported clade (99%). Based on tef-1α , the Serbian isolate appeared as a separate, well-supported clade (97%) that was basal to other poorly resolved, polyphyletic clades containing European A. gallica isolates. It is speculated that the unidentified Armillaria isolate from Serbia could represent an evolutionary ancestral state because of its separate, basal position compared with other clades comprising polyphyletic European A. gallica isolates. Alternatively, this unidentified Serbian isolate could represent an unusual hybrid because of its high-level sequence heterogeneity, represented by multiple two-nucleotide codes, within tef-1α. Further characterization is needed to confirm the taxonomic status and ecological/evolutionary significance of this unique, unknown Armillaria isolate from Serbia. [ABSTRACT FROM AUTHOR]

A survey to identify ophiostomatoid fungi that infect wounds on native Norwegian and Swedish broadleaved trees was undertaken during summer 2004. A fungus resembling a species of Sporendocladia was commonly isolated from the exposed cambium and inner bark of wounds. Morphological examination and comparisons of DNA sequence data for the ITS and 5.8 S regions of the rRNA gene region led to its identification as Sporendocladia bactrospora. Pathogenicity trials on young Populus tremula and Betula pubescens trees showed that S. bactrospora is capable of causing lesions on these trees. There have been few previous reports of S. bactrospora, and in most cases, these have been as saprophytes on wood. In contrast, results of this study show that it is a common inhabitant of freshly made wounds on native broadleaved trees in Scandinavia, and it appears to contribute to staining of wood. [ABSTRACT FROM AUTHOR]

Ceratocystis polonica and Heterobasidion parviporum are important fungal pathogens in Norway spruce ( Picea abies). Tree susceptibility to these pathogens with respect to phenology was studied using artificial fungal inoculations at six stages of bud development, and assessed by measuring phloem necroses in the stems of 2- and 8-year-old trees. Tree capacity for resistance was assessed by measuring phloem nonstructural carbohydrates at each stage. Phloem necroses were significantly larger in trees with fungal versus control inoculations and increased significantly over time. Changes in nonstructural carbohydrates occurred in the trees; a significant decline in starch and a slight but significant increase in total sugars occurred over time. These results suggest that susceptibility to fungal pathogens and carbohydrate levels in the stems of the trees were related to fine-scale changes in bud development. A trade-off may occur between allocation of starch (the major fraction of the stem carbohydrate pool) to bud development/shoot growth versus defence of the stem. Previous tests of plant defence hypotheses have focused on herbivory on plants growing under different environmental conditions, but the role of phenology and the effect of pathogens are also important to consider in understanding plant resource allocation patterns. [ABSTRACT FROM AUTHOR]

A rapid increase in the frequency of Dutch elm disease (DED), a wilting disease of elm trees caused by bark-beetle vectored fungi, was observed in the early 1990s on several wych elm stands around Oslofjord, southern Norway. To examine the current status of the disease and its impacts on elm population, disease frequency and size distribution of elms were recorded at four locations. Northern parts of Lier, a municipality most affected by DED in Norway 15 years ago, showed in the survey season 4% disease frequency, whereas 13.8% of trees were dead, the dead trees having accumulated over several years in the unmanaged stands. In southern parts of the municipality the mean disease and death percentages were 1.9 and 2.4%. Compatible with their low disease incidence in early 1990s, the other two areas now examined, municipality of Larvik and district of Grenland, showed comparably low frequency of DED. Northern part of Lier showed significantly higher overall density of elm trees per hectare than the other examined areas, and also the small elms below 5 cm in d.b.h. were most frequent in this region. In contrast, the density of large trees was lower in northern Lier than in the other examined areas. These data suggest that regeneration of the tree is not prohibited owing to the disease but that the large trees have been locally reduced in frequency as a result of DED. The superior general density of elm trees in northern Lier, owing to the exceptionally rich soil in the warm southern slopes of the region,> may have contributed to the rapid increase of DED in the area 15 years ago and to the subsequent settlement of the disease outbreak as a chronic stage. [ABSTRACT FROM AUTHOR]

The causative agent of dieback on European ash ( Fraxinus excelsior) was first described as Chalara fraxinea based on cultural morphology because no sexual stage of the fungus was known. Later, based on culturing of ascospores of a candidate teleomorph, morphological comparison and nuclear ribosomal internal transcribed spacer sequencing, the sexual stage of C. fraxinea was assigned as Hymenoscyphus albidus, a native and widespread species in Europe. Recently, the morphological species concept of H. albidus was shown to cover two species that cannot be separated from each other based on teleomorph characters, but which can be distinguished by several DNA markers. As a result, the strains causing ash dieback were reassigned as H. pseudoalbidus. The closely related H. albidus is presumably a non-pathogenic endophyte, but pathogenicity tests to confirm this hypothesis have not yet been performed. Genotyping of herbarium specimens has shown that H. pseudoalbidus was present in Switzerland for at least a decade prior to the epidemic outbreak in Europe. The origin of the ash dieback pathogen, and the general importance of correct pathogen identification to development of effective disease control, are discussed. [ABSTRACT FROM AUTHOR]

Dieback of European ash ( Fraxinus excelsior), caused by the ascomycete Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea), started around 1992 in Poland and has since then spread over large geographical areas. By November 2010, the disease had been recorded in 22 European countries. The gradual expansion and high intensity of the ash dieback epidemic in Europe may suggest that H. pseudoalbidus is an invasive alien organism. In Norway, ash dieback was first reported in spring 2008, and a survey in early summer of the same year revealed that the disease had spread over large parts of the southern and eastern regions of the country. The distance from the southernmost to the northernmost infected stands was, at that time, about 400 km. Some old necrotic lesions were also observed, indicating that the ash dieback pathogen is likely to have been present in Norway since at least 2006. In 2009, a spore sampler was installed in a diseased ash stand at Ås, South-Eastern Norway. Sampling started in late July and continued until late September. Large numbers of ascospores resembling those of H. pseudoalbidus were observed, with the maximum number of spores occurring from the end of July to mid-August. The deposition of ascospores occurred mainly between 6 and 8 a.m. Ascospores are most likely to be the primary source initiating host infections and responsible for the rapid recent spread of H. pseudoalbidus in Europe. [ABSTRACT FROM AUTHOR]

Aims: To demonstrate that a thorough characterization and virulotyping of Escherichia coli strains isolated from sheep over time leads to new insights into ovine E. coli potentially becoming human pathogens through horizontal gene transfer. Methods and Results: One hundred and fifty E. coli isolates from two sheep, sampled over 3 weeks, were characterized by serotyping, virulotyping, genotyping using multiple locus variable number tandem repeats analysis (MLVA) and susceptibility to phage infection in vitro. The 35 MLVA profiles and the serotype and virulotypes of the strains were closely associated. Many MLVA profiles differed in one locus independent of serotypes. Escherichia coli isolates of the same serotype or virulotype had identical or very similar MLVA profiles. No transductants that incorporated the bacteriophages were found in vivo, but six E. coli isolates were susceptible to the phage infection in vitro. Changes in MLVA profiles were seen after acquisition of Stx phages in vitro only. Conclusions: The sheep carried Stx phage susceptible E. coli that possessed virulence markers associated with human pathogenicity. Changes in bacterial genomes by phage transfer may complicate outbreak source investigations. Serotype has to be taken into account when evaluating strain relationships by MLVA. Significance and Impact of the Study: Sheep carry E. coli that encode for virulence markers and belong to serogroups known to be human pathogens. In addition, a selection of isolates was found to be susceptible to horizontal transfer of Shiga toxin genes by means of bacteriophages in vitro, and the transfer resulted in a discernible change of the MLVA patterns of E. coli. [ABSTRACT FROM AUTHOR]

Aim: To determine whether a Shiga toxin 2 (Stx2)-encoding phage from Escherichia coli O157:H7 could be transmitted to commensal E. coli in a ruminant host without adding a specific recipient strain. Methods and Results: Sheep were inoculated with an E. coli O157:H7 strain containing an Stx2-encoding bacteriophage (Φ3538) in which a chloramphenicol-resistant gene, cat, is inserted into stx2. A total of 149 faecal samples were sampled and analysed for detection and quantification of E. coli O157:H7 and presumptive transductants. Phage Φ3538 (Δ stx2:: cat) was demonstrated to be transduced to an ovine E. coli O175:H16 at one occasion. Conclusions: The study demonstrates an in vivo transduction in sheep from an E. coli O157:H7 strain to an ovine E. coli O175:H16. A functional Stx2-encoding phage was incorporated into the host’s DNA. Significance and Impact of the Study: This is the first in vivo stx phage transduction study reported in which a recipient strain was not fed to the test animals. We suggest that the access to susceptible hosts is one main limiting factor for transduction to occur in the intestine. [ABSTRACT FROM AUTHOR]

Summary In a greenery trial of Nobel fir (Abies procera ) in western Norway, most branches had good quality until 1994 when 31% of the branches were discarded. Since then a high level of losses has been noted and 87% were discarded in 2000. A survey in 1998 revealed that the needle blight fungus Delphinella abietis was the main cause. It seems that frost did not initiate or maintain the disease in the stand, but generally low temperatures may have been important. Discarded branches with the fungus have been left on the ground and have probably been important in maintaining a high level of attack. [ABSTRACT FROM AUTHOR]

The pathogenicity and growth of six cultures of the bark-beetle-associated blue-stain fungus Ceratocystis polonica were compared. Four were subcultures, established at different times, of the same isolate (80–53/7), and at least one of these was suspected to have lost its pathogenicity to Norway spruce (Picea abies) during subculturing. Two other cultures (93–208/115 and 94–169/13) were pathogenic. The pathogenicity of all cultures to Norway spruce was compared in three inoculation experiments, using (i) massive inoculation (≈144 inoculations per tree), (ii) low-density inoculation (14 inoculations per tree) of larger trees and (iii) single inoculation of 2-year-old seedlings. Fungal growth rates were tested in vitro on malt agar, and on malt agar under oxygen-deficient conditions. Isolates 93–208/115 and 94–169/13 and one of the subcultures of isolate 80–53/7 were pathogenic in all three inoculation experiments. Two other subcultures were nonpathogenic, while the last subculture was intermediate. The pathogenic cultures had higher growth rates on malt agar than the nonpathogenic and intermediate cultures, and they also grew better under oxygen-deficient conditions. These results demonstrate that blue-stain fungi may lose their pathogenicity after serial vegetative transfers on artificial substrates. The results are also consistent with the hypothesis that rapid growth and the ability to grow in oxygen-poor environments are important pathogenicity factors in blue-stain fungi. [ABSTRACT FROM AUTHOR]

Examines the induction of disease resistance in Norway spruce by necrotizing fungi. Effects of pre-inoculation with the root fungus Heterobasidion annosum, Nectria fuckeliana or a pathogenic strain of Ceratocystis polonica; Correlation between phloem necroses and extent of host symptoms.

The pine shoot beetles Tomicus minor and T. piniperda are common in the Nordic countries. Of these, T. piniperda may attack and kill living but severely stressed trees, whereas T. minor has never been reported to be individually responsible for killing live trees. Both species are associated with blue-stain fungi: T. minor with Ophiostoma canum and T. piniperda with Leptographium wingfieldii and Ophiostoma minus. The growth of these fungi was studied in phloem and sapwood of live Scots pine trees, on malt agar, and on malt agar under oxygen-deficient conditions. Leptographium wingfieldii was more virulent (i.e. caused more extensive host symptoms) grew more quickly on malt agar, and was less affected by oxygen-deficient growth conditions than either O. minus or O. canum. Ophiostoma canum was least virulent. In low-density inoculations it induced lesions similar to those induced by sterile control inoculations; it grew very slowly on malt agar and stopped growing after ≈30 mm under oxygen-deficient conditions. Ophiostoma minus was intermediate in all respects. The different virulence of the blue-stain fungi associated with the two pine shoot beetles may explain the lower level of aggressiveness in T. minor. [ABSTRACT FROM AUTHOR]

Summary Three clones of Norway spruce (Picea abies) were studied for their response to mass-inoculation with the blue-stain fungus Ceratocystis polonica. The effect of different pretreatments (fungal inoculation and wounding) before mass-inoculation was investigated for their possible role in an acquired resistance reaction. Pretreated trees showed enhanced resistance to the subsequent mass-inoculation relative to control trees that received no pretreatment. Furthermore, the fungal colonization of inoculated trees was less than that of wounded trees. The phenolic content of the bark, analysed by RP-HPLC, was compared in trees receiving different treatments. Trees inoculated with C. polonica had higher average concentration of (+)-catechin, taxifolin and trans-resveratrol than wounded trees. Both inoculated and wounded trees had higher average concentrations of these compounds than control trees. The effect of the phenolic extract of Norway spruce bark on the growth of the root rot fungus Heterobasidion annosum and the blue-stain fungi C. polonica and Ophiostoma penicillatum were investigated in vitro. Heterobasidion annosum was not negatively affected, and the extracts had fungistatic effects on the blue-stain fungi. The growth of O. penicillatum was more inhibited than the growth of the more aggressive C. polonica. [ABSTRACT FROM AUTHOR]

Norway spruce trees (Picea abies (L.) Karst.) pretreated by wounding and fungal infection showed highly enhanced resistance to a subsequent challenge inoculation with the pathogenic bluestain fungus Ceratocystis polonica (Siem.) C. Moreau. This is the first time the effectiveness of the constitutive and inducible defenses has been shown to depend on prior wounding and infection in conifers, although such acquired resistance has previously been found in several angiosperms. Trees that were pretreated with a combination of 12 bark wounds (1.6 × 10 cm), four fungal inoculations and four sterile inoculations 1–15 days before mass inoculation with C. polonica at 400 inoculations per square meter over a 0.8 m stem section had significantly shorter necroses in the phloem, less bluestained sapwood, and less dead cambium than untreated control trees. Pretreatment with four fungal or sterile inoculations alone did not lead to enhanced resistance. Pretreatment by bark wounding alone seemed to provide an intermediate degree of resistance compared to bark wounding, fungal inoculations and sterile inoculations combined. All trees had a marked increase in the number of resin ducts in the year of inoculation compared with previous years, suggesting that formation of traumatic resin ducts play an important role in the development and maintainance of enhanced resistance. [ABSTRACT FROM PUBLISHER]

This article focuses on interactions of two decay fungi Heterobasidion annosum and Stereum sanguinolentum with two non-decay fungi and bacteria, following inoculation into stems of 16 healthy Picea abies. All the microbes used in inoculations were of spruce origin. Growth and survival of microbes were followed over the subsequent five years. The stems were effectively colonized by the bacteria. Of the inoculated microbes, Ascocoryne cyclichnium and the bacteria had successfully invaded almost all the trees.

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The occurence of Armillaria species was assessed in Norway, enabling the northern-most distribution of this genus to be determined in Europe. Four Armillaria species were found in Norway. Armillaria borealis was the most common species occurring on woody vegetation to the permafrost zone (ca. 69°N). Armillaria cepistipes was present in southern and central Norway, but was not found further than 66°N. Armillaria solidipes and Armillaria gallica were rare, found at only one locality each; 59°40′ and 59°32′, respectively. Armillaria species were found on 14 hosts, but there was no significant difference between occurrence of A. borealis and A. cepistipes on declining and dead trees. Phylogenetic analyses separated each species into separate clades. All isolates of A. borealis, except one, and most isolates of A. solidipes were in separate clades. However, a subclade within the A. borealis clade was formed of two A. ostoyae and one A. borealis isolates. Two small A. cepistipes genets were found in a declining oak stand. [ABSTRACT FROM AUTHOR]

In September 2001, the uredinia sori of rust were found on alder trees in As parish, Akershus, in south-eastern Norway. Further examination of these sori, which always occurred on the lower side of the infected leaves, revealed urediniospores, which were uniformly echinulate and which had apical spines. From this morphology, Melampsoridium hiratsukanum was identified. Melampsoridium betulinum is also known to occur on A. glutinosa in As. A germination test with fresh urediniospores of M. hiratsukanum revealed four to six bizonate pores, similar to those reported for M. betulinum. However, this rust lacks spines in the apical region of its urediniospores and it is the presence of spines in the apical region that distinguishes these rust species.