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

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Vi har tatt i bruk nye metoder innen plantemetabolomikk for å påvise og identifisere forsvarsstoff i gran og presenterer her noen resultater fra dette arbeidet. Hvert fjerde tre i skogen er angrepet av råtesopp. Rotråtesopp angriper treet gjennom rota og spiser opp kjerneveden på sin vei oppover i stammen. Den delen av trestokken som er angrepet av rotråte kan ikke lenger brukes til byggematerialer og skognæringen lider store tap. Kan de soppangrepne trestammene brukes til andre formål? Vi vet grana produserer en mengde forsvarsstoffer som en reaksjon på soppangrepet. Kan forsvarsstoffene utnyttes til å lage nye plantevernmidler, trebeskyttelsesmidler eller legemidler?

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European ash (Fraxinus excelsior), a keystone species with wide distribution and habitat range in Europe, is threatened at a continental scale by an invasive alien ascomycete, Hymenoscyphus fraxineus. In its native range of Asia, this fungus is a leaf endophyte with weak parasitic capacity and robust saprobic competence in local ash species that are closely related to European ash. In European ash, H. fraxineus has a similar functional role as in Asia, but the fungus also aggressively kills shoots, resulting in crown dieback and tree death. H. fraxineus is a typical invasive species, as its spread relies on high propagule pressure. While crown dieback of European ash is the most obvious symptom of ash dieback, the annual colonization of ash leaves is a crucial key dependency for the invasiveness of H. fraxineus, since its fruiting bodies are formed on overwintered leaf vein tissues in soil debris. Leaves of European ash host a wide range of indigenous epiphytes, endophytes, facultative parasites and biotrophic fungi, including Hymenoscyphus albidus, a relative of H. fraxineus that competes for the same sporulation niche as the invader. At face value, leaves of European ash are colonized by a large and diverse indigenous mycobiome. In order to understand why this invader became successful in Europe, we discuss and summarize the current knowledge of diversity, seasonal dynamics and traits of H. fraxineus and indigenous fungi associated with leaves of European ash.

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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.

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Dieback of European ash, caused by the ascomycete Hymenoscyphus fraxineus originating from Asia, has rapidly spread across Europe, and is threatening this keystone tree at a continental scale. High propagule pressure is characteristic to invasive species. Consistently, the enormous production of windborne ascospores by H. fraxineus in an ash forest with epidemic level of disease obviously facilitates its invasiveness and long distance spread. To understand the rate of build-up of propagule pressure by this pathogen following its local introduction, during 2011–2017 we monitored its sporulation at a newly infested ash stand in south-western Norway characterized with mild winters and cool summers. We also monitored the propagule pressure by Hymenoscyphus albidus, a non-pathogenic native species that competes for the same sporulation niche with H. fraxineus. During the monitoring period, crown condition of ash trees had impaired, and 20% of the dominant trees were severely damaged in 2017. H. fraxineus showed an exponential increase in spore production between 2012 and 2015, followed by drastic decline in 2016 and 2017. During 2011–2013, the two Hymenoscyphus species showed similar sporulation level, but thereafter spores of H. albidus were no longer detected. The data suggest that following local introduction, the population of H. fraxineus reaches rapidly an exponential growth stage if the local weather conditions are favorable for ascomata maturation across years. In the North Atlantic climate, summer temperatures critically influence the pathogen infection pressure, warm summers allowing the population to grow according to its biotic potential, whereas cold summers can cause a drastic decline in propagule pressure.

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Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H.fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H.fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H.fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.

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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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Askeskuddsjuke forårsakes av en liten sopp, Hymenoscyphus fraxineus, på norsk kalt askeskuddbeger. Sjukdommen har i løpet av kort tid spredd seg over store deler av Europas askeskoger. Soppen er en fremmed art som trolig stammer fra Asia hvor den er assosiert med asketrær som er nær beslektet med europeisk ask. Det er særlig unge trær som er utsatt, men også eldre trær kan drepes over tid. Mye er uklart angående hvordan soppen infiserer, men sporene spres med vind til bladene på asketrær. Dersom soppen klarer å vokse forbi bladfestet før bladfall vil den i løpet av vinteren angripe greinene. Typiske symptomer er sår i barken og døde skudd.

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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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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.

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Fokus i denne rapporten er på skogbehandlingen, og på maksimering av verdiproduksjon. En forutsetning for dette er høy sagtømmerproduksjon, samtidig som det er viktig å vurdere arealets totale tømmerverdi. Følgende forutsetninger ligger til grunn for rapporten: Bevaring av biologisk mangfold og ivaretakelse av andre flerbrukshensyn Det legges til grunn at all skogbehandling utføres i henhold til dagens lovverk og frivillige sertifiseringsordninger, og at dette ivaretar hensyn til biologisk mangfold og andre flerbrukshensyn. Vi går derfor ikke inn på betydningen ulik skogbehandling vil ha for biologisk mangfold eller andre flerbrukshensyn, eller tilpasninger av skogbehandlingen for dette. Bærekraftig skogbruk i klimasammenheng Miljødirektoratet mfl. (2016) drøfter vern eller bruk av skog som klimatiltak. I rapporten konkluderes det med at det ikke er grunnlag for å vektlegge vern av norsk skog som klimatiltak. En forutsetning er at det drives et bærekraftig skogbruk i klimasammenheng. Dette ble definert som følger: «Bærekraftig skogbruk i klimasammenheng innebærer at skogens produktivitet og evne til å lagre karbon ikke forringes, og at karbonbeholdninger ikke reduseres permanent.» (Miljødirektoratet mfl. 2016). Vi forutsetter gjennom rapporten at skogbehandling drives bærekraftig i tråd med denne definisjonen, uten at vi går nærmere inn på betydningen og eventuelle nødvendige tilpasninger. Skogbehandling for å motvirke klimaendringer Skogbehandling som motvirker klimaendringer, for eksempel ved å øke karbonopptaket, vil i mange tilfeller være i samsvar med skogbehandling for maksimal verdiproduksjon, men ikke alltid. Vi har i denne rapporten kun fokusert på verdiproduksjon, og betydning av skogbehandling på ulike karbonbeholdninger er ikke vurdert. Driftstekniske forhold («hvordan ta ut tømmeret») Driftskostnader vil være av stor betydning for skogeiers økonomiske resultat, og både de endringer vi allerede ser og forventede klimaendringer er forventet å gi større driftstekniske utfordringer. I denne rapporten ser vi imidlertid utelukkende på den betydning skogbehandlingen vil ha for antatt verdiproduksjon.

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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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Askeskuddsjuke har spredd seg med rekordfart i Europa og i Norge. Ask (Fraxinus excelsior) er meget sensitiv for denne nye sjukdommen. De aller fleste områder i Europa med dette treslaget er nå infisert med sekksporesoppen askeskuddbeger (Hymenoscyphus fraxineus) som forårsaker sjukdommen. I Norge ble askeskuddsjuke første gang registrert i 2008 og allerede da ble den funnet over store deler av Østlandet og Sørlandet. Deretter har askeskuddsjuke spredd seg nordover på Vestlandet i gjennomsnitt 51 km per år. I 2016 ble det nordligste funnet registrert i Aure kommune nær grensa til Trøndelag.

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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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During post glacial colonization, loss of genetic diversity due to leading edge effects may be attenuated in forest trees because of their prolonged juvenile phase, allowing many migrants to reach the colonizing front before populations become reproductive. The northern range margins of temperate tree taxa in Europe are particularly suitable to study the genetic processes that follow colonization because they have been little affected by northern refugia. Here we examined how post glacial range dynamics have shaped the genetic structure of common ash (Fraxinus excelsior L.) in its northern range compared to its central range in Europe. We used four chloroplast and six nuclear microsatellites to screen 42 populations (1099 trees), half of which corresponded to newly sampled populations in the northern range and half of which represented reference populations from the central range obtained from previously studies. We found that northern range populations of common ash have the same chloroplast haplotypes as south-eastern European populations, suggesting that colonization of the northern range took place along a single migration route, a result confirmed by the structure at the nuclear microsatellites. Along this route, diversity strongly decreased only in the northern range, concomitantly with increasing population differentiation and complex population substructures, a pattern consistent with a leading edge colonization model. Our study highlights that while diversity is maintained in the central range of common ash due to broad colonizing fronts and high levels of gene flow, it profoundly decreases in the northern range, where colonization was unidirectional and probably involved repeated founder events and population fluctuations. Currently, common ash is threatened by ash dieback, and our results on northern populations will be valuable for developing gene conservation strategies.

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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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Råte påfører norske skogeiere og skogbruket årlige tap estimert til rundt 100 mill. NOK (Solheim og Stamnes, upubl.). Basert på den siste nasjonale råteundersøkelsen i granskog (Huse m.fl. 1994), var det mest råte i granas naturlige utbredelsesområde (Aust-Agder, Østlandet og Midt-Norge opp til Saltfjellet), hvor i gjennomsnitt nesten hvert fjerde tre var råteinfisert ved slutthogst. I enkelte granbestand kan råtefrekvensen ved slutthogst komme opp mot 60- 70 %. I skogreisingsstrøk (Vest-Agder, Vestlandet og Nord-Norge nord for Saltfjellet) var bare opptil hvert tiende hogstmodent grantre råteinfisert. I 2014 ble det utført en liten spørreundersøkelse for å kartlegge hvor bevisste de forskjellige aktørene i skogbruket er på råteproblemet, om de mener problemet har endret seg de senere årene og i hvilken grad det brukes forebyggende kontrolltiltak mot råte.

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In Europe, common ash (Fraxinus excelsior) is being decimated because of the invasive fungus Hymenoscyphus fraxineus. In its native range in Asia this ascomycete is considered a harmless leaf associate of F. mandshurica and F. chinensis subsp. rhynchophylla. Field observations from Europe suggest that there is speciesspecific variation in disease susceptibility among European and North American Fraxinus species, but a wider comparison at the genus level has been missing so far. We assessed disease symptoms and pathogen apothecium development in 17 Fraxinus species from Asia, Europe and North America exposed to high infection pressure in a Danish arboretum. We also tested their susceptibility to pathogen infection through controlled stem and leaf inoculations and subsequently assessed the level of pathogen DNA by a qPCR assay. The results suggested the presence of a phylogenetic signal in disease susceptibility where closely related Asian, European and North American species in section Fraxinus had relatively high levels of H. fraxineus DNA in the leaves and supported high production of apothecia. Leaves from some North American species also contained relatively high levels of H. fraxineus DNA, supported moderate production of apothecia and developed lesions—stating the need to avoid introduction of H. fraxineus to North America.

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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.

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Tree harvest and different harvesting methods may affect the soil carbon (C) pool in forest ecosystems. In con- ventional stem-only timber harvesting (SOH), branches and tops that are left in the forests may contribute to the build-up of the soil carbon pool. In whole-tree harvesting (WTH), inputs of organic matter from branches and tops are strongly reduced. We established field experiments at Gaupen, SE and Vindberg, SW Norway, to study the short-term effects of SOH and WTH on processes affecting the accumulation and loss of soil C. Logging residues on the WTH plots were collected in piles that were removed after 6 months, rendering two sub treatments (WTH- pile and WTH-removal areas). We weighed selected trees and logging residues, surveyed understorey biomass production, quantified pre-harvest soil C and nutrient pools down to 30 cm. Soil respiration was measured and soil water sampled monthly during the growing season, while temperature and moisture were measured continuously. Organic and mineral horizons were incubated at different temperatures to estimate potential C and N mineraliza- tion, and deep sequencing of the ITS2 barcode region of fungal DNA was performed on the samples. Litterbags were deployed in the SOH plots. The logging residues amounted to 2.2-2.4 kg C m-2 At Gaupen, the mean in situ soil respiration rates increased following harvest with all treatments, but were significantly higher in WTH-pile and SOH relative to the WTH- removal areas in the first year as well as the fourth year of treatment. The former rates included aboveground decomposing needles and twigs but excluded coarser branches. The observed increase in the WTH-removal areas may be related to decomposing roots, as well as to increased C mineralization partly due to the higher soil tem- peratures following harvest. Soil temperature was the single most important factor explaining the variability in soil respiration rates over all treatments. At Vindberg, a decrease in soil respiration was observed with all treatments in the second and third years following harvest. At both sites, decomposition of logging residues from needles was more rapid relative to twigs and fine roots. The decomposing residues released a substantial amount of nitrogen which was gradually reflected in the soil water at 30 cm soil depth. A considerable increase in the NO3-N concen- tration also in the WTH-removal areas in the second year following harvest suggests an increase in N availability from decomposing fine roots and/or soil organic matter. The increased N availability in the WTH-removal areas was supported by results from short term lab incubations of undisturbed soil from the forest floor. The changes in the WTH-removal areas were also reflected in the soil fungal diversity: saprophytic ascomycetes on decaying plant material showed a striking increase in all treatments. For the WTH-removal areas, this may, again, be related to the increased input of root litter; however, the decrease in mycorrhizal basidiomycete species and the vigorous increase of ascomycetes following harvest may also affect the C mineralization of soil organic matter.

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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.

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Polymerase chain reaction (PCR), a biochemical technology that generates copies of a particular DNA sequence, has revolutionized life sciences. Thirty years after its development in 1983, PCR is a standard and indispensable technique in medical and biological research for a variety of applications. Compared to many other fields, novel PCR applications and sequencing platforms have been rather slowly adopted by research groups engaged in wood protection. Regarding laboratory and field experiments for testing the efficacy of wood treatments, this opinion article discusses the potential of 2nd generation PCR applications and sequencing platforms for profiling 1) the growth and activity of wood decay microbes upon feeding and detoxification of treated wood, and 2) for recording the successional changes that take place in microbial community along with progress of wood decay.

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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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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.

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Local climate conditions have a major influence on the biological decomposition of wood. To examine the influence of different temperature regimes on wood decay caused by the brown rot fungus Postia placenta in wood with differing natural durability, sapwood (sW) and heartwood (hW) of Scots pine, inoculated mini-blocks were incubated for up to 10 weeks at temperatures conducive or above optimal to wood decay. We profiled mass loss (ML) and wood composition, and accompanying changes in wood colonization and transcript level regulation of fungal candidate genes. The suppressive effect of suboptimal temperature on wood decay caused by P. placenta appeared more pronounced in Scots pine hW with increased durability than in sW with low decay resistance. The differences between sW and hW were particularly pronounced for cultures incubated at 30°C: unlike sW, hW showed no ML, poor substrate colonization and marker gene transcript level profiles indicating a starvation situation. As brown rot fungi show considerable species-specific variation in temperature optima and ability to mineralize components that contribute to wood durability, interactions between these factors will continue to shape the fungal communities associated to wood in service.

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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.

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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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Arter av sopp og insekter som introduseres til nye områder kan forårsake omfattende tredød og store endringer i skogøkosystemer. Introduksjoner i andre deler av verden viser tydelig hvilket skadepotensial slike arter har. Skog og landskap søker å være i forkant med forskning som kan møte utfordringen fra nye arter i norske skoger.

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• 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.

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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.

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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.

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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.

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Twenty cypress accessions were tested for freezing tolerance. After freezing to −15°C, differences among cypress accessions were tested by measuring electrolyte leakage and chlorophyll fluorescence. Based on these data, cypress accessions showing contrasting freezing tolerance were subjected to transcript profiling of candidate genes upon the development of cold hardening, with the ultimate goal of providing a scientific basis for selecting/breeding cypress genotypes with higher tolerance to low temperature. Nine different cypress genes were selected: a heat shock protein, a putative chaperonin, a chlorophyll-binding protein, a serine/threonine protein kinase, a putative exonuclease, a dehydrin, and three senescenceassociated proteins. Transcript levels of these genes were profiled during cold hardening under controlled conditions using real-time reverse-transcription-polymerase chain reaction. While the genes showed regulation patterns common to both cypress accessions, in the case of chaperonin, exonuclease, and some senescence-associated proteins, clonal differences in gene regulation were found. The potential relationship of these differences with cold tolerance is discussed.

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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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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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Askeskuddsjuke er på frammarsj i Norge og i Europa. I 2010 var det imidlertid ikke så dramatisk som året før, men på våre overvåkingsflater har vi registrert en liten nedgang i kronetetthet. Så det er all grunn til fortsatt bekymring. Det er stor forskningsaktivitet i mange land, og nå vet vi også hvilken sopp som er årsaken. I tillegg begynner det å komme rapporter om andre askearter som er mottakelige for sjukdommen.

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Enkelte mener å ha observert at det var mindre skader på asketrær i 2010 enn året før og tror at bekymringen for askens framtid er betydelig overdreven. På våre overvåkingsflater har vi i 2010 registrert en liten nedgang i kronetetthet, og vi ser også at askeskuddsjuken er på stadig frammarsj både i Norge og i Europa ellers. Så det er all grunn til fortsatt bekymring.

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Traditional wood preservatives based on biocides are effective against wood-deteriorating organisms because of their toxicity. By contrast, modified woods are non-toxic by definition. To investigate the efficiency of various wood modifications, quantitative real-time polymerase chain reaction (qPCR) was used to profile the DNA amounts of the white-rot fungus Trametes versicolor (L.) [Lloyd strain CTB 863 A] during an 8-week-long growth period in treated Pinus sylvestris (L.) sapwood. The studied wood was modified by acetylation, furfurylation, and thermal treatment. The traditional wood preservatives bis-(N-cyclohexyldiazeniumdioxy)-copper (Cu-HDO) and chromated copper arsenate (CCA) were used as references, whereas untreated P. sylvestris (L.) sapwood served as a control. The maximum levels of fungal DNA in native wood occurred at the end of the experiment. For all wood treatments, the maximum fungal DNA level was recorded after an incubation period of 2 weeks, followed by a decline until the end of the trial. For the preservative-treated woods, Cu-HDO showed the lowest level of fungal DNA throughout the experiment, indicating that exploratory hyphal growth is limited owing to the phytotoxicity of the treatment. The other treatments did not inhibit the exploratory hyphal growth phase. We conclude that qPCR studies of hyphal growth patterns within wood should provide a powerful tool for evaluating and further optimizing new wood protection systems.

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Rotkjuke, Heterobasidion annosum s.l. er et stort problem i de nordlige barskogsområder. I Norge er hele 20 prosent av grantrærne angrepet av denne skadesoppen. Dette fører ikke bare til at tømmeret blir ubrukelig til de fleste formål, men også til frigjøring av mye CO2 som ellers ville vært bundet i trærne. Rotkjuka angriper via rotkontakter. Dette skjer ofte via stubber av felte nabotrær etter hogst eller tynning. Soppen ødelegger stammen opp til tolv meter fra bakkenivå.

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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.

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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.

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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.

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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.

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Våren 2002 oppdaget flere skogeiere at en del av granplantene som ble plantet ut høsten før hadde toppavdøing. Samtidig registrerte planteskolebestyrere at noen av granplantene som skulle plantes ut hadde visnesymptomer. Ved Skogforsk ble disse plantene undersøkt ved hjelp av tradisjonelle og moderne molekylære teknikker.

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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.

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*Strawberry Fragaria × ananassa (cv. Korona) was inoculated with Botrytis cinerea by dipping berries in a conidial suspension. *Colonization by the pathogen was monitored using real-time PCR, ELISA and ergosterol assays, the first showing the highest sensitivity. The expression of pathogen B-tubulin and six polygalacturonases (Bcpg1-6) and three host defence genes (polygalacturonase-inhibiting protein (FaPGIP) and two class II chitinases) were monitored using real-time RT-PCR. *The maximum transcript levels of the host defence genes occurred at 16 h postinoculation (hpi) at the presumed initial penetration stage. The unique transcript profile of Bcpg2 over the 96-h incubation time and its high transcript levels relative to those of the other Bcpgs at 8-24 hpi suggest that the gene has a specific role in the penetration stage. *Bcpg1 was expressed constitutively at a relatively high level in actively growing mycelia throughout the experimental period. Comparison of the transcript profiles indicated that Bcpg1 and Bcpg3-6 were coordinately regulated.

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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.

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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.

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Rhizoctonia solani was frequently isolated in the Italian Alps from ursery-grown European beech (Fagus sylvatica) seedlings displaying symptoms of cotyledon rot. Koch?s postulates were verified and mode of infection of the associated isolates was investigated with light and scanning electron microscopy. Population structure of the pathogen was investigated by scoring the anastomosis reaction type in pairings between different isolates from the same seedbed. One pathogen genotype showed a large distribution area within the seedbed, this implying that the inoculum had been building up in the seedbed over a longer time period. Hyphal anastomosis tests and sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA indicated that the pathogen belongs to AG 2-1 of R. solani. ITS sequence analysis indicates that the isolates from beech are closely related to R. solani isolates causing a disease on tulip. The striking similarities between the two diseases are discussed.

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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.

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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.

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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.

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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.

Sammendrag

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.

Sammendrag

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.

Sammendrag

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.

Sammendrag

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.