Halvor Solheim

Research Professor

(+47) 920 33 663
halvor.solheim@nibio.no

Place
Ås H8

Visiting address
Høgskoleveien 8, 1433 Ås

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Abstract

Political action can reduce introductions of diseases caused by invasive forest pathogens (IPs) and public support is important for effective prevention. The public’s awareness of IP problems and the acceptability of policies aiming to combat these pathogens were surveyed in nine European countries (N = 3469). Although awareness of specific diseases (e.g., ash dieback) varied, problem awareness and policy acceptability were similar across countries. The public was positive towards policies for informational measures and stricter standards for plant production, but less positive towards restricting public access to protected areas. Multilevel models, including individual and country level variables, revealed that media exposure was positively associated with awareness of IP problems, and strengthened the link between problem awareness and policy acceptability. Results suggest that learning about IPs through the media and recognizing the associated problems increase policy acceptability. Overall, the study elaborates on the anthropogenic dimension of diseases caused by IPs.

Abstract

In Norway the common ash (Fraxinus excelsior L.) has its northernmost distribution in Europe. It grows along the coastal range as small fragmented populations. The first occurrence of ash dieback caused by Hymenoscyphus fraxineus in Norway was reported in 2008. At that time, the disease had already spread through large areas of southern and south-eastern parts of Norway. Since then the disease continued spreading with a speed of about 50- 60 km per year along the western coastal range. To monitor the disease development over time, we established eight permanent monitoring plots in south-eastern and western Norway in 2009 and 2012, respectively. In all plots tree mortality was high, especially among the youngest trees in south-eastern Norway. The extent of crown damage has continually increased in all diameter classes for both regions. In 2009, 76.8 % of all trees on the five monitoring plots in south-eastern Norway were considered to be healthy or slightly damaged, and only 8.9 % to be severely damaged. In 2015, 51.7 % were dead, 13.5 % severely damaged and only 25.7 % remained healthy or slightly damaged. To assess the infection pressure and spore dispersal patterns of the pathogen, we used a Burkard volumetric spore sampler placed in an infested ash stand in southern Norway. We examined the airborne ascospores of H. fraxineus and H. albidus captured on the sampling tape microscopically and with real-time PCR assays specific to these fungi. We detected very few ascospores of H. albidus, whereas ascospores of H. fraxineus dominated throughout entire sampling periods of 2009, 2010 and 2011. Spore discharge occurred mainly between the hours of 5 and 8 a.m., though the distinctive sporulation had yearly variation between 5-7 a.m. We observed the same diurnal pattern throughout the entire sampling period, with a seasonal peak in spore liberation between mid-July and midAugust, after which the number of ascospores decreased substantially. Similar diurnal patterns were observed throughout the sampling period except that after mid-August the number of trapped ascospores substantially decreased. To compare the genetic pattern of common ash in the northern and central ranges of Europe we analyzed the Norwegian samples together with available samples from central Europe by using chloroplast and nuclear microsatellite markers. We found that the northern range of common ash was colonized via a single migration route that originated in eastern or south-eastern Europe with little influence originating from other southern or western European refugia. In the northern range margins, genetic diversity decreased and population differentiation increased, coherent with a post-glacial colonization history characterized by founder events and population fluctuations. Based on our findings we discuss the future management and conservational implications.

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Abstract

Dieback of European ash (Fraxinus excelsior L.), a disease caused by the ascomycete Hymenoscyphus fraxineus (previously referred to as H. pseudoalbidus or Chalara fraxinea), was first observed in Poland in the early 1990ies, and is currently present almost throughout the entire distribution area of European ash. The characteristic symptoms of the disease include dead shoots with necrotic lesions in the bark and discoloration of xylem and pith but the seasonal dynamics of pathogen spread in shoot tissues remain poorly understood. To investigate whether the internal spread of the fungus involves season-specific patterns, saplings with necrotic bark lesions in 1-2 -year-old stem regions were collected during 2014-2015 at time intervals in spring, summer, autumn and winter at several localities in western Ukraine and at two localities in south-eastern Norway. Tissuespecific presence of H. fraxineus was determined by a highly sensitive quantitative real-time PCR assay that is specific to DNA of H. fraxineus. The relatively high proportion of bark samples positive for H. fraxineus in the saplings collected during spring provides support to a model that H. fraxineus can be a primary causative agent of bark lesions and that other fungi may eventually replace it in old infection areas.

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Abstract

High biodiversity is regarded as a barrier against biological invasions. We hypothesized that the invasion success of the pathogenic ascomycete Hymenoscyphus fraxineus threatening common ash in Europe relates to differences in dispersal and colonization success between the invader and the diverse native competitors. Ash leaf mycobiome was monitored by high-throughput sequencing of the fungal internal transcribed spacer region (ITS) and quantitative PCR profiling of H. fraxineus DNA. Initiation of ascospore production by H. fraxineus after overwintering was followed by pathogen accumulation in asymptomatic leaves. The induction of necrotic leaf lesions coincided with escalation of H. fraxineus DNA levels and changes in proportion of biotrophs, followed by an increase of ubiquitous endophytes with pathogenic potential. H. fraxineus uses high propagule pressure to establish in leaves as quiescent thalli that switch to pathogenic mode once these thalli reach a certain threshold – the massive feedback from the saprophytic phase enables this fungus to challenge host defenses and the resident competitors in mid-season when their density in host tissues is still low. Despite the general correspondence between the ITS-1 and ITS-2 datasets, marker biases were observed, which suggests that multiple barcodes provide better overall representation of mycobiomes.

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Abstract

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.

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Abstract

Armillaria possesses several intriguing characteristics that have inspired wide interest in understanding phylogenetic relationships within and among species of this genus. Nuclear ribosomal DNA sequence– based analyses of Armillaria provide only limited information for phylogenetic studies among widely divergent taxa. More recent studies have shown that translation elongation factor 1-α (tef1) sequences are highly informative for phylogenetic analysis of Armillaria species within diverse global regions. This study used Neighbor-net and coalescence-based Bayesian analyses to examine phylogenetic relationships of newly determined and existing tef1 sequences derived from diverse Armillaria species from across the Northern Hemisphere, with Southern Hemisphere Armillaria species included for reference. Based on the Bayesian analysis of tef1 sequences, Armillaria species from the Northern Hemisphere are generally contained within the following four superclades, which are named according to the specific epithet of the most frequently cited species within the superclade: (i) Socialis/Tabescens (exannulate) superclade including Eurasian A. ectypa, North American A. socialis (A. tabescens), and Eurasian A. socialis (A. tabescens) clades; (ii) Mellea superclade including undescribed annulate North American Armillaria sp. (Mexico) and four separate clades of A. mellea (Europe and Iran, eastern Asia, and two groups from North America); (iii) Gallica superclade including Armillaria Nag E (Japan), multiple clades of A. gallica (Asia and Europe), A. calvescens (eastern North America), A. cepistipes (North America), A. altimontana (western USA), A. nabsnona (North America and Japan), and at least two A. gallica clades (North America); and (iv) Solidipes/Ostoyae superclade including two A. solidipes/ostoyae clades (North America), A. gemina (eastern USA), A. solidipes/ostoyae (Eurasia), A. cepistipes (Europe and Japan), A. sinapina (North America and Japan), and A. borealis (Eurasia) clade 2. Of note is that A. borealis (Eurasia) clade 1 appears basal to the Solidipes/Ostoyae and Gallica superclades. The Neighbor-net analysis showed similar phylogenetic relationships. This study further demonstrates the utility of tef1 for global phylogenetic studies of Armillaria species and provides critical insights into multiple taxonomic issues that warrant further study.

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Abstract

The goal of this study was to assess the long-term effects of partial harvesting and supplementary soil scarification on the frequency of root and butt rot in managed uneven-sized Norway spruce stands. Frequency of rot and the population structure of the rot fungi were assessed on 1353 stumps after clear-cutting 21 years after a selection harvesting experiment. The initial experiment was comprised of three harvest strength (low, intermediate and high) of single-tree selection, removing approximately 25, 45 and 65% of the stand basal area. Uncut control plots were established at the same time. Supplementary soil scarification was applied in subplots within the single-tree selection plots, using a medium-sized excavator. After clear-cutting the stumps were analyzed with respect to rot caused by Heterobasidion parviporum, Armillaria spp., Stereum sanguinolentum as well as other rot fungi. Rot caused by Armillaria spp. was most common (8.6% of the stumps), while infection by H. parviporum (2.9%) or S. sanguinolentum (3.0%) was less frequent. The group “other rot” (5.4%) comprised 21 identified taxa, each occurring in 1–15 stumps. Significantly lower rot frequencies were found for the uncut control (16.3%) and intermediate harvest strength (15.7%), compared with low harvest strength (23.6%). A rot frequency of 21.0% was found in the high harvest strength. In two of three harvest strengths, the rot frequency was higher than for the uncut control. As the observed rot frequencies did not increase consistently with increasing harvest strength, the results do not completely support the initial expectations of increased rot after single-tree selection compared with the uncut control. However, since the probability of rot in individual stumps on plots treated with single-tree selection was significantly affected by the distance to the nearest strip road (H. parviporum) as well as dependent on the size of and distance to the nearest stump of trees cut during the experimental harvest (H. parviporum, S. sanguinolentum and total rot), it is evident that the single-tree selection harvesting was partially responsible for some of the observed rot. One of the selection criteria in the initial harvest was a sanitary removal of trees of poor vitality. Varying degrees of sanitation felling may therefore have offset the effects of new infections in wounds or spread of rot fungi through adjacent stumps. Supplementary soil scarification in small gaps of the residual stand had no significant effect on the frequency of rot, suggesting that such treatment may be used to facilitate regeneration in uneven-sized spruce stands on similar sites.

Abstract

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.

Abstract

Ash dieback, caused by the ascomycete Hymenoscyphus fraxineus, has been spreading throughout Europe since the early 1990s, threatening European ash at a continental scale. Little is known about the development of the disease in individual forest trees and in different age classes. In this study we monitored ash dieback on trees of different diameter classes in five permanent plots in ash stands in south-eastern Norway from 2009 to 2016, and from 2012 to 2016 in three plots in western Norway with a shorter disease history. Our results showed that more than 80% of the youngest and more than 40% of the intermediate future crop trees in the plots in south-eastern Norway were dead by 2016, while the disease development in large, dominant trees was slower. Although less damage has been observed in the plots in western Norway, the trend for the juvenile trees is the same as in south-eastern Norway with rapidly increasing damage and mortality. Most dead trees in south-eastern Norway were found at sites with high soil moisture and showed symptoms of root-rot caused by Armillaria species. Infected trees, both young and old ones, are weakened by the disease and appear to be more susceptible to other, secondary pathogens, especially under unfavourable site conditions.

Abstract

Ash dieback, caused by the ascomycete Hymenoscyphus fraxineus, was first observed in the eastern and southernmost Norway in 2008. Based on the age of stem bark lesions, it was concluded that the fungus had arrived to the region no later than 2006. Since 2008 the annual spread of the disease northwards along the west coast of Norway has been monitored. The registration was done each year during early summer around a disease frontier recorded in the previous year. The occurrence of necrotic bark lesions in the previous-year shoots and dieback of these shoots, and isolation of H. fraxineus from the discoloured wood associated with necrotic bark lesions were used as signs of ash dieback. These records indicate an annual spread of ash dieback in the range between 25 km and 78 km, and a mean annual spread of 51 km. The cause of the spread is discussed.

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Abstract

Species of Leptographium are generally characterized by mononematous conidiophores and are commonly associated with bark beetles and weevils. These species are responsible for sapstain and in some cases serious diseases on a range of primarily coniferous trees. In comparison with coniferous trees, the occurrence of Leptographium species on hardwood trees has been poorly studied in Europe. During a survey of ophiostomatoid fungi on various tree species in Norway and Poland, three unusual species, which fit the broader morphological description of Leptographium spp., were found in association with Scolytus ratzeburgi, Dryocoetes alni and Trypodendron domesticum on a variety of hardwoods, and from wounds on Tilia cordata. Phylogenetic analyses of sequence data for three gene regions (ITS2-LSU, β-tubulin, and TEF1-α) showed that these Leptographium species are phylogenetically closely related to each other and form a well-supported lineage that included Grosmannia grandifoliae and Leptographium pruni. The first species could be distinguished from the other Leptographium species based on conidiophores arising from spiral hyphae, chlamydospore-like structures and a hyalorhinocladiella-like synanamorph in culture. The second species differs from the previous one by having distinctly shorter conidiophores and smaller conidia. This species also produces a well-developed sporothrix-like synanamorph with denticulate conidiogenous cells. Based on these unusual morphological characteristics and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium trypodendri sp. nov. and L. betulae sp. nov. are provided. The third group of isolates belonged to Grosmannia grandifoliae, representing the first report of this species outside of the USA. The newly defined G. grandifoliae complex is the first species complex in Leptographium s.l. consisting of only hardwood-infecting species.

Abstract

The root rot pathogens in Norway spruce (Picea abies) Heterobasidion ssp. cause substantial loss in carbon sequestered in forest and economic revenue for forest owners. To facilitate strategic breeding planning for increased resistance against this pathogen in particular, the blue stain fungus Endoconidiophora polonica, growth and wood quality traits (wood density and spiral grain), we estimated additive genetic parameters, correlations and the potential response from selection. Parameters were estimated from a progeny trial series established at two sites (25 years from planting) and their parents in a seed orchard (43 years from grafting). A standard half-sib analysis based on progenies and a parent–offspring regression was used for estimation of heritabilities. Resistance against the pathogens was measured as lesion length under bark after inoculations in phloem. Heritability values varied with site and estimation procedure from 0.06 to 0.33, whereas the phenotypic variance (as CV P ) is high and fairly stable around 40–50 %. Heritability values for wood density and spiral grain in the same material varied from 0.32 to 0.63. The highest heritability values were generally obtained from parent–offspring regression. There is no evidence of resistance traits being genetically correlated with growth or wood quality traits. Wood density is negatively correlated with stem diameter. Implications for breeding are discussed.

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Abstract

Dothistroma needle blight (DNB) is one of the most important diseases of pine. Although its notoriety stems from Southern Hemisphere epidemics in Pinus radiata plantations, the disease has increased in prevalence and severity in areas of the Northern Hemisphere, including Europe, during the last two decades. This increase has largely been attributed to expanded planting of susceptible hosts, anthropogenic dispersal of the causative pathogens and changes in climate conducive to disease development. The last comprehensive review of DNB was published in 2004, with updates on geographic distribution and host species in 2009. Importantly, the recognition that two species, Dothistroma septosporum and D. pini, cause DNB emerged only relatively recently in 2004. These two species are morphologically very similar, and DNA-based techniques are needed to distinguish between them. Consequently, many records of host species affected or geographic location of DNB prior to 2004 are inconclusive or even misleading. The objectives of this review were (i) to provide a new database in which detailed records of DNB from 62 countries are collated; (ii) to chart the current global distribution of D. septosporum and D. pini; (iii) to list all known host species and to consider their susceptibility globally; (iv) to collate the published results of provenance trials; and (v) to consider the effects of site factors on disease incidence and severity. The review shows that DNB occurs in 76 countries, with D. septosporum confirmed to occur in 44 and D. pini in 13. There are now 109 documented Pinaceae host taxa for Dothistroma species, spanning six genera (Abies, Cedrus, Larix, Picea, Pinus and Pseudotsuga), with Pinus being the dominant host genus, accounting for 95 host taxa. The relative susceptibilities of these hosts to Dothistroma species are reported, providing a resource to inform species choice in forest planting. Country records show that most DNB outbreaks in Europe occur on Pinus nigra and its subspecies. It is anticipated that the collaborative work described in this review will both underpin a broader global research strategy to manage DNB in the future and provide a model for the study of other forest pathogens.

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Abstract

Two species of blue-stain fungi with similar morphologies, Ophiostoma brunneo-ciliatum and Ophiostoma clavatum, are associates of bark beetles infesting Pinus spp. in Europe. This has raised questions whether they represent distinct taxa. Absence of herbarium specimens and contaminated or mistakenly identified cultures of O. brunneo-ciliatum and O. clavatum have accentuated the uncertainty regarding their correct identification. The aim of this study was to reconsider the identity of European isolates reported as O. brunneo-ciliatum and O. clavatum by applying DNA-based identification methods, and to provide appropriate type specimens for them. Phylogenetic analyses of the ITS, βT, TEF-1α and CAL gene sequences revealed that the investigated isolates represent a complex of seven cryptic species. The study confirmed that ITS data is insufficient to delineate species in some Ophiostoma species clusters. Lectotypes and epitypes were designated for O. clavatum and O. brunneo-ciliatum, and three new species, Ophiostoma brunneolum, Ophiostoma macroclavatum and Ophiostoma pseudocatenulatum, are described in the newly defined O. clavatum-complex. The other two species included in the complex are Ophiostoma ainoae and Ophiostoma tapionis. The results suggest co-evolution of these fungi in association with specific bark beetles. The results also confirm the identity of the fungus associated with the pine bark beetle Ips acuminatus as O. clavatum, while O. brunneo-ciliatum appears to be mainly associated with another pine bark beetle, Ips sexdentatus.

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Abstract

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

Globally, billions of tons of carbon sequestered in trees are annually recycled back to the atmosphere through wood decomposition by microbes. In Norway, every fifth Norway spruce shows at final harvest infection by pathogenic white-rot fungi in the genera Heterobasidion and Armillaria. As these fungi can mineralize all components of wood, we predicted that they have a significant carbon footprint. Gas samples taken from infected stems were analyzed for CO2 and CH4 concentrations, and wood samples from different parts of the decay columns were incubated under hypoxic (4% O2) and anoxic laboratory conditions. In spring and summer the stem concentrations of CO2 were generally two times higher in trees with heartwood decay than in healthy trees. For most of the healthy trees and trees with heartwood decay, mean stem concentrations of CH4 were comparable to ambient air, and only some Armillaria infected trees showed moderately elevated CH4. Consistently, low CH4 production potentials were recorded in the laboratory experiment. Up-scaling of CO2 efflux due to wood decay in living trees suggests that the balance between carbon sequestration and emission may be substantially influenced in stands with high frequency of advanced root and stem heartwood decay.

Abstract

Ari M. Hietala, Volkmar Timmermann, Isabella Børja & Halvor Solheim Norwegian Forest and Landscape Institute. PO Box 115, 1431 Ås, Norway: ari.hietala@skogoglandskap.no Owing to the Gulf Stream, the northernmost European populations of several tree species are found in Norway. Common ash (Fraxinus excelsior), the only native ash species in Norway, is present in the lowlands in the southeastern part with continental climate and in southern and southwestern coastal regions with North Atlantic climate up to Central Norway. The current standing volume of ash in Norway is ca 3 mill m3 (broadleaved trees in total 220 mill m3). The first documentation of Ash Dieback (ADB) is from 2008 from a nursery in the southeastern part of the country. A survey later that year showed that dieback symptoms were present over a distance of nearly 400 km in the southeastern region. In addition to nurseries and forests, ADB symptoms were observed on roadside, alley, garden and park trees. Based on the presence of old ADB-like stem lesions detected in 2008, the pathogen must have arrived to Norway no later than 2006. In 2008, the Norwegian Food Safety Authority laid down regulations with the aim of preventing further spread of ADB. These regulations divide the country into quarantine, observation and infection-free zones, and prohibit the export of ash seedlings, seed and wood from the quarantine zone. Despite of these regulations, the disease spread rapidly along the western coast in the period between 2009 and 2013, and currently only the ash stands in Central Norway are free of the disease. The rapid spread of the disease in Norway is obviously due to airborne dispersal of pathogen ascospores. In our experimental stand in SE Norway the number of pathogen fruit bodies can be as high as 10,000 per m2 in the peak season, mid-July to mid-August. During the early morning hours the amount of pathogen ascospores at a diseased stand can exceed 100,000 ascospores per m3 air. The first symptoms of the disease, necrotic lesions on leaf blade and petiole, appear typically during the first two weeks of August in SE Norway. To observe long-term impacts of ADB, eight monitoring plots have been established in continental and North Atlantic climate zones. In SE Norway with the oldest disease history, above 60 % of the trees with a breast height diameter (BHD) below 12.5 cm have so far died or suffer from severe defoliation, 1/3 of the larger trees being affected to a similar degree. The proportions of healthy (no signs of defoliation) small and larger trees are 20% and 37%, respectively. In SW Norway with more recent disease history a similar trend is observed but the proportion of dead trees is still small. As a consequence of ADB, the Norwegian nurseries no longer grow ash seedlings. There are currently no practical control options for the disease in forestland. Several European countries have reported that even at heavily diseased ash stands there are often some ash trees that show little symptoms. This may be due to genetic variation between trees in disease resistance, a hypothesis that is currently being investigated in several European projects. Thus implementation of forest management practices that eliminate ash could have a negative effect as survival of the tree ultimately depends on selection of trees with increased disease resistance. Bibliography for Ari M. Hietala Ari M. Hietala is a Senior Forest Pathologist at the Norwegian Forest and Landscape Institute, which is a primarily government funded organisation providing scientific research and services to government, non-governmental and commercial organisations. He has worked with a range of fungal root and shoot diseases occurring on broadleaved trees and conifers indigenous to the Nordic countries. Ari and the rest of the group participate currently in several European consortia engaged in ash dieback research.

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Abstract

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 5.8S 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.

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A large database of invasive forest pathogens (IFPs) was developed to investigate the patterns and determinants of invasion in Europe. Detailed taxonomic and biological information on the invasive species was combined with country-specific data on land use, climate, and the time since invasion to identify the determinants of invasiveness, and to differentiate the class of environments which share territorial and climate features associated with a susceptibility to invasion. IFPs increased exponentially in the last four decades. Until 1919, IFPs already present moved across Europe. Then, new IFPs were introduced mainly from North America, and recently from Asia. Hybrid pathogens also appeared. Countries with a wider range of environments, higher human impact or international trade hosted more IFPs. Rainfall influenced the diffusion rates. Environmental conditions of the new and original ranges and systematic and ecological attributes affected invasiveness. Further spread of established IFPs is expected in countries that have experienced commercial isolation in the recent past. Densely populated countries with high environmental diversity may be the weakest links in attempts to prevent new arrivals. Tight coordination of actions against new arrivals is needed. Eradication seems impossible, and prevention seems the only reliable measure, although this will be difficult in the face of global mobility.

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Abstract

The relative frequency of Therrya fuckelii and T. pini fruiting on dead branches of Scots pine was investigated in southern Norway by examining lightning-damaged and wind-fallen trees, randomly collected branches and Nordic herbarium collections of these ascomycetes representing the order Rhytismatales. Ascus, ascospore, and subhymenium characteristics were used as criteria for species identification, while a sequence analysis of ITS rDNA gene cluster was performed to compare the relatedness of the species to each other and to corresponding fungal sequences available at the NCBI GenBank Sequence Database. In a few cases, the two Therrya species co-occurred on the same branch, but in general, whether field or herbarium material, T. fuckelii was clearly more common than T. pini.Within the Nordic countries, both species occurred throughout the natural distribution area of Scots pine. The ITS rDNA sequence of T. pini strains was 91% similar to T. fuckelii strains, the differences locating both within the internal transcribed spacers ITS1 and ITS2 and the 5.8 S rDNA gene. More variation in the ITS1-5.8S-ITS2 sequence was observed among T. pini than T. fuckelii samples; genetic implications of this finding are discussed. Upon sequence analysis, we discovered that a T. pini sequence has been deposited in the NCBI GenBank under a false identity. We emphasize the importance of co-examining strains that originate from mature fruit bodies with fully developed morphologic features as reference samples.

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Abstract

The pathogenic white-rot basidiomycete Heterobasidion irregulare is able to remove lignin and hemicellulose prior to cellulose during the colonization of root and stem xylem of conifer and broadleaf trees. We identified and followed the regulation of expression of genes belonging to families encoding ligninolytic enzymes. In comparison with typical white-rot fungi, the H. irregulare genome has exclusively the short-manganese peroxidase type encoding genes (6 short-MnPs) and thereby a slight contraction in the pool of class II heme-containing peroxidases, but an expansion of the MCO laccases with 17 gene models. Furthermore, the genome shows a versatile set of other oxidoreductase genes putatively involved in lignin oxidation and conversion, including 5 glyoxal oxidases, 19 quinone-oxidoreductases and 12 aryl-alcohol oxidases. Their genetic multiplicity and gene-specific regulation patterns on cultures based on defined lignin, cellulose or Norway spruce lignocellulose substrates suggest divergent specificities and physiological roles for these enzymes. While the short-MnP encoding genes showed similar transcript levels upon fungal growth on heartwood and reaction zone (RZ), a xylem defense tissue rich in phenolic compounds unique to trees, a subset of laccases showed higher gene expression in the RZ cultures. In contrast, other oxidoreductases depending on initial MnP activity showed generally lower transcript levels on RZ than on heartwood. These data suggest that the rate of fungal oxidative conversion of xylem lignin differs between spruce RZ and heartwood. It is conceivable that in RZ part of the oxidoreductase activities of laccases are related to the detoxification of phenolic compounds involved in host-defense. Expression of the several short-MnP enzymes indicated an important role for these enzymes in effective delignification of wood by H. irregulare.

Abstract

Common juniper (Juniperus communis) hosts not many pests or pathogens, but recently increasing needle blight has been observed in Norway. During a survey the needle blight was recorded in many parts of southern Norway but not above 550 m a.s.l., and it has been found both in forests, pastures and gardens. Trees are affected differently; some trees seem to be unaffected, while other trees may be killed. The cause of the disease is a fungus in the family Mycosphaerellaceae hitherto not reported from Norway. In forest pathology literature it has been named Stigmina juniperina, but also Asperisporium juniperinum. However, based on results of molecular sequence analyses it is proposed here that a more appropriate name should be Passalora juniperina (Georgescu & Badea) H. Solheim comb. Nov.

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Abstract

Shoot dieback disease of European ash caused by the ascomycete Hymenoscyphus pseudoalbidus threatens ash on a continental scale. A spore sampler placed in a diseased ash forest in Southern Norway, coupled with microscopy and DNA-based fungal species-specific real-time PCR assays, was employed to profile diurnal and within-season variation in infection pressure by ascospores of H. pseudoalbidus and the potentially co-existing non-pathogenic Hymenoscyphusalbidus. Hymenoscyphus pseudoalbidus was found to be predominant in the stand. Massive simultaneous liberation, by active discharge of pathogen ascospores in the morning, peaked in mid-Jul. to mid-Aug. Accumulation of pathogen DNA on leaflets of current-year leaves reached a high level plateau phase before appearance of autumn coloration, suggesting that pathogen establishment in leaves is terminated before the onset of leaf senescence.

Abstract

Winter damage caused by frost is frequently observed on common ash (Fraxinus excelsior) in Norway. In spring 2007, extensive winter damage most likely camouflaged ash dieback caused by Chalara fraxinea. In 2008, ash dieback caused by C. fraxinea had spread to large areas in the southern part of Norway. The disease was widespread in forests and nurseries, but also on roadside trees, and in gardens and parks. In 2009, the disease had spread to new areas; about 30 km into Rogaland county in southwestern Norway and also further into some valleys in southeastern Norway.

Abstract

We compared gene expression in Norway spruce secondary phloem (bark) and developing xylem (sapwood) in response to the necrotrophic pathogen Heterobasidion parviporum, wounding and methyl jasmonate (MeJ). The pathogen induced systemic and local up-regulation of PaPX3, PaPX2 and PaChi4 in both bark and sapwood that returned to constitutive levels as the plants recovered from the infection, whereas the local responses to MeJ were similar in both tissues but was longer lasting for PaPX3 and PaChi4. Genes involved in lignin biosynthesis (PaPAL1, PaPAL2, PaC4H3/5 and PaHCT1) were up-regulated locally in the bark in response to pathogen and wounding whereas MeJ induced a similar but stronger local response. The ethylene biosynthesis related transcripts PaACO and PaACS did not increase in response to MeJ treatment or the pathogen, however it increased both locally and systemically as a response to wounding in the sapwood. These results demonstrate that the local and systemic host responses to pathogen infection and wounding largely correspond and reveal striking similarities between the local response to a necrotroph, wounding and MeJ treatment in both bark and living wood.

Abstract

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.

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Abstract

• Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. • We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. • A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. • Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.

Abstract

Background: NB-LRR resistance proteins are involved in recognizing pathogens and other exogenous stressors in plants. Resistance proteins are the first step in induced defence responses and a better understanding of their regulation is important to understand the mechanisms of plant defence. Much of the post-transcriptional regulation in plants is controlled by microRNAs (miRNA). We examined the expression of five Norway spruce miRNA that may regulate NB-LRR related transcripts in secondary phloem (bark) of resistant Norway spruce after wounding and inoculation with the necrotrophic blue stain fungus Ceratocystis polonica. Results: The plants of this clone recovered from both the pathogen inoculations and wounding alone. We found local and systemic induction of the resistance marker genes PaChi4, PaPAL and PaPX3 indicative of an effective induced host defence response. There were minor local and systemic changes in the expression of five miRNAs and 21 NB-LRRs between healthy and treated plants. Only five putative NB-LRRs (PaLRR1, PaLRR3, PaLRR14, PaLRR15 and PaLRR16) showed significant increases greater than two-fold as a local response to C. polonica. Of all NB-LRRs only PaLRR3, the most highly differentially regulated NB-LRR, showed a significant increase also due to wounding. The five miRNAs showed indications of an initial local and systemic down-regulation at day 1, followed by a later increase up to and beyond the constitutive levels at day 6. However, the initial down-regulation was significant only for miR3693 and miR3705. Conclusions: Overall, local and systemic expression changes were evident only for the established resistance marker genes and PaLRR3. The minor expression changes observed both for the followed miRNAs and their predicted NB-LRR targets suggest that the expression of most NB-LRR genes are maintained close to their constitutive levels in stressed and healthy Norway spruce plants.

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Abstract

The ascomycete fungus Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea) is responsible for ash dieback currently expanding over large parts of Europe. Our objective was to investigate the genetic structure of H. pseudoalbidus and to examine its relationship to the species H. albidus, known as a saprotroph. The study comprised 181 isolates of H. pseudoalbidus collected within the diseased area, 17 H. albidus isolates from six apothecia, collected outside the diseased area in Norway, and nine apothecia of H. pseudoalbidus collected in Sweden. By analysis of microsatellite markers developed for this study, combined with AP-PCR using the M13 primer, we demonstrated sexual heterothally in H. pseudoalbidus, detected high gene flow and low geographic structure of the H. pseudoalbidus population and found indications of a founder effect. Also, substantial genetic differences were detected between the two species of fungi; only four of seven microsatellite markers developed for H. pseudoalbidus were amplified for H. albidus, and no alleles were shared among the species. Furthermore, AP-PCR banding patterns were distinctly different for the two species. We conclude that even though the two fungi have a similar habitat and are morphologically virtually identical, they do not share a recent common ancestor.

Abstract

Aspen trees are exposed to a range of attackers and employ varied strategies to reduce their impact. The diversity of responses may have importance for resistance properties at the stand level, and justifies the search for varied defensive strategies in natural populations. We used transcriptomic tools to evaluate diverse responses at the gene activity level in Populus tremula in response to wounding, and to inoculation with two pathogenic fungi (Melampsora magnusiana vs Ceratocystis sp.) that differ in life style (biotroph vs necrotroph) and host tissue requirement (live leaf vs dead wood tissues). Two aspen genotypes from the SwAsp collection with differences in growth and phenolic composition were used to study differences in resistance properties. High defence gene induction, high growth and elevated defence properties toward the biotroph appeared to support each other in this study exemplified in the more resistant SwAsp clone, whereas the more susceptible SwAsp clone was much less responsive to infections, and displayed more symptoms when infected with M. magnusiana. Interestingly, in the more resistant clone wounding gave greater systemic activity of selected candidate genes than when combined with the necrotroph, suggesting that this pathogen has some ability to suppress the induction or translocation of the systemic defence signal in this particular clone.

Abstract

The GH61 represents the most enigmatic Glycoside Hydrolase Family (GH) regarding putative enzymatic activity and importance in cellulose degradation. Heterobasidion irregulare is a necrotizing pathogen and white rot fungus, causing enormous damages in conifer forests.The genome of H. irregulare allowed identification of ten HiGH61 genes. qRT-PCR analysis separate the HiGH61 members into two groups; one that show up regulation on lignocellulosic substrates and another that show either down regulation or constitutive expression. This grouping suggests that the fungus relates different sets of GH61s for different substrates, like in the various stages of necrotizing and saprophytic growth on the host.One HiGH61 showed up to 17000 fold increase on spruce heartwood suggesting a pivotal role in cellulose decomposition during saprophytic growth. Sequence analysis of these genes reveals that all GH61s but one possess the conserved metal binding motif predicted to be essential for activity.The sequences also divide into groups having either an insert near the N-terminus or an insert near the second catalytic histidine, which both may represent extensions of the substrate binding surface. Three HiGH61s encode cellulose-binding modules (CBM1), indicating direct targeting of crystalline cellulose, two being up regulated on pure cellulose.There was a common substrate-specific induction patterns of the HiGH61s with several reference cellulolytic and hemicellulolytic GHs, this taken together with their low levels on media lacking lignocellulose, reflect the concerted nature of cell wall polymer degradation.

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Abstract

A 1149 bp genomic fragment corresponding to the 5' non-coding region of the PgD1 (Picea glauca Defensin 1) gene was cloned, characterized, and compared with all Arabidopsis thaliana defensin promoters. The cloned fragment was found to contain several motifs specific to defence or hormonal response, including a motif involved in the methyl jasmonate reponse, a fungal elicitor responsive element, and TC-rich repeat cis-acting element involved in defence and stress responsiveness. A functional analysis of the PgD1 promoter was performed using the uidA (GUS) reporter system in stably transformed Arabidopsis and white spruce plants. The PgD1 promoter was responsive to jasmonic acid (JA), to infection by fungus and to wounding. In transgenic spruce embryos, GUS staining was clearly restricted to the shoot apical meristem. In Arabidopsis, faint GUS coloration was observed in leaves and flowers and a strong blue colour was observed in guard cells and trichomes. Transgenic Arabidopsis plants expressing the PgD1::GUS construct were also infiltrated with the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. It caused a suppression of defensin expression probably resulting from the antagonistic relationship between the pathogen-stimulated salicylic acid pathway and the jasmonic acid pathway. It is therefore concluded that the PgD1 promoter fragment cloned appears to contain most if not all the elements for proper PgD1 expression and that these elements are also recognized in Arabidopsis despite the phylogenetic and evolutionary differences that separates them.

Abstract

Pathogen challenge of tree sapwood induces the formation of reaction zones with antimicrobial properties such as elevated pH and cation content. Many fungi lower substrate pH by secreting oxalic acid, its conjugate base oxalate being a reductant as well as a chelating agent for cations. To examine the role of oxalic acid in pathogenicity of white-rot fungi, we conducted spatial quantification of oxalate, transcript levels of related fungal genes, and element concentrations in heartwood of Norway spruce challenged naturally by Heterobasidion parviporum. In the pathogen-compromised reaction zone, upregulation of an oxaloacetase gene generating oxalic acid coincided with oxalate and cation accumulation and presence of calcium oxalate crystals. The colonized inner heartwood showed trace amounts of oxalate. Moreover, fungal exposure to the reaction zone under laboratory conditions induced oxaloacetase and oxalate accumulation, whereas heartwood induced a decarboxylase gene involved in degradation of oxalate. The excess level of cations in defense xylem inactivates pathogen-secreted oxalate through precipitation and, presumably, only after cation neutralization can oxalic acid participate in lignocellulose degradation. This necessitates enhanced production of oxalic acid by H. parviporum. This study is the first to determine the true influence of white-rot fungi on oxalate crystal formation in tree xylem.

Abstract

Heterobasidion parviporum, a common pathogenic white-rot fungus in managed Norway spruce forests in northern and central Europe, causes extensive decay columns within stem heartwood of the host tree. Infected trees combat the lateral spread of decay by bordering the heartwood with a fungistatic reaction zone characterized by elevated pH and phenol content. To examine the mode of fungal feeding in the reaction zone of mature Norway spruce trees naturally infected by H. parviporum, we conducted spatial proWling of pectin and hemicellulose composition, and established transcript levels of candidate fungal genes encoding enzymes involved in degradation of the diVerent cell wall components of wood. Colonized inner heartwood showed pectin and hemicellulose concentrations similar to those of healthy heartwood, whereas the carbohydrate proWles of compromised reaction zone, irrespective of the age of fungal activity in the tissue, indicated selective fungal utilization of galacturonic acid, arabinose, xylose and mannose. These data show that the rate of wood decay in the reaction zone is slow. While the up-regulation of genes encoding pectinases and hemicellulases preceded that of the endoglucanase gene during an early phase of fungal interaction with xylem defense, the manganese peroxidase gene showed similar transcript levels during diVerent phases of wood colonization. It seems plausible that the reaction zone components of Norway spruce interfere with both lignin degradation and the associated co-hydrolysis of hemicelluloses and pectin, resulting in a prolonged phase of selective decay.

Abstract

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

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.

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Abstract

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

During a survey conducted in August 2009 in northern Norway, symptoms typical for red band needle blight (1) were observed in four young Scots pine (Pinus sylvestris) stands. The stands, less than 15 years old, were located in humid sites near rivers in Bardu and Målselv municipalities, Troms County. Many of the oldest needles (2- to 3- years-old) in the lower part of young trees were partially or completely brown, but still attached, and red bands could be observed. Aggregations of conidial stromata were often seen in the red bands. Conidia were hyaline, smooth, thin walled, and filiform, 1.9 to 2.6 μm wide and 12 to 36 μm long....

Abstract

In forest soils, saprotrophic, necrotrophic and ectomycorrhizal fungi are involved in carbon cycling. Heterobasidion annosum, white rot necrotrophic fungi, is known to decompose wood lignocellulose by secreting a broad range of oxidative enzymes. The genome H. annosum s.l. was sequenced by JGI to a 8.23X coverage and assembled into 39 scaffolds with a total size of 33.7 Mb covering more than 98% of the whole genome. Based on the genome sequence we have characterized gene families coding for enzymes known to participate in conversion of wood lignin: multicopper oxidases (MCOs, 18 genes) as laccases (Lcc), class II peroxidases (8 genes) as manganese peroxidases (MnP), glyoxal oxidases (5 genes, GLOX), quinone-reducing oxidoreductases (19 genes, QOR) and GMC oxidoreductases (12 genes) as aryl alcohol oxidases (AAO). We studied the genomic organisation and phylogeny of these genes as well as their expression using qRT-PCR. Comparative and phylogenetic analyses of genes coding for enzymes involved in wood lignin conversion and decomposition (i.e. lignin-modifying class II peroxidases) reveal differences between white- and brown-rot, necrotrophic and saprotrophic wood-decaying basidiomycetes. Transcript profiling using qRT-PCR revealed that some transcripts were very abundant in lignin-rich media, in cellulose-rich media, in wood or in the free-living mycelium grown liquid culture, suggesting specific functions of these genes, which need to be studied further.

Abstract

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.

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Abstract

Background: Tree-killing bark beetles (Coleoptera, Scolytinae) are among the most economically and ecologically important forest pests in the northern hemisphere. Induction of terpenoid-based oleoresin has long been considered important in conifer defense against bark beetles, but it has been difficult to demonstrate a direct correlation between terpene levels and resistance to bark beetle colonization. Methods: To test for inhibitory effects of induced terpenes on colonization by the spruce bark beetle Ips typographus (L.) we inoculated 20 mature Norway spruce Picea abies (L.) Karsten trees with a virulent fungus associated with the beetle, Ceratocystis polonica (Siem.) C. Moreau, and investigated induced terpene levels and beetle colonization in the bark. Results: Fungal inoculation induced very strong and highly variable terpene accumulation 35 days after inoculation. Trees with high induced terpene levels (n = 7) had only 4.9% as many beetle attacks (5.1 vs. 103.5 attacks m22) and 2.6% as much gallery length (0.029 m m22 vs. 1.11 m m22) as trees with low terpene levels (n = 6). There was a highly significant rank correlation between terpene levels at day 35 and beetle colonization in individual trees. The relationship between induced terpene levels and beetle colonization was not linear but thresholded: above a low threshold concentration of ,100 mg terpene g21 dry phloem trees suffered only moderate beetle colonization, and above a high threshold of ,200 mg terpene g21 dry phloem trees were virtually unattacked. Conclusion/Significance: This is the first study demonstrating a dose-dependent relationship between induced terpenes and tree resistance to bark beetle colonization under field conditions, indicating that terpene induction may be instrumental in tree resistance. This knowledge could be useful for developing management strategies that decrease the impact of tree-killing bark beetles.

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Abstract

Introduction: Windstorm is one of the most destructive environmental disturbance factors on forests, but its influence on conifer defense chemistry and susceptibility to insects and diseases is not well understood. Materials and methods: We selected groups of 10 Norway spruce trees with short leaders, leaning stems, or no apparent damage 17 months after the largest storm ever recorded in Sweden. Trees were mass-inoculated with Ceratocystis polonica, a virulent blue stain fungus associated with the spruce bark beetle (Ips typographus) to estimate tree resistance. Terpene and phenolic composition in the bark was analyzed by gas chromatography–mass spectrometry, two-dimensional gas chromatography, and liquid chromatography. Results: In contrast to our hypothesis, the results showed that trees with no apparent damage were more susceptible to C. polonica inoculation than short-leader and leaningstem trees. Chemical composition also differed between trees in different damage classes. (+)-3-carene and two unidentified stilbenes were higher, and taxifolin glycoside was lower in trees without apparent damage than in the others. The relative amount of (−)-α-pinene was negatively correlated, whereas (+)-3-carene, sabinene, (−)-germacrene D, thunbergol and two unidentified stilbenes were positively correlated with fungal performance. Conclusions: These results suggested that wind damage had increased resistance level of short-leader and leaning trees to C. polonica inoculation, and that change in terpene and phenolic composition in the bark could be at least partly responsible for the induced resistance. Different possible explanations for this unexpected finding are discussed.

Abstract

Plants are exposed to a variety of pathogens in their natural habitats. To understand the key processes of defense responses in aspen (Populus tremulae) at the transcript level two clones C72 and C23 with differential level of resistance from the SwAsp collection were inoculated with a foliar rust (Melampsora magnusiana Wagnar). Leaf samples were collected from adjacent areas of the inoculation site to examine the long distance (systemic) defense responses at day1, day3 and day14 post treatments. We performed microarray experiments on the biothrophic interaction, on comparison with the healthy controls we found that the two clones respond in a widely different fashion to the rust. Clone C23 showed almost no response to biotroph after 24 hours while clone 72 gave a clear defense response to the pathogen. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) showed a significant differential expression patterns in susceptible and resistant colnes. Chitinase, cinnamic acid reductase and the iaa genes showed signification up-regulation in resistant clone. The level of expression was 5.9 delta threshold cycles in chitinase gene at day14. Data analysis from extracted total phenolics and condensed tannins verify the results of cDNA arrays and qRT-PCR.

Abstract

Development in surface mould growth on painted/unpainted wooden claddings and acting climatic factors were investigated over a period of 3 years. Eight wood substrates, including modified, preservative-treated and untreated wood, were tested in combination with three types of paint: (1) water-borne alkyd modified acrylic paint without fungicide; (2) solvent-borne alkyd paint without fungicide; and (3) ICP (internal comparison product). One set of samples was exposed unpainted. The samples were tested according to a modified version of EN 927-3. A logistic regression model was fitted to the data. The degree of mould growth varied with exposure time, coating typology, wood substrate, temperature and relative humidity. Exposure time and coating typology contributed most to the model. After 3 years of outdoor exposure unpainted panels and panels coated with solvent-borne paint without fungicide had more mould growth than panels coated with ICP and water-borne paint without fungicide. Unpainted oil/copper–organic preservative-treated claddings had higher resistance to mould growth than other unpainted wood substrates. Coated untreated pine and coated acetylated pine were more susceptible to mould growth than other coated wooden substrates.

Abstract

The genome H. annosum s.l. was sequenced by JGI to a 8.23X coverage and assembled into 39 scaffolds with a total size of 33.7 Mb covering more than 98% of the whole genome. Based of genome sequence we annotated a number of genes for fungal enzymes that are believed to participate in lignin degradation, including: laccases (Lcc18 genes), manganese peroxidases (MnP8 genes) and hydrogen peroxide forming enzymes such as glyoxal oxidases (GLOX5 genes), quinone oxidoreductases (QOR17 genes) and aryl alcohol oxidases (AAO16 genes), which is in concordance with these gene family sizes observed in other sequenced white-rot fungi. We studied the genomic organisation and phylogeny of these genes as well as their expression using NimbleGen arrays and qRT-PCR. Transcript profiling using whole-genome oligo arrays and qRT-PCR revealed that some transcripts were very abundant in lignin-rich media (Lcc5 15, MnP2, GLOX4, QOR2 10, AAO9), in cellulose-rich media (lcc2, 7 16, MnP3 4, GLOX3, QOR4 6, AAO2, 7 10), in wood (Lcc3, MnP4, QOR2, GLOX1, AAO10) or in the free-living mycelium grown liquid culture (Lcc1, 3, 10 13), suggesting specific functions of these genes, which need to be studied further.

Abstract

Microbial disfigurement of coated wooden surfaces is considered a major maintenance concern and will shorten the aesthetic service life of wooden facades. The effect of the physical surface structure of a paint film when applied on wood may have an impact on the susceptibility to mould growth. Six model paints were formulated to give the following physical surface structures: glossy, matt, soft, hard, hydrophobic, and a film with air inclusion. The model paints and a standard paint, with and without fungicide, were applied on panels of Norway spruce (Picea abies L Karst.) and exposed outdoors for nearly three years according to a modified version of EN 927-3. A logistic regression model was fit to the data, and the degree of mould growth varied with exposure time and type of paint. Hard model paint was significantly more susceptible than the other model paints and had a performance close to the standard paint without fungicide. Soft model paint provided the best performance, with the least mould growth. Temperature, relative humidity, and precipitation did not significantly contribute to the model. (C) 2010 Elsevier Ltd. All rights reserved.

Abstract

Ophiostoma spp. include important pathogens of trees and causal agents of sapstain. These fungi infect wounds on trees and are typically carried by insects, especially bark beetles. Ophiostoma spp. on coniferous hosts in the Northern Hemisphere are well-known. However, other than for the serious pathogens O. ulmi and O. novo-ulmi, very little research has been done on the occurrence of this group of fungi on native broad-leaved trees, especially in the Nordic countries. In this study, surveys were conducted in several areas of Norway to isolate Ophiostoma spp. associated with wounds on native broad-leaved trees belonging to the genera Betula, Fagus, Quercus, Sorbus and Tilia. Morphological studies and comparisons of DNA sequences for the ITS, 5.8S and part of the beta-tubulin gene regions were used to confirm the identity of the fungi collected. Ophiostoma spp., and especially their Pesotum anamorphs, were common on wounds on the trees sampled. In most cases, they were associated with wood stain. Ophiostoma spp. collected included predominantly O. quercus, O. borealis sp. nov., and O. denticiliatum. The results of this study emphasize that the diversity of Ophiostoma spp. on broad-leaved trees is still incompletely understood in Norway and other European countries.

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Abstract

The blue-stain fungus Ceratocystis resinifera colonizes wounds on living Picea spp. and other conifers in Europe and North America. Little is known regarding the pathogenicity of this fungus and consequently, four Norwegian C. resinifera isolates were inoculated on to Norway spruce (Picea abies) using two different techniques. These included single-point inoculations on young trees (two inoculations per tree on 14-year-old trees) and mass-inoculations on older trees (∼200 inoculations per tree on 34-year-old trees). In both experiments, C. resinifera induced minor symptoms that in most cases did not differ significantly from inoculation with sterile agar. The virulent blue-stain fungus C. polonica, which was inoculated for comparative purposes, induced extensive symptoms, causing 83% dead cambium circumference and 82% blue-stained sapwood, and long necrotic lesions in the phloem. The results suggest that C. resinifera is non-pathogenic or only mildly pathogenic to Norway spruce and does not present a threat to these trees.

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Abstract

Constitutive and inducible terpene production is involved in conifer resistance against bark beetles and their associated fungi. In this study 72 Norway spruce (Picea abies) were randomly assigned to methyl jasmonate (MJ) application, inoculation with the bluestain fungus Ceratocystis polonica, or no-treatment control. We investigated terpene levels in the stem bark of the trees before treatment, 30 days and one year after treatment using GC–MS and two-dimensional GC (2D-GC) with a chiral column, and monitored landing and attack rates of the spruce bark beetle, Ips typographus, on the trees by sticky traps and visual inspection. Thirty days after fungal inoculation the absolute amount and relative proportion of (+)-3-carene, sabinene, and terpinolene increased and (+)-α-pinene decreased. Spraying the stems with MJ tended to generally increase the concentration of most major terpenes with minor alteration to their relative proportions, but significant increases were only observed for (−)-β-pinene and (−)-limonene. Fungal inoculation significantly increased the enantiomeric ratio of (−)-α-pinene and (−)-limonene 1 month after treatment, whereas MJ only increased that of (−)-limonene. One year after treatment, both MJ and fungal inoculation increased the concentration of most terpenes relative to undisturbed control trees, with significant changes in (−)-β-pinene, (−)-β-phellandrene and some other compounds. Terpene levels did not change in untreated stem sections after treatment, and chemical induction by MJ and C. polonica thus seemed to be restricted to the treated stem section. The enantiomeric ratio of (−)-α-pinene was significantly higher and the relative proportions of (−)-limonene were significantly lower in trees that were attractive to bark beetles compared to unattractive trees. One month after fungal inoculation, the total amount of diterpenes was significantly higher in putative resistant trees with shorter lesion lengths than in putative susceptible trees with longer lesions. Thus, terpene composition in the stem bark may be related to resistance of Norway spruce against I. typographus and C. polonica.

Abstract

Constitutive and inducible terpene production is involved in conifer resistance against bark beetles and their associated fungi. In this study 72 Norway spruce (Picea abies) were randomly assigned to methyl jasmonate (MJ) application, inoculation with the bluestain fungus Ceratocystis polonica, or no-treatment control. We investigated terpene levels in the stem bark of the trees before treatment, 30 days and one year after treatment using GC–MS and two-dimensional GC (2D-GC) with a chiral column, and monitored landing and attack rates of the spruce bark beetle, Ips typographus, on the trees by sticky traps and visual inspection. Thirty days after fungal inoculation the absolute amount and relative proportion of (+)-3-carene, sabinene, and terpinolene increased and (+)-α-pinene decreased. Spraying the stems with MJ tended to generally increase the concentration of most major terpenes with minor alteration to their relative proportions, but significant increases were only observed for (-)-β-pinene and (-)-limonene. Fungal inoculation significantly increased the enantiomeric ratio of (-)-α-pinene and (-)-limonene 1 month after treatment, whereas MJ only increased that of (-)-limonene. One year after treatment, both MJ and fungal inoculation increased the concentration of most terpenes relative to undisturbed control trees, with significant changes in (-)-β-pinene, (-)-β-phellandrene and some other compounds. Terpene levels did not change in untreated stem sections after treatment, and chemical induction by MJ and C. polonica thus seemed to be restricted to the treated stem section. The enantiomeric ratio of (-)-α-pinene was significantly higher and the relative proportions of (-)-limonene were significantly lower in trees that were attractive to bark beetles compared to unattractive trees. One month after fungal inoculation, the total amount of diterpenes was significantly higher in putative resistant trees with shorter lesion lengths than in putative susceptible trees with longer lesions. Thus, terpene composition in the stem bark may be related to resistance of Norway spruce against I. typographus and C. polonica.

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Abstract

The anamorph genus Leptographium Lagerberg and Melin includes species that are typically bark beetleassociated fungi, with teleomorphs in Grosmannia. During a survey of ophiostomatoid fungi in Norway, two unusual species, that fit the broader morphological description of Leptographium, were isolated directly from the rootfeeding beetles, Dryocetes authographus and Hylastes cunicularius, as well as from roots infested by these insects. The first of these could be distinguished from other described species based on a sparse sporulation, black spore drops and chlamydospores in older cultures. This species also produces a Hyalorhinocladiella synanamorph. The second species was characterised by distinctly curved conidia. Based on these unusual morphological characteristics and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium chlamydatum sp. nov. and L. curvisporum sp. nov. are provided.

Abstract

European ash (Fraxinus excelsior), also known as common ash, occurs naturally inland in lower areas of southeastern Norway and along the southern coast of the country. It is important both as a forest and ornamental tree. During the last decade, dieback has become a disastrous disease on F. excelsior in many European countries. The anamorphic fungus Chalara fraxinea T. Kowalski (1), described for the first time from dying ash trees in Poland, is now considered the cause of ash dieback (2). In May of 2008, C. fraxinea was isolated from 1.5 m high diseased F. excelsior in a nursery in Østfold County in southeastern Norway. Symptoms included wilting, necrotic lesions around leaf scars and side branches, and discoloration of the wood. From symptomatic branches, small pieces (approximately 1 cm3) were excised in the transition area between healthy and discolored wood. After surface sterilization (10 s in 70% ethanol + 90 s in NaOCl), the pieces were air dried for 1 min in a safety cabinet, cut into smaller pieces, and placed on media. The fungus was isolated on potato dextrose agar (PDA) and water agar (WA). On PDA, the cultures were tomentose, light orange, and grew slowly (21 mm mean colony diameter after 2 weeks at room temperature). Typical morphological features of C. fraxinea developed in culture. Brownish phialides (14.8 to 30.0 [19.5] × 2.5 to 5.0 [4.1] μm, n = 50) first appeared in the center of the colonies on the agar plugs that had been transferred. The agar plugs were 21 days old when phialides were observed. Abundant sporulation occurred 3 days later. Conidia (phialospores) extruded apically from the phialides and formed droplets. Conidia measured 2.1 to 4.0 (3.0) × 1.4 to 1.9 (1.7) μm (n = 50). The first-formed conidia from each phialide were different in size and shape from the rest by being longer (6 μm, n = 10) and more narrow in the end that first appeared at the opening of the phialide. Internal transcribed spacer sequencing confirmed that the morphological identification was correct (Accession No. EU848544 in GenBank). A pathogenicity test was carried out in June of 2008 by carefully removing one leaf per plant on 10 to 25 cm high F. excelsior trees (18 trees) and placing agar plugs from a 31-day-old C. fraxinea culture (isolate number 10636) on the leaf scars and covering with Parafilm. After 46 days, isolations were carried out as described above from discolored wood that had developed underneath necrotic lesions in the bark and subsequently caused wilting of leaves. All the inoculated plants showed symptoms, and C. fraxinea was successfully reisolated. No symptoms were seen on uninoculated control plants (eight trees) that had received the same treatment except that sterile PDA agar plugs had been used.

Abstract

To identify differentially expressed genes of the white-rot fungus Heterobasidion parviporum subtractive cDNA libraries were constructed using suppressive subtraction hybridization (SSH) technique with RNA extracted from an advanced stage of decay area and from colonization front next to the reaction zone of the stem of a mature Norway spruce trees. Besides cytochrome P450s and proteins with unknown function, the SSH libraries constructed contained genes involved in basic cellular processes, andcell wall degradation. To examine the role of selected candidate genes three trees, showing a variable degree of wood decay, were used for real-time RT-PCR profiling of candidate genes. In the decay transition areas the study revealed activity centers that showed remarkable similarity in the transcript profiles of monitored genes.

Abstract

The genome sequence of the conifer rot root pathogen Heterobasidion annosum was generated at JGI with 8.23 X coverage. The nuclear genome assembles in 39 scaffolds of total 33.7 Mbp estimated to cover 98.1% of the complete genome. We predicted 12,270 genes with an average length of 1,617 bp and exon number and length of 5.54 and 250 bp respectively. About 50% (5999) of the predicted genes could be validated by EST support with the 40,807 EST´s generated with in the project. The genome has a GC content of 52.0% and very little repetitive sequences with 2,895 SSR per mega base. The physical genome is congruent with the genetic linkage map, and most of the linkage groups have been possible to anchor to the 18 largest scaffolds.

Abstract

Clonal variation towards resistance has been observed in Norway spruce Heterobasidion annosum s.l. (H.a). H.a. is the main cause of root rot and has a severe economic impact on an economically important conifer tree species. Annual financial losses are in the hundreds of millions of Euros annually. Less susceptible clones appear to have an efficient system of recognizing the pathogen and initiating early defense signalling events. Active defense responses can be started locally and transmitted systemically. This work focus on the expression both spatially (systemically) and temporally in this pathosystem. Two-year-old, somatic saplings of the Norway spruce clone were challenged with H.a., wounded, methyl jasmonate painted and compared to untreated controls and ninety plants were used for the experiment. Stem samples were collected at 1, 3, 6 and 13 days post inoculation (d.p.i). The stem of the saplings were divided into sections along its length and the bark and wood separated from each other at time of collection. In order to see local response an area of 1cm including the site of inoculation was collected, while the spatial (systemic) response was assessed in sections collected at distances of 3 and 6cm away from the site of inoculation. The separated bark and wood were analysed for differential gene expression by qRT-PCR, and the results from peroxidases (PaPX3 and PaPX2) and a chitinase (PaChi4) are presented. Both local and systemic up- and down-regulation were observed at the transcriptional level in both bark and wood, up to 2000 fold local increase in expression was observed for PaChi4.

Abstract

Due to the great economic losses caused by the root and butt-rot pathogen Heterobasidion annosum, development of efficient control measures is warranted. H. annosum a necrotroph colonize the Norway spruce from inside and is responsible for 100s of millions of Euros losses annually. Considerable clonal variation has been recorded for Norway spruce in resistance towards H. annosum, but the defence mechanisms contributing to host resistance against both necrotrophic and biotrophic fungi remain poorly understood. The recent genome sequencing of Populus has made the genus a model to facilitate tree genetics. Genome-wide transcript profiling of Populus tremula upon pathogen attack will now be used, and homologues of Norway spruce genes to defence genes up-regulated in Populus will be identified. Two aspen clones (23 and 72) from the SwAsp collection (Luquez 2007) were used in the present study. Plants were propagated from tissue culture and kept in greenhouse under un-manipulated conditions. To study the host defence mechanisms, the rust (Melampsora magnusiana Wagner) and a bluestain fungus were used as a biotrophic and necrotrophic fungus respectively. Melampsora spores solution was applied to the underside of the leaf. To control for sectoriality six leaves were infected on each plant. To ensure high humidity and avoid cross contamination, plastic bags were wrapped around infected leaves. Leaves above infected leaves (10, 9, and 8) were harvested respectively 1, 3 and 14 days after inoculation. Initial results from microarray data indicate a clear separation between two Aspen clone (23 and 72) lines. For line 23 the response to biotroph and necrotroph seems to be similar. Whereas the response for line 72 is divergent for the treatments as they go in opposite direction. The controls show that there is an initial difference in the 2 lines (controls are separated). What are the genes that make biotrohic and necrotrophic treatment of 72 look so different will be worked out from microarray data. Differential expression of defense genes in biotrohic and necrotrophic treatment will be verified further with quantitative real time PCR. Chemical analysis of Aspen leaves gave less phenolic compounds as plants were kept in greenhouse. HPLC will be carried out after reaching some conclusion from microarray data analysis.

Abstract

Ophiostomatoid fungi were isolated from Scolytus ratzeburgi infesting Betula pendula and B. pubescens in Norway. Fungi were identified based on morphology, DNA sequence comparison for two gene regions and phylogenetic analyses. The most abundant fungus was Ophiostoma karelicum, suggesting a specific relationship between the fungus, the vector insect and the host tree. Our results suggest that O. karelicum occurs across the geographic range of S. ratzeburgi and its close relatedness to the Dutch elm disease fungi suggests that it could be important if introduced into other parts of the world. Other fungi, only occasionally isolated from S. ratzeburgi, were identified as O. quercus and a novel taxon, described here as O. denticiliatum sp. nov.

Abstract

Two mature clones of Norway spruce (Picea abies (L.) Karst.) shown to have different level of resistance towards inoculation of Heterobasidion parviporum were compared with respect to spatiotemporal expression of transcripts related to biosynthesis of lignin, stilbenes and other phenolic compounds in response to fungal inoculation and physical wounding. Both clones responded to H. parviporum and physical wounding at transcriptional and chemical levels. Taxifolin, detected in the resistant clone only, increased in concentration following both wounding and inoculation. Concentrations of stilbenoid glucosides were highest in the susceptible clone. Following wounding or inoculation, concentrations of these glucosides increased in the susceptible clone, and quantities of their corresponding aglycones increased dramatically in both clones close to the treatment point. Significant changes in transcription were detected over the entire lesion length for all transcripts, and only the changes in a few transcripts indicated a response to inoculation with H. parviporum differing from that caused by wounding alone. The resistant clone had higher basal concentrations of lignin (LTGA) compared to the susceptible clone; concentrations increased in both clones after wounding and wounding plus inoculation treatments, but remained consistently higher in the resistant clone, suggesting higher lignin levels in the cell walls compared to the susceptible clone. In addition, the transcript level in the same clones was also measured the following year and we saw indications of primed defences for a number of gene products likely resulting from the inoculations performed 12 months prior.

Abstract

The root-rot causing fungus Heterobasidion annosum senso lato is the most devastating pathogen of conifers in Europe. This pathogen enter Norway spruce trees trough the roots and colonizes the tree from within, growing as a saprophyte when established within the dead heartwood and acting as a necrotroph when in contact with living host tissue. The genome of this devastating pathogen has now been sequenced in collaboration with JGI and gene annotation is ongoing and genomic work is currently in progress (Stenlid et al. work in progress). We have worked with the host Norway spruce from a molecular perspective for more than ten years. Twenty percent of the trees in Norwegian spruce stands tend to be infected and this pathogen that can colonize ten meters up inside the trunk. The tree have defences against this pathogen and the attack can be fought off by the bark and living wood but not by the hearthwood. The tree has a unique defense against this internal attack by forming a reaction zone; in this case the host defense is directed inwardly by the still living sapwood toward the central colonized wood. We have in the last years studied the host responses to infection in Norway spruce clones at the transcriptional level and found that the speed of recognition and spatial defense signalling appears to be the hallmarks of trees with high degree of resistance. We strive to study both partners in this pathosystem from a molecular perspective, and are using suppressive subtractive hybridization (SSH) followed by Real-Time RT PCR verification to look at differentially expressed genes(Yakovlev et al. 2008). In addition the colonization profiles are followed on extracted gDNA using quantitative Real-Time PCR (Hietala et al. 2009).

Abstract

In natural conditions plants are continuously exposed to number of pathogens both biotrophs and necrotrophs. To understand their defense response at the transcript level two clones C72 and C23 with differential level of resistance from the SwAsp collection were inoculated with a biotroph (Melampsora magnusiana Wagnar) and necrotroph (Ceratocysis spp.) and compared to wounded and healthy controls. Samples were collected in leaves and areas some distance away from the inoculation site to examine the long distance (systemic) defense responses at day, day3 and day14 post treatments. We performed microarray experiments on the necrotrophic and biothrophic interaction compared with the healthy controls and found that the two clones respond in widely different fashions to the treatments applied. Clone C23 showed almost no response to biotroph and necrotroph inoculations after 24 hours while clone 72 gave a clear defense response to both pathogens. We are now in the process of verifying these results and looking at additional time-points using qRT-PCR.

Abstract

The inhibitory effect of methanol bark extracts from six deciduous and three coniferous European tree species were bioassayed against eight fungi from the different damage categories, brown rot, white rot, canker and blue-stain. This is the first report providing data on the antifungal activity of several Europaen tree species against fungi within these damage categories. Generally the decay fungi were more inhibited by the bark extracts than the blue-stain fungi, while the lowest inhibition was found among the cancer fungi. The main pattern found between the fungal groups in relation to the bark extracts in this study is believed to be caused by the route of ingress. Acer platanoides bark extract proved to be the most effcient bark extract tested, significantly reducing the growth rate of all tested fungi. Betula pubescens bark extract generally gave the weakest reduction in growth rate. In this study, the conifer bark extracts were in general more active against the canker and blue stain ascomycete fungi than the deciduous trees extracts.

Abstract

Climate change may influence in a worse manner for the forests in various ways. Some pathogens may increase their importance and new may arrive. Root and butt rot is the most serious problem in Norway spruce forests. In mean more than every fourth tree is infested when harvested. Dryer summers may give increased frequency of rot caused by Heterobasidion. In addition Armillaria spp may gain change in weather condition both as root rot and in connection with a syndrome together with drought and bark beetles. More unstable winter climate may give increase of Gremmeniella attack on Scots pine. Longer and warmer growth season will give many pathogens better condition. Among those is Ophiostoma novo-ulmi causing Dutch elm disease which is lasting in south eastern Norway at a rather low frequency and the volume of elm is not lower than for 15 years ago. In which way the newly introduced Chalara fraxinea will behave in Norway is uncertain, but a better growth season will probably also influence on the possibility to be spread all over Norway where ash are growing.

Abstract

Ips typographus is economically most important insect pest of mature spruce in Eurasia. Normally, it prefers to reproduce in dead and/or dying trees, but following large-scale storm disturbances, its outbreaks kill waste areas of living stands. One factor triggering such epidemics is a surplus of broken and uprooted trees with non-existent, or weak, defence....

Abstract

Induced reactions in the phloem is a basic mechanism of conifer resistance to bark beetle and their associated fungi (1,2). Previous research has proved that certain doses of Ceratocystis polonica infection or methyl jasmonate (MeJA) application could induce acquired resistance and decrease subsequent fungal or bark beetle colonization (3,4,5). To study the induced chemical changes after fungal infection and MeJA application in the phloem of mature Norway spruce, three groups, each of 24 P. abies trees of similar size, were chosen in Tönnersjöheden, southern Sweden, in May 2006. The three groups were then inoculated with C. polonica, sprayed with MeJA, or used as untreated control, respectively. Phloem samples were taken twice from each tree: on the same day as treatment and 1 mo later. The terpene composition of all the samples was analyzed by GC-MS, and the enantiomeric compositions of α-pinene, β-pinene, and limonene were analyzed by 2D-GC (6). The result indicated that both MeJA application and C. polonica infection had certain effects on the terpene composition. C. polonica infection significantly increased the biosynthesis of 3-carene, sabinene, and terpinolene. Both mean absolute amounts and relative amounts of these monoterpenes increased in samples from fungus inoculated trees, similar to what is observed in Scots pine after Leptographium wingfieldii inoculation (7). MeJA application increased the absolute amount of α-pinene, β-pinene, limonene, and some other major terpenes, but it did not change the relative amount of these terpenes. However, neither MeJA application nor fungal infection changed the enantiomeric compositions of α-pinene, β-pinene, and limonene in the phloem of Norway spruce.

Abstract

The root-rot causing fungus Heterobasidion annosum sensu lato is the most devastating pathogen of conifers in Europe. This pathogen enters Norway spruce through the roots and can colonize the tree from within, growing as a saprophyte when established within the dead heartwood and acting as a necrotroph when in contact with living host tissue. Despite the high incidence of damage, trees have defences against this pathogen in the bark and living wood. Furthermore, spruce has a defense against internal attack by forming a reaction zone, in this case the host defense is directed inwardly by the still living sapwood toward the central colonized heartwood. We have studied the host responses to infection in Norway spruce clones at the transcriptional level and found that the speed of recognition and that spatial defense signalling appears to be the hallmarks of trees with high degree of resistance...

Abstract

The difficulty in subculturing biotrophic fungi complicates etiological studies related to the associated plant diseases. By employing internal transcribed spacer rDNA-targeted quantitative real-time polymerase chain reaction, we now show that the heteroecious rust Thekopsora areolata, commonly associated in natural conditions to sapling shoots and cones of Norway spruce and leaves of wild bird cherry, frequently infects nurserygrown seedlings of the conifer. A spatial sampling scheme was used to investigate seedlings and saplings of Norway spruce showing phloem necrosis: the highest concentration of DNA of T. areolata was recorded in the area with necrotic phloem. The separate analysis of bark and wood tissues suggested that the initial spread of the rust to healthy tissues neighboring the infection site takes place in the bark. A Phomopsis species found to coexist with T. areolata in several seedlings showed very high DNA levels in the upper part of the lesion, and even in the visually healthy proximal tissues above the lesions, which indicates that the ascomycete, most probably a secondary invader following primary infection by T. areolata, has a latent stage during early host colonization. We hypothesize that this hemibiotrophic mode of infection contributes to the successful coexistence of Phomopsis with a biotrophic rust.

Abstract

Due to the great economic losses caused by the root and butt-rot pathogen Heterobasidion annosum, developments of efficient control measures are warranted. H. annosum a necrotroph colonize the Norway spruce from inside and is responsible of 10-13 millions Euros losses in Norway alone. Considerable clonal variation has been recorded for Norway spruce in resistance towards H. annosum, but the defence mechanisms contributing to host resistance remain poorly understood. The recent genome sequencing of Populus has made the genus a model to facilitate tree genetics. Genome-wide transcript profiling of Populus tremula upon pathogen attack will now be used, and homologues of Norway spruce genes to defence genes up-regulated in Populus will be identified. Populus-Phellinus tremula pathosystem is selected as P. tremula behaves like H. annosum.

Abstract

The Norwegian Monitoring Programme for Forest Damage (OPS) has since its start registered damage to selected trees. The aim of the registrations has been to explain variations in crown density and crown colour. In answer to international requests, the Norwegian Forest and Landscape Institute has prepared a short guide to the determination of the most common forms of damage found in Norwegian forests...

Abstract

The root-rot causing fungus Heterobasidion annosum senso lato is the most devastating pathogen of conifers in Europe. This pathogen enter Norway spruce trees trough the roots and colonizes the tree from within, growing as a saprophyte when established within the dead heartwood and acting as a necrotroph when in contact with living host tissue. Twenty percent of the trees in Norwegian spruce stands tend to be infected and this pathogen that can colonize ten meters up inside the tree trunk, decaying the silvicultural most valuable part of the tree. Despite this high incidence of damage the tree has efficient defences against this pathogen and the attack is eventually fought off if present in the bark or living wood. The tree also has a defense against this internal attack (by Heterobasidion established in the heartwood expanding and invading outward toward the living sapwood) by forming a reaction zone; in this case the host defense is directed inwardly by the still living sapwood toward the central colonized wood. We have in the last years studied the host responses to infection in Norway spruce clones at the transcriptional level and found that the speed of recognition and that spatial defense signalling appears to be the hallmarks of trees with high degree of resistance. We strive to study both partners in this pathosystem from a molecular perspective, and are now focusing on the pathogen and what fungal gene-products are being expressed during the colonization of the heartwood compared to those expressed close to the active host defense (reaction zone) using suppressive subtractive hybridization (SSH) followed by Real-Time RT PCR analysis. In addition the colonization profiles were followed on extracted gDNA using quantitative Real-Time PCR.

Abstract

The area of wood protection is in a period of change. New tools are needed to understand the mode of action, and to further improve the new wood protection systems. A set of useful tools are found among the molecular methods. This paper presents an overview of some of the tools available, and the methods are exemplified by papers within the frame of wood protection issues. However, there is still a great unexplored potential within the field of wood protection by the use of various molecular methods. The majority of the work using molecular methods has been performed on species identification issues and within species variation. This paper lists some new promising molecular methods for wood protection issues and a presentation of a new project. The new project will help to gain some new knowledge about how the fungal decay processes are affected by different wood modification systems.

Abstract

In winter 2000-2001, there was a serious outbreak of Gremmeniella abietina Morelet in southeastern Norway. During the outbreak, we noted that injured Scots pine trees (Pinus sylvestris L.) developed secondary buds in response to the fungus attack, and we decided to study the relationship between injury, appearance of secondary buds and recovery of the trees thereafter. For this purpose, 143 trees from 10 to 50 years of age were chosen and grouped into crown density classes. Injury was assessed in detail, and buds were counted before bud burst in the spring of 2002. In addition, a subset of 15 trees was followed through the summer of 2002 to assess recovery. All injured trees developed secondary buds, with a clear overweight of dormant winter buds in proportion to interfoliar buds. Healthy control trees did not develop secondary buds at all. The secondary buds appeared predominantly on the injured parts of the tree; interfoliar buds in particular developed just beneath the damaged tissue. Most of the secondary buds died during the winter of 2001-2002, mainly because the fungus continued to spread after the first outbreak. Many of the remaining buds developed shoots with abnormal growth during the summer. Secondary buds may help trees to recover from Gremmeniella attacks, but this strategy may fail when the fungus continues to grow and injure the newly formed buds and shoots.

Abstract

In spring 2002, extensive damage was recorded in southeast Norway on nursery-grown Norway spruce seedlings that had either wintered in nursery cold storage or had been planted out in autumn 2001. The damage was characterised by a top shoot dieback. Two visually distinct types of necroses were located either on the upper or lower part of the 2001-year-shoot. Isolations from the upper stem necroses rendered Gremmeniella abietina, while Phomopsis sp. was isolated mostly from the from the lower stem necroses. RAMS (random amplified microsatellites) profiling indicated that the G. abietina strains associated with diseased nursery seedlings belonged to LTT (large-tree type) ecotype, and inoculation tests confirmed their pathogenicity on Norway spruce seedlings. Phomopsis sp. was not pathogenic in inoculation tests, this implying it may be a secondary colonizer. We describe here the Gremmeniella - associated shoot dieback symptoms on Norway spruce seedlings and conclude that the unusual disease outburst was related to the Gremmeniella epidemic caused by the LTT type on large pines in 2001. The role of Phomopsis sp. in the tissue of diseased Norway spruce seedlings is yet unclear.

Abstract

To identify chemical resistant markers induced by fungal or mechanical injury, young trees of Scots pine (Pinus sylvestris) were subjected to inoculations of blue stain fungi associated with the pine shoot beetles Tomicus piniperda and T. minor. Among the 20 trees selected for chemical analyses, 16 were divided into four groups: one as control and three were pretreated by wounding only, or by inoculation with either the blue stain fungus Leptographium wingfieldii or Ophiostoma canum.Four wk after pretreatment, all 16 pretreated trees were mass-inoculated with L. wingfieldii. The absolute and relative amounts, as well as the enantiomeric compositions of monoterpene hydrocarbons in the phloem, were determined via a small sample of the phloem before and after the pretreatment and mass inoculation, by using two-dimensional gas chromatography (2D GC) and GC-mass spectrometry (MS).After mass inoculation, the absolute amounts of most of the monoterpenes decreased in the phloem sampled 20 cm from the fungal infection, and were higher in the phloem sampled within the infected reaction zone.The relative amounts of both ()--pinene and ()-limonene increased in phloem samples taken 20 cm above the fungal inoculation in the preinoculated trees compared with phloem sampled from the remaining four control trees. The enantiomeric compositions of -pinene and limonene changed, after fungal growth, at defined distances from the inoculation site: the proportion of the ()-enantiomers was highest in the phloem sampled 20 cm from the fungal inoculation.Four wk after pretreatment, monoterpene production in the phloem at the site of inoculation was more enhanced by L. wingfieldii than by O. canum. However, the different virulence levels of the fungi did not affect the enantiomeric composition of the monoterpenes. The biosynthesis of monoterpene enantiomers is discussed in relation to induced pathogen resistance.

Abstract

Norway spruce (Picea abies (L.) Karst.) has a natural distribution in the northern parts of Europe and Asia and is economically the most important tree species grown in the Nordic countries. A common threat to Norway spruce is the basidiomyceteous fungus Heterobasidion parviporum Niemelä and Korhonen. H. parviporum mainly attacks Norway spruce, although Siberian fir (Abies sibirica Ledeb.) and Scots pine (Pinus sylvestris L.) occasionally get infected. One obstacle to studying host/pathogen interaction in conifers has been the limited availability of mature clones for controlled inoculations, as genetic variation within the host material and the lack of replicates complicate interpretation of the results. Somatic embryogenesis, rooted cuttings, and tissue cultures may provide solutions for this problem. Tissue cultures from mature Norway spruce trees have been proposed as a possible model system for assessing resistance toward fungal pathogens. Recent data on chitinase isoform activity in the Norway spruce/H. parviporum pathosystem are encouraging; clonal variation was observed in the isoforms affected by inoculation, and the isoforms showing increased band intensity following bark inoculation by H. parviporum were also induced in the inoculated tissue cultures of the corresponding clones. To investigate the biological relevance of tissue cultures in host-pathogen interaction studies, transcript levels of selected host and pathogen genes in tissue cultures of Norway spruce were compared to those in bark of 33-year-old ramets of the same clones upon challenge by the pathogenic fungus H. parviporum. Similar transcript profiles of the pathogen and host genes were observed in both tissues, this supporting the use of tissue cultures as experimental material for the pathosystem. Higher transcript levels of the host genes phenylalanine ammonia lyase, peroxidase, and glutathione-S-transferase were observed in the more resistant clone #589 than in the less resistant clone #409 during the early stages of colonization. The most striking difference between the spruce clones was related to gene transcript levels of a class IV chitinase, which showed a continuous increase in clone #409 over the experimental period, with a possible association of this gene product to programmed cell death. Several of the fungal genes assayed were differentially expressed during colonization, including putative glutathione-S-transferases, laccase, cellulase, cytochrome P450 and superoxide dismutase genes. The transcriptional responses suggest an important role for the antioxidant systems of both organisms.

Abstract

In field experiments, clones of Norway spruce [Picea abies (L.) Karst.] showed different degrees of resistance against pathogenic fungi inoculated into the bark that correlate with differences in polyphenolic parenchyma (PP) cells of the bark. Cells of spruce callus cultures, particularly towards the callus surface, resemble PP cells and this study looks at changes in callus cells during infection and the relative resistance of cultures from clones of low (weak) or high (strong) resistance to fungal infection. Callus cultures, initiated from trees with different resistance, were co-inoculated with Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. Callus cells from strong clones resemble PP cells of bark tissue from strong clones, having more polyphenolic bodies, while callus cells from weak clones are more similar to PP cells from those clones, which have less extensive phenolic bodies. Callus cultures from trees with weak resistance were more quickly overgrown by both species of pathogenic fungi than cultures from trees with strong resistance. Callus cells of infected cultures showed changes similar to activated PP cells of bark, including enhanced accumulation of polyphenolics. Phenolic bodies were more numerous and more extensive (larger and denser) in callus cells of strong versus weak clones under all conditions. Thus, callus cells may perform similar functions in defense as PP cells in the bark. Callus from trees of varying resistance seem to reflect the relative resistance of the trees from which they are derived, and this study indicates that some mechanisms of resistance can be studied using callus from trees of different resistance.

Abstract

Fungi cause serious problems in wood utilization, and environmentally benign wood protection is required as an alternative to traditional chemicals. Chitosan has shown promising antimicrobial properties against several microorganisms. In this study, we present the characterization of and antifungal properties of a commercial chitosan formulation developed for impregnation of wood.A broad range of chemical and mycological methods were used to evaluate the uptake, fixation, and antifungal properties of chitosan for wood preservation. The results show that the higher the uptake of chitosan the lower the relative recovery of chitosan in wood after leaching, and the higher the molecular weight of chitosan the higher the recovery.Chitosan with high molecular weight proved to be more efficient against decay fungi than chitosan with low molecular weight. The fungi tested on chitosan-amended nutrient agar medium were totally inhibited at 1% (w/v) concentration.In decay studies using small wood blocks, 4.8% (w/v) chitosan concentration gave the best protection against brown rot fungi.

Abstract

This paper describes the use of quantitative real-time PCR for monitoring colonization of birch wood (Betula pubescens) by the white-rot fungus Trametes versicolor in an EN113 decay experiment. The wood samples were harvested after 4, 8, 12, 16 and 20 weeks of incubation.The mass loss was in the range of 440%. Chitin and ergosterol assays were conducted for comparison. Second-order polynomial fits of the mass loss of decayed wood versus chitin, ergosterol and DNA gave correlations (r2) of 0.87, 0.61 and 0.84, respectively. Compared to the other two assays employed, real-time PCR data correlated best with the relative mass loss of decayed samples 48 weeks after inoculation, while the saturation and decline of DNA-based estimates for fungal colonization 1620 weeks after inoculation indicated that the DNA assay is not suited for quantification purposes in the late stages of decay.The impact of conversion factors, extraction efficiency, inhibitory compounds and background levels in relation to the three detection assays used is discussed.

Abstract

In spring 2002, extensive damages were recorded in southeast Norway on nursery-grown Norway spruce seedlings that had either wintered in nursery cold storage or had been planted out in autumn 2001. The damages were characterised by leader shoot dieback and necroses on the upper or lower part of the 2001-year-shoot. Gremmeniella abietina and Phomopsis sp. were frequently isolated from the diseased seedlings. RAMS (random amplified microsatellites) profiling indicated that the G.abietina strains associated with diseased nursery seedlings belonged to LTT (large-tree type) ecotype, and inoculation tests confirmed their pathogenicity on Norway spruce. Based on sequence analysis of the internal transcribed spacer (ITS) regions of ribosomal DNA, the Phomopsis strains associated with diseased seedlings do not represent any characterized Phomopsis species associated with conifers. Phomopsis sp. was not pathogenic in inoculation tests, this implying it may be a secondary colonizer. ITS-based real-time PCR assays were developed in order to detect and quantify Gremmeniella and Phomopsis in the nursery stock. We describe here the Gremmeniella - associated shoot dieback symptoms on Norway spruce seedlings and conclude that the unusual disease outburst was related to the Gremmeniella epidemic caused by the LTT type on large pines in 2001.

Abstract

During a period of 2 years and 3 months (1 January 2001 - 20 March 2003) Mycoteam had 3161 consultations in buildings in southern Norway, 1428 revealing damage from decay fungi. One consultation often revealed several occurrences of fungi, and the total number of occurrences of decay fungi was 3434. Thirty-five different species/genera/groups of decay fungi were recorded. During this period brown rot was more frequent (77.4 %) than soft rot (19.2 %) and white rot (3.4 %). Coniophora puteana (16.3 %) and Serpula lacrymans (16 %) were the most frequently identified species. Different species of the genus Antrodia were recorded in 18.4 % of the occurrences, while the group Corticiaceae accounted for 5.7 % and soft rot for 15.8 %. Investigations of damaged structural parts of buildings showed that decay fungi were most common in walls (18.3 %). Floor damage accounted for 13.4 % of the damaged structures and roofs for 8.8 %. Nearly all species and groups of the investigated fungi were most common indoors. Gloeophyllum sepiarium on the other hand was most common outdoors, and Dacrymyces stillatus was exclusively found outdoors. The Norwegian data were compared with published time series data from Denmark (1946-66, 1966-71, 1974-75, 1982) and Finland (1978-84, 1985-88). S. lacrymans and C. puteana were the most frequent species in these datasets too. Antrodia spp. were also common in the Finnish reports, but barely recorded (as identified species) in Denmark. In both the Danish and the Finnish data, damage to floors is the most frequently recorded structural damage in buildings.

Abstract

The genus Leptographium was described in 1927 and currently includes 48 species, with L. lundbergii as the type species. In recent years, the taxonomic status of L. lundbergii has not been uniformly agreed upon and it has been the topic of considerable debate. The problem was compounded by the absence of a type specimen, and the species was epitypified at a later stage. Unfortunately, the whereabouts of the epitype is now unknown. In 1983, Wingfield & Marasas described L. truncatum, which is morphologically similar to L. lundbergii. Based on DNA comparisons and similarities in their morphology, this fungus was reduced to synonymy with L. lundbergii. The loss of the type specimen as well as variation in the morphology of strains identified as L. lundbergii prompted us to re-examine the taxonomic status of this species. A number of strains from various geographic areas were studied. These include a strain of L. lundbergii deposited at CBS by Melin in 1929 (CBS 352.29) as well as the ex-type strain of L. truncatum. The strains were compared based on morphology and comparison of multiple gene sequences. Three genes or genic regions, ITS2 and part of the 28S gene, partial â-tubulin and partial elongation factor 1-α were compared. Strains currently identified as L. lundbergii, represented a complex of species. Strains initially described as L. truncatum clustered separately from other L. lundbergii strains, could be distinguished morphologically and should be treated as a distinct taxon. L. lundbergii is provided with a new and expanded description based on a neotype designated for it. A third group was also identified as separate from the main L. lundbergii clade and had a distinct Hyalorhinocladiella-type anamorph, described here as H. pinicola sp.nov.

Abstract

The anatomical defense responses in stems of Norway spruce (Picea abies) clones of different resistance to pathogenic fungi were characterized over time and distance from small mechanical wounds or wounds inoculated with the root rot fungus Heterobasidion annosum. Common responses for both treatments included division of ray parenchyma and other cells in the cambial zone, accumulation of phenolic inclusions in ray parenchyma cells, activation of phloem parenchyma (PP) cells, and formation of traumatic resin ducts (TDs) in the xylem. TD formation occurred synchronously from a tangential layer of cells, or symplasmic domain, within the zone of xylem mother cells. TD induction is triggered by a signal, which propagates a developmental wave in the axial direction at about 2.5cm per day. TDs are formed at least 30cm above single inoculations within 16–36days after inoculation. The size and number of TDs is attenuated further away from the inoculation site, indicating a dose-dependent activity leading to TD development. Compared to sterile wounding, fungal inoculation gave rise to more and larger TDs in all clones, and multiple rows of TDs in weak clones. Fungal inoculation also induced the formation of more new PP cells, increasing the number of PP cells in the phloem in the year of inoculation up to 100%. TD and PP cell formation was greater in susceptible compared to resistant clones and after fungal versus sterile inoculation. Potential mechanisms responsible for this variable response are discussed.

Abstract

Genetic variation in three multiallelic loci was analysed with Temperature Gradient Gel Electrophoresis in order to assess the genetic population structure of Venturia tremulae var. tremulae in order to understand the evolutionary potential of the pathogen against resistance breeding. Also the identification of the fungus was verified with molecular analysis of reference isolates. The Fst and Gst values were very low indicating no substructuring or restrictions to gene flow between Fennoscandian populations of V. tremulae. The results imply high epidemiological efficiency of the fungus and therefore continuous breeding programme should be implemented for Venturia resistance of aspen.

Abstract

Chitosan, a derivate of the natural amino polysaccharide chitin, has proven effective as a potential environmentally benign antimicrobial component. Few studies have focused on chitosan applied to wood against wood inhabiting and decaying fungi.In these screening studies several mycological experiments were performed to screen chitosan as a potential wood protecting agent. Growth studies on chitosan-amended media showed total inhibition of Poria placenta, Coriolus versicolor and Aspergillus niger using 1% w/v concentration.Chitosan with high average molecular weight (MW) was more efficient against mould and staining fungi than chitosan with low MW. Agar plate leaching tests showed only a small leaching effect using a 5% concentration on A. niger and P. placenta. Decay testing with P. placenta demonstrated efficacy using 5% and 2.5% concentrations in unleached samples. Leaching decreased the efficacy of chitosan and further investigations are needed to improve the fixation in wood.

Abstract

Introduction: The objectives of the present study were to monitor H. annosum colonization rate (Hietala et al., 2003) and expression of host chitinases in clonal Norway spruce material with differing resistances. Transcript levels of three chitinases, representing classes I, II and IV, were monitored with real-time PCR.Material and MethodsInoculation experiment: Ramets of two 32 -year-old clones differing in resistance were employed as host material. Inoculation and wounding was performed. A rectangular strip containing phloem and cambium, with the inoculation site in the middle, was removed 3, 7 and 14 days after inoculation.Quantification of fungal colonizationMultiplex real-time PCR detection of host and pathogen DNA was performed (Hietala et al., 2003). Quantification of gene expression: Chitinase levels were monitored with Singleplex real-time PCR.Results and ConclusionsThe colonization profiles provided by the quantitative multiplex real-time PCR procedure (Hietala et al., 2003), when combined with spatial and temporal transcript profiling of 3 chitinases, provide a useful basis for identifying defense related genes, and for assessing their impact on pathogen colonization rates.Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. Fourteen days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for the strong clone (589), but had progressed further into the host tissue in the weak (409) clone.Transcript levels of the class II and IV chitinases increased following wounding or inoculation, while the transcript level of the class I chitinase declined following these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in 589 than in similar sites in 409 three days after inoculation, suggesting that the clones differ in the rate of chitinase-related signalperception.

Abstract

We have monitored the H. annosum colonization rate and expression of host chitinases in Norway spruce material with differing resistances. Transcript levels of three chitinases, representing classes I, II and IV, were monitored with real-time PCR. Ramets of two 32 -year-old clones differing in resistance were employed as host material and inoculation and wounding was performed. Quantification of fungal colonization: Multiplex real-time PCR detection of host and pathogen DNA was performed. Chitinase transcript levels were also monitored with real-time PCR. Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. Fourteen days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for the strong clone (589), but had progressed further into the host tissue in the weak clone (409). Transcript levels of the class II and IV chitinases increased following wounding or inoculation, while the transcript level of the class I chitinase declined following these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in 589 than in similar sites in 409 three days after inoculation, suggesting that the clones differ in the rate of chitinase-related signal perception. The spatiotemporal accumulation patterns obtained for the two clones used are consistent with their resistance classifications, these warranting further and more detailed studies on these chitinases.

Abstract

Pathogen colonization and transcript levels of three host chitinases,putatively representing classes I, II, and IV, were monitored with real-time PCR after wounding and bark infection by Heterobasidion annosum in 32-year-old trees of Norway spruce (Picea abies) with low (clone 409) or high (clone 589) resistance to this pathogen. Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. At 14 days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for clone 589 but had progressed further into the host tissue in clone 409. Transcript levels of the class II and IV chitinases increased after wounding or inoculation, but the transcript level of the class I chitinase declined after these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in clone 589 than in similar sites in clone 409 3 days after inoculation. This difference was even more pronounced 2 to 6 mm away from the inoculation point, where no infection was yet established, and suggests that the clones differ in the rate of chitinase-related signal perception or transduction. At 14 days after inoculation, these transcript levels were higher in clone 409 than in clone 589, suggesting that the massive upregulation of class II and IV chitinases after the establishment of infection comes too late to reduce or prevent pathogen colonization.

Abstract

A multiplex real-time PCR assay was developed to monitor the dynamics of the Picea abies-Heterobasidion annosum pathosystem. Tissue cultures and 32-year-old trees with low or high resistance to this pathogen were used as the host material. Probes and primers were based on a laccase gene for the pathogen and a polyubiquitin gene for the host.The real-time PCR procedure was compared to an ergosterol-based quantification method in a tissue culture experiment, and there was a strong correlation product moment correlation coefficient, 0.908) between the data sets. The multiplex real-time PCR procedure had higher resolution and sensitivity during the early stages of colonization and also could be used to monitor the host.In the tissue culture experiment, host DNA was degraded more rapidly in the clone with low resistance than in the clone with high resistance. In the field experiment, the lesions elicited were not strictly proportional to the area colonized by the pathogen.Fungal colonization was more restricted and localized in the lesion in the clone with high resistance, hereas in the clone with low resistance, the fungus could be detected until the visible end of the lesion. Thus, the real-time PCR assay gives better resolution than does the traditionally used lesion length measurement when screening host clones for resistance.

Abstract

A quantitative multiplex real-time PCR procedure was developed to monitor the dynamics in Norway spruce (Heterobasidion annosum) pathosystem. The assay reliably detected down to 1 pg of H. annosum DNA and 1 ng of host DNA in multiplex conditions. As a comparative method for quantifying fungal colonization,we applied the ergosterol assay. There was a very high correlation between the results obtained with the two methods, this strengthening the credibility of both assays. The advantages and disadvantages of these assays are discussed.

Abstract

Determining the level of pathogenic fungi and other microorganisms during colonization of the host is central in phytopathological studies. A direct way to monitor fungal hyphae within the host is microscopic examination, but chitin and ergosterol-levels are commonly used to indirectly measure the amount of fungus present. Recently real-time PCR technology is being used to follow infection agents in host tissues. We study the molecular basis of host defense responses, using the coniferous host Norway spruce infected with the pathogen Heterobasidion parviporum as the experimental system. This basidiomycete and the closely related pathogen H. annosum are the major root rot causing pathogens in conifers. To screen host material for differential resistance towards H. parviporum, it is a necessity to quantify the fungal colonization of the host tissues. Therefore, we aimed to develop and compare the sensitivity of a real-time PCR to an ergosterol based method for determining the rate of colonization, and applied the methods to rank the infection level of the pathogen on the spruce clones 053 and 589. We developed a quantitative multiplex real-time PCR procedure that reliably detecting down to 1pg H. parviporum DNA and 1ng host DNA. There was a very high correlation between the fungal-biomass/total-biomass and fungal-DNA/total-DNA rankings obtained with ergosterol and real-time PCR, strengthening the credibility of both methods. Based on both ergosterol and real-time PCR, it was clear that the clone 053 was hosting more fungal biomass than clone 589. The results indicate that this real-time procedure can be a useful method to screen different spruce material for their relative resistance to the pathogen H. parviporum.

Abstract

In a greenery trial of Noble fir 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

Determining the level of pathogenic fungi and other microorganisms during colonization of the host is central in phytopathological studies. A direct way is to monitor fungal hyphae by microscopic examination, but indirect chitin and ergosterol-based assays have been among the most applied methods in determining fungal biomass within host tissues. Recently real-time technology is increasingly receiving attention as a way to follow infection agents in host tissues.We study the molecular basis of host defense responses, using the coniferous host Norway spruce (Picea abies) infected with the basidomycete Heterobasidion annosum as the experimental system. This basidiomycete is the major root rot causing pathogens in conifers of all age classes.In order to screen host material for differential resistance towards H.annosum for both scientific and commercial reasons, it is a necessity to reliably quantify the fungal colonization of the host tissues. Therefore, the aim of this study was to develop and compare the sensitivity of a real-time PCR assay to an ergosterol based method for determining the rate of colonization by H.annosum in inoculated spruce material. We also applied the methods to rank the infection level of the pathogen on the spruce tissue culture clones.We were able to develop a quantitative multiplex real-time PCR procedure that reliably detecting down to 1pg H.annosum DNA and 1ng host DNA in DNA extracted from infected tissues. There was a very high correlation between the fungal-biomass/total-biomass and fungal DNA-total DNA rankings obtained with ergosterol and real-time PCR respectively, strengthening the credibility of both methods.Based on both ergosterol and real-time PCR, it was clear that some spruce clones were faster and more heavily infected than others. These results indicate that both ergosterol and this real-time procedure can be useful methods to screen different spruce material for their relative resistance to the pathogen H.annosum.

Abstract

One of our main interests is to learn about the molecular basis of host defense responses, using the coniferous host Norway spruce infected with the pathogen Heterobasidion parviporum as the experimental system. This basidiomycete and the closely related pathogen H. annosum are the major root rot causing pathogens in conifers.To screen host material for differential resistance towards H. parviporum, it is a necessity to quantify the fungal colonization of the host tissues. Therefore, we aimed to develop and compare the sensitivity of a real-time PCR to an ergosterol based method for determining the rate of colonization. We developed a quantitative multiplex real-time PCR procedure that reliably detecting down to 1pg H. parviporum DNA and 1ng host DNA.There was a very high correlation between the fungal-biomass/total-biomass and fungal-DNA/total-DNA rankings obtained with ergosterol and real-time PCR, strengthening the credibility of both methods. The results indicate that this real-time procedure can be a useful method to screen different spruce material for their relative resistance to the pathogen H. parviporum.

Abstract

Tree resistance to the patogenic blue stain fungus Ceratocystis polonica was studied in a monoclonal stand of Norway spruce (Picea abies [L] Karst.) in relation to tree social status and diameter at breast height (DBH). The DBH distribution of the 33-year-old stand ranged from 5 to 35 cm. There were clear differences in tree height between the suppressed (DBH 7.4-10.3 cm), co-dominant (DBH 11.8-17.4 cm) and dominant (DBH 18.6-23.9 cm) tree classes. The resistance was tested by mass inoculating trees with a low (400 inoculations m-2, 60 cm inoculation belt) or high (400 inoculations m-2, 120 cm inoculation belt) dosage. The small, suppressed trees were more susceptible to inoculation than the co-dominant and dominant trees, based on amount of blue-stained and occluded sapwood, lesion length, and dead cambium/phloem. A threshold in tree social status or tree size might be important in the overall resistance to fungal infection.

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Abstract

Tree resistance to the patogenic blue stain fungus Ceratocystis polonica was studied in a monoclonal stand of Norway spruce (Picea abies [L] Karst.) in relation to tree social status and diameter at breast height (DBH). The DBH distribution of the 33-year-old stand ranged from 5 to 35 cm. There were clear differences in tree height between the suppressed (DBH 7.4-10.3 cm), co-dominant (DBH 11.8-17.4 cm) and dominant (DBH 18.6-23.9 cm) tree classes. The resistance was tested by mass inoculating trees with a low (400 inoculations m-2, 60 cm inoculation belt) or high (400 inoculations m-2, 120 cm inoculation belt) dosage. The small, suppressed trees were more susceptible to inoculation than the co-dominant and dominant trees, based on amount of blue-stained and occluded sapwood, lesion length, and dead cambium/phloem. A threshold in tree social status or tree size might be important in the overall resistance to fungal infection.

Abstract

In 1996, 7000 ha of pine forests were defoliated by the pine looper Bupalus piniaria in south-western Sweden.The susceptibility of trees of different defoliation classes (0, 30, 60, 90 and 100% defoliation) to beetle-vectored blue-stain fungi was tested in inoculation experiments.Forty and 120-year-old Scots pine trees were inoculated with `single\", i.e. a few inoculations of Leptographium wingfieldii and Ophiostoma minus, two blue-stain fungi associated with the pine shoot beetle Tomicus piniperda. The young trees were also \"mass\" inoculated with L. wingfieldii at a density of 400 inoculation points per m2 over a 60 cm stem belt.Host tree symptoms indicated that only trees with 90100% defoliation were susceptible to the mass inoculation.Single inoculations did not result in any consistent differences in fungal performance between trees of different defoliation classes, regardless of inoculated species or tree age class.Leptographium wingfieldii produced larger reaction zones than O. minus, and both species produced larger lesions in old than in young trees.As beetle-induced tree mortality in the study area occurred only in totally defoliated stands, mass inoculations seem to mimic beetle-attacks fairly well, and thus seem to be a useful tool for assessing host resistance.As even severely defoliated pine trees were quite resistant, host defence reactions in Scots pine seem to be less dependent on carbon allocation than predicted by carbon-based defence hypotheses.

Abstract

Genetic associations between initiation of embryogenic tissue (ET) and susceptibility to the phytopathogenic fungi Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. in Norway spruce have been studied by initiating ET from zygotic embryos of mature seeds collected from 19 clones tested for susceptibility to the pathogens in a clonal field trial.Initiation frequencies varied significantly among clones (families), ranging from 12 to 56%. The family variance component accounted for more than 40% of the total variance in initiation frequency of ET. The estimates of broad-sense heritability of fungus susceptibility of the clones ranged from 0.12 for length of phloem necrosis after low-density inoculation with H. annosum to 0.55 for blue-stained sapwood after mass inoculation with C. polonica.None of the susceptibility measures showed any phenotypic correlation with initiation of embryogenic tissue. Genetic correlations and their standard errors were estimated by bootstrapping. Two measures of fungal susceptibility correlated genetically with initiation of ET; estimated at 0.58 for lesion length after inoculation with C. polonica and 0.29 for H. annosum lesion length. A better measure of susceptibility, blue-stained sapwood following inoculation with C. polonica, was not correlated with initiation of ET.

Abstract

We have studied how callus cultures from two clones of Norway spruce influence the growth of two pathogens, Ceratocystis polonica and Heterobasidion annosum, when co-cultivated in vitro. In field experiments, trees of clone 409 were susceptible to both fungi, whereas clone 589 was less affected. Callus was cultured on medium containing cytokinins (benzylaminopurine, kinetin) and with or without auxin (2,4-dichlorphenoxy acetic acid). For co-cultivation with fungus, one piece of callus was placed towards the edge of each Petri dish. One and 14 days after inoculation with callus the dishes were co-inoculated with the fungus. Both clones strongly stimulated the initial growth of both fungi. Clone 589 inhibited the growth of both fungi when the fungi were inoculated one day after the callus. When the callus was cultured on medium without auxin for 14 days before co-inoculation clone 589 strongly inhibited the growth of both fungi, whereas clone 409 inhibited H. annosum only.

Abstract

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

Norway spruce trees, exposed to sub-lethal attacks by the bark beetle Ips typographus, form distinct necrotic and resin-soaked reaction zones in the bark and outer sapwood within which the beetles and their associated fungi are enveloped and arrested. In these trees a ring of axial resin ducts regularly form in the stem xylem. These ducts are included in a false annual ring with narrow tracheids. A similar ring of resin ducts is seen in trees artificially administered a sub-lethal inoculation dose of the blue-stain fungus Ceratocystis polonica, a pathogenic associate of I. typographus. Here we report preliminary studies on the development of traumatic axial ducts in stems of Norway spruce after beetle and fungus invasion, and some cytochemical aspects related to the formation of phenolics and terpenes. We suggest that traumatic resin ducts furnish the reaction zones with materials toxic to both beetles and fungi, and that increased numbers of ducts may render trees more resistant to attack.