Fungal succession in decomposing ash leaves colonized by the ash dieback pathogen Hymenoscyphus fraxineus or its harmless relative Hymenoscyphus albidus.

Introduction: The ascomycete Hymenoscyphus fraxineus, originating from Asia, is currently threatening common ash (Fraxinus excelsior) in Europe, massive ascospore production from the saprotrophic phase being a key determinant of its invasiveness. Methods: To consider whether fungal diversity and succession in decomposing leaf litter are affected by this invader, we used ITS-1 metabarcoding to profile changes in fungal community composition during overwintering. The subjected ash leaf petioles, collected from a diseased forest and a healthy ash stand hosting the harmless ash endophyte Hymenoscyphus albidus, were incubated in the forest floor of the diseased stand between October 2017 and June 2018 and harvested at 2-3-month intervals. Results: Total fungal DNA level showed a 3-fold increase during overwintering as estimated by FungiQuant qPCR. Petioles from the healthy site showed pronounced changes during overwintering; ascomycetes of the class Dothideomycetes were predominant after leaf shed, but the basidiomycete genus Mycena (class Agaricomycetes) became predominant by April, whereas H. albidus showed low prevalence. Petioles from the diseased site showed little change during overwintering; H. fraxineus was predominant, while Mycena spp. showed increased read proportion by June. Discussion: The low species richness and evenness in petioles from the diseased site in comparison to petioles from the healthy site were obviously related to tremendous infection pressure of H. fraxineus in diseased forests. Changes in leaf litter quality, owing to accumulation of host defense phenolics in the pathogen challenged leaves, and strong saprophytic competence of H. fraxineus are other factors that probably influence fungal succession. For additional comparison, we examined fungal community structure in petioles collected in the healthy stand in August 2013 and showing H. albidus ascomata. This species was similarly predominant in these petioles as H. fraxineus was in petioles from the diseased site, suggesting that both fungi have similar suppressive effects on fungal richness in petiole/rachis segments they have secured for completion of their life cycle. However, the ability of H. fraxineus to secure the entire leaf nerve system in diseased forests, in opposite to H. albidus, impacts the general diversity and successional trajectory of fungi in decomposing ash petioles.

The Native Hymenoscyphus albidus and the Invasive Hymenoscyphus fraxineus Are Similar in Their Necrotrophic Growth Phase in Ash Leaves.

The populations of European ash and its harmless fungal associate Hymenoscyphus albidus are in decline owing to ash dieback caused by the invasive Hymenoscyphus fraxineus, a fungus that in its native range in Asia is a harmless leaf endophyte of local ash species. To clarify the behavior of H. albidus and its spatial and temporal niche overlap with the invasive relative, we used light microscopy, fungal species-specific qPCR assays, and PacBio long-read amplicon sequencing of the ITS1-5.8S-ITS2 region to examine fungal growth and species composition in attached leaves of European ash. The plant material was collected from a healthy stand in central Norway, where ash saplings in late autumn showed leaflet vein necrosis like that commonly related to H. fraxineus. For reference, leaflet samples were analyzed from stands with epidemic level of ash dieback in southeastern Norway and Estonia. While H. albidus was predominant in the necrotic veins in the healthy stand, H. fraxineus was predominant in the diseased stands. Otherwise, endophytes with pathogenic potential in the genera Venturia (anamorph Fusicladium), Mycosphaerella (anamorph Ramularia), and Phoma, and basidiomycetous yeasts formed the core leaflet mycobiome both in the healthy and diseased stands. In necrotic leaf areas with high levels of either H. albidus or H. fraxineus DNA, one common feature was the high colonization of sclerenchyma and phloem, a region from which the ascomata of both species arise. Our data suggest that H. albidus can induce necrosis in ash leaves, but that owing to low infection pressure, this first takes place in tissues weakened by autumn senescence, 1-2 months later in the season than what is characteristic of H. fraxineus at an epidemic phase of ash dieback. The most striking difference between these fungi would appear to be the high fecundity of H. fraxineus. The adaptation to a host that is phylogenetically closely related to European ash, a tree species with high occurrence frequency in Europe, and the presence of environmental conditions favorable to H. fraxineus life cycle completion in most years may enable the build-up of high infection pressure and challenge of leaf defense prior to autumn senescence.

The Relationship between Fungal Diversity and Invasibility of a Foliar Niche-The Case of Ash Dieback.

European ash (Fraxinus excelsior) is threatened by the invasive ascomycete Hymenoscyphus fraxineus originating from Asia. Ash leaf tissues serve as a route for shoot infection but also as a sporulation substrate for this pathogen. Knowledge of the leaf niche partitioning by indigenous fungi and H. fraxineus is needed to understand the fungal community receptiveness to the invasion. We subjected DNA extracted from unwashed and washed leaflets of healthy and diseased European ash to PacBio sequencing of the fungal ITS1-5.8S-ITS2 rDNA region. Leaflets from co-inhabiting rowan trees (Sorbus aucuparia) served as a reference. The overlap in leaflet mycobiomes between ash and rowan was remarkably high, but unlike in rowan, in ash leaflets the sequence read proportion, and the qPCR-based DNA amount estimates of H. fraxineus increased vigorously towards autumn, concomitant with a significant decline in overall fungal richness. The niche of ash and rowan leaves was dominated by epiphytic propagules (Vishniacozyma yeasts, the dimorphic fungus Aureobasidion pullulans and the dematiaceous hyphomycete Cladosporium ramotenellum and H. fraxineus), and endophytic thalli of biotrophs (Phyllactinia and Taphrina species), the indigenous necrotroph Venturia fraxini and H. fraxineus. Mycobiome comparison between healthy and symptomatic European ash leaflets revealed no significant differences in relative abundance of H. fraxineus, but A. pullulans was more prevalent in symptomatic trees. The impacts of host specificity, spatiotemporal niche partitioning, species carbon utilization profiles and life cycle traits are discussed to understand the ecological success of H. fraxineus in Europe. Further, the inherent limitations of different experimental approaches in the profiling of foliicolous fungi are addressed.

Propagule Pressure Build-Up by the Invasive Hymenoscyphus fraxineus Following Its Introduction to an Ash Forest Inhabited by the Native Hymenoscyphus albidus.

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.

Fungal diversity and seasonal succession in ash leaves infected by the invasive ascomycete Hymenoscyphus fraxineus.

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.

Novel Hydraulic Vulnerability Proxies for a Boreal Conifer Species Reveal That Opportunists May Have Lower Survival Prospects under Extreme Climatic Events.

Top dieback in 40-60 years old forest stands of Norway spruce [Picea abies (L.) Karst.] in southern Norway is supposed to be associated with climatic extremes. Our intention was to learn more about the processes related to top dieback and in particular about the plasticity of possible predisposing factors. We aimed at (i) developing proxies for P 50 based on anatomical data assessed by SilviScan technology and (ii) testing these proxies for their plasticity regarding climate, in order to (iii) analyze annual variations of hydraulic proxies of healthy looking trees and trees with top dieback upon their impact on tree survival. At two sites we selected 10 tree pairs, i.e., one healthy looking tree and one tree with visual signs of dieback such as dry tops, needle shortening and needle yellowing (n = 40 trees). Vulnerability to cavitation (P 50) of the main trunk was assessed in a selected sample set (n = 19) and we thereafter applied SilviScan technology to measure cell dimensions (lumen (b) and cell wall thickness (t)) in these specimen and in all 40 trees in tree rings formed between 1990 and 2010. In a first analysis step, we searched for anatomical proxies for P 50. The set of potential proxies included hydraulic lumen diameters and wall reinforcement parameters based on mean, radial, and tangential tracheid diameters. The conduit wall reinforcement based on tangential hydraulic lumen diameters ((t/b ht)(2)) was the best estimate for P 50. It was thus possible to relate climatic extremes to the potential vulnerability of single annual rings. Trees with top dieback had significantly lower (t/b ht)(2) and wider tangential (hydraulic) lumen diameters some years before a period of water deficit (2005-2006). Radial (hydraulic) lumen diameters showed however no significant differences between both tree groups. (t/b ht)(2) was influenced by annual climate variability; strongest correlations were found with precipitation in September of the previous growing season: high precipitation in previous September resulted in more vulnerable annual rings in the next season. The results are discussed with respect to an "opportunistic behavior" and genetic predisposition to drought sensitivity.

Effects of prolonged drought on the anatomy of sun and shade needles in young Norway spruce trees.

Predicted increases in the frequency and duration of drought are expected to negatively affect tree vitality, but we know little about how water shortage will influence needle anatomy and thereby the trees' photosynthetic and hydraulic capacity. In this study, we evaluated anatomical changes in sun and shade needles of 20-year-old Norway spruce trees exposed to artificial drought stress. Canopy position was found to be important for needle structure, as sun needles had significantly higher values than shade needles for all anatomical traits (i.e., cross-sectional needle area, number of tracheids in needle, needle hydraulic conductivity, and tracheid lumen area), except proportion of xylem area per cross-sectional needle area. In sun needles, drought reduced all trait values by 10-40%, whereas in shade needles, only tracheid maximum diameter was reduced by drought. Due to the relatively weaker response of shade needles than sun needles in drought-stressed trees, the difference between the two needle types was reduced by 25% in the drought-stressed trees compared to the control trees. The observed changes in needle anatomy provide new understanding of how Norway spruce adapts to drought stress and may improve predictions of how forests will respond to global climate change.

DNA quantification of basidiomycetous fungi during storage of logging residues.

The demand for bioenergy caused an increased use of logging residues, branches and treetops that were previously left on the ground after harvesting. Residues are stored outdoors in piles and it is unclear to what extent fungi transform this material. Our objective was to quantify the amount of wood degrading fungi during storage using quantitative real-time PCR (qPCR) to detect basidiomycetous DNA in logging residues, a novel approach in this field. We found that the qPCR method was accurate in quantifying the fungal DNA during storage. As the moisture content of the piled logging residues decreased during the storage period, the fungal DNA content also decreased. Scots pine residues contained more fungal DNA than residues from Norway spruce. Loose piles had generally more fungal DNA than bundled ones.

Effect of thinning on anatomical adaptations of Norway spruce needles.

Conifers and other trees are constantly adapting to changes in light conditions, water/nutrient supply and temperatures by physiological and morphological modifications of their foliage. However, the relationship between physiological processes and anatomical characteristics of foliage has been little explored in trees. In this study we evaluated needle structure and function in Norway spruce families exposed to different light conditions and transpiration regimes. We compared needle characteristics of sun-exposed and shaded current-year needles in a control plot and a thinned plot with 50% reduction in stand density. Whole-tree transpiration rates remained similar across plots, but increased transpiration of lower branches after thinning implies that sun-exposed needles in the thinned plot were subjected to higher water stress than sun-exposed needles in the control plot. In general, morphological and anatomical needle parameters increased with increasing tree height and light intensity. Needle width, needle cross-section area, needle stele area and needle flatness (the ratio of needle thickness to needle width) differed most between the upper and lower canopy. The parameters that were most sensitive to the altered needle water status of the upper canopy after thinning were needle thickness, needle flatness and percentage of stele area in needle area. These results show that studies comparing needle structure or function between tree species should consider not only tree height and light gradients, but also needle water status. Unaccounted for differences in needle water status may have contributed to the variable relationship between needle structure and irradiance that has been observed among conifers.

Stand age and fine root biomass, distribution and morphology in a Norway spruce chronosequence in southeast Norway.

We assessed the influence of stand age on fine root biomass and morphology of trees and understory vegetation in 10-, 30-, 60- and 120-year-old Norway spruce stands growing in sandy soil in southeast Norway. Fine root (< 1, 1-2 and 2-5 mm in diameter) biomass of trees and understory vegetation (< 2 mm in diameter) was sampled by soil coring to a depth of 60 cm. Fine root morphological characteristics, such as specific root length (SRL), root length density (RLD), root surface area (RSA), root tip number and branching frequency (per unit root length or mass), were determined based on digitized root data. Fine root biomass and morphological characteristics related to biomass (RLD and RSA) followed the same tendency with chronosequence and were significantly higher in the 30-year-old stand and lower in the 10-year-old stand than in the other stands. Among stands, mean fine root (< 2 mm) biomass ranged from 49 to 398 g m(-2), SLR from 13.4 to 19.8 m g(-1), RLD from 980 to 11,650 m m(-3) and RSA from 2.4 to 35.4 m(2) m(-3). Most fine root biomass of trees was concentrated in the upper 20 cm of the mineral soil and in the humus layer (0-5 cm) in all stands. Understory fine roots accounted for 67 and 25% of total fine root biomass in the 10- and 120-year-old stands, respectively. Stand age had no affect on root tip number or branching frequency, but both parameters changed with soil depth, with increasing number of root tips and decreasing branching frequency with increasing soil depth for root fractions < 2 mm in diameter. Specific (mass based) root tip number and branching density were highest for the finest roots (< 1 mm) in the humus layer. Season (spring or fall) had no effect on tree fine root biomass, but there was a small and significant increase in understory fine root biomass in fall relative to spring. All morphological characteristics showed strong seasonal variation, especially the finest root fraction, with consistently and significantly higher values in spring than in fall. We conclude that fine root biomass, especially in the finest fraction (< 1 mm in diameter), is strongly dependent on stand age. Among stands, carbon concentration in fine root biomass was highest in the 30-year-old stand, and appeared to be associated with the high tree and canopy density during the early stage of stand development. Values of RLD and RSA, morphological features indicative of stand nutrient-uptake efficiency, were higher in the 30-year-old stand than in the other stands.

Etiology and real-time polymerase chain reaction-based detection of gremmeniella- and phomopsis-associated disease in norway spruce seedlings.

ABSTRACT In spring 2002, an unusual disease outburst was recorded on Norway spruce seedlings in southeast Norway. Extensive damage was recorded on 1- and 2-year-old Norway spruce seedlings that either had wintered in nursery cold storage or had been planted out in autumn 2001. The damage was characterized by leader shoot dieback and stem necroses on the upper or lower part of the shoot from 2001. Gremmeniella abietina and a Phomopsis sp. frequently were isolated from the diseased seedlings. Internal transcribed spacer (ITS) ribosomal (r)DNA sequence analysis and random amplified microsatellites profiling indicated that the G. abietina strains associated with diseased nursery seedlings belonged to the large-tree type (LTT) ecotype of the European race of G. abietina var. abietina, and inoculation tests confirmed their pathogenicity on Norway spruce. Based on ITS rDNA sequence analysis, the Phomopsis strains associated with diseased seedlings did not represent any characterized Phomopsis spp. associated with conifers. The Phomopsis sp. was not pathogenic in inoculation tests, indicating that it may be a secondary colonizer. ITS-based real-time polymerase chain reaction assays were developed in order to detect and quantify G. abietina and Phomopsis in the nursery stock. We describe here the G. abietina-associated shoot dieback symptoms on Norway spruce seedlings and conclude that the unusual disease outburst likely was related to the G. abietina var. abietina epidemic caused by the LTT on large Scots pines in 2001.