Oomycete Soil Diversity Associated with Betula and Alnus in Forests and Urban Settings in the Nordic-Baltic Region.

This study aimed to determine the differences and drivers of oomycete diversity and community composition in alder- and birch-dominated park and natural forest soils of the Fennoscandian and Baltic countries of Estonia, Finland, Lithuania, Norway, and Sweden. For this, we sequenced libraries of PCR products generated from the DNA of 111 soil samples collected across a climate gradient using oomycete-specific primers on a PacBio high-throughput sequencing platform. We found that oomycete communities are most affected by temperature seasonality, annual mean temperature, and mean temperature of the warmest quarter. Differences in composition were partly explained by the higher diversity of Saprolegniales in Sweden and Norway, as both total oomycete and Saprolegniales richness decreased significantly at higher longitudes, potentially indicating the preference of this group of oomycetes for a more temperate maritime climate. None of the evaluated climatic variables significantly affected the richness of Pythiales or Peronosporales. Interestingly, the relative abundance and richness of Pythiales was higher at urban sites compared to forest sites, whereas the opposite was true for Saprolegniales. Additionally, this is the first report of Phytophthora gallica and P. plurivora in Estonia. Our results indicate that the composition of oomycetes in soils is strongly influenced by climatic factors, and, therefore, changes in climate conditions associated with global warming may have the potential to significantly alter the distribution range of these microbes, which comprise many important pathogens of plants.

Vaccination of Elms against Dutch Elm Disease-Are the Associated Epiphytes and Endophytes Affected?

Dutch elm disease (DED) is causing extensive mortality of ecologically and culturally valuable elm trees (Ulmus spp.). Treatment of elms with the biological vaccine Dutch Trig® has been found to provide effective protection against DED by stimulating the defensive mechanisms of the trees. We hypothesized that the same mechanisms could also affect non-target organisms associated with elms. We explored the possible effects of vaccination on epiphytes (mainly lichens) and fungal endophytes living in the bark and young xylem of treated elms. Epiphyte cover percentage was assessed visually using a grid placed on the trunks, and a culture-based approach was used to study endophytes. Epiphyte cover was lower on the trunks of vaccinated trees as compared with unvaccinated trees, but the difference was not statistically significant. The presence of slow-growing and uncommon endophytes seemed to be reduced in continuously vaccinated elms; however, the highest endophyte diversity was found in elms four years after cessation of the vaccination treatments. Our findings suggest that although vaccination may shape epiphyte and endophyte communities in elms, its impacts are not straightforward. More detailed studies are, therefore, needed to inform the sustainable application of the vaccine as a part of the integrated management of DED.

Core endophytic mycobiome in Ulmus minor and its relation to Dutch elm disease resistance.

The core microbiota of plants exerts key effects on plant performance and resilience to stress. The aim of this study was to identify the core endophytic mycobiome in U. minor stems and disentangle associations between its composition and the resistance to Dutch elm disease (DED). We also defined its spatial variation within the tree and among distant tree populations. Stem samples were taken i) from different heights of the crown of a 168-year-old elm tree, ii) from adult elm trees growing in a common garden and representing a gradient of resistance to DED, and iii) from trees growing in two distant natural populations, one of them with varying degrees of vitality. Endophyte composition was profiled by high throughput sequencing of the first internal transcribed spacer region (ITS1) of the ribosomal DNA. Three families of yeasts (Buckleyzymaceae, Trichomeriaceae and Bulleraceae) were associated to DED-resistant hosts. A small proportion (10%) of endophytic OTUs was almost ubiquitous throughout the crown while tree colonization by most fungal taxa followed stochastic patterns. A clear distinction in endophyte composition was found between geographical locations. By combining all surveys, we found evidence of a U. minor core mycobiome, pervasive within the tree and ubiquitous across locations, genotypes and health status.

Beneficial and pathogenic plant-microbe interactions during flooding stress.

The number and intensity of flood events will likely increase in the future, raising the risk of flooding stress in terrestrial plants. Understanding flood effects on plant physiology and plant-associated microbes is key to alleviate flooding stress in sensitive species and ecosystems. Reduced oxygen supply is the main constrain to the plant and its associated microbiome. Hypoxic conditions hamper root aerobic respiration and, consequently, hydraulic conductance, nutrient uptake, and plant growth and development. Hypoxia favours the presence of anaerobic microbes in the rhizosphere and roots with potential negative effects to the plant due to their pathogenic behaviour or their soil denitrification ability. Moreover, plant physiological and metabolic changes induced by flooding stress may also cause dysbiotic changes in endosphere and rhizosphere microbial composition. The negative effects of flooding stress on the holobiont (i.e., the host plant and its associated microbiome) can be mitigated once the plant displays adaptive responses to increase oxygen uptake. Stress relief could also arise from the positive effect of certain beneficial microbes, such as mycorrhiza or dark septate endophytes. More research is needed to explore the spiralling, feedback flood responses of plant and microbes if we want to promote plant flood tolerance from a holobiont perspective.

Aspen Leaves as a "Chemical Landscape" for Fungal Endophyte Diversity-Effects of Nitrogen Addition.

Abiotic and biotic factors may shape the mycobiome communities in plants directly but also indirectly by modifying the quality of host plants as a substrate. We hypothesized that nitrogen fertilization (N) would determine the quality of aspen (Populus tremula) leaves as a substrate for the endophytic fungi, and that by subjecting the plants to N, we could manipulate the concentrations of positive (nutritious) and negative (antifungal) chemicals in leaves, thus changing the internal "chemical landscape" for the fungi. We expected that this would lead to changes in the fungal community composition, in line with the predictions of heterogeneity-diversity relationship and resource availability hypotheses. To test this, we conducted a greenhouse study where aspen plants were subjected to N treatment. The chemical status of the leaves was confirmed using GC/MS (114 metabolites, including amino acids and sugars), LC/MS (11 phenolics), and UV-spectrometry (antifungal condensed tannins, CTs), and the endophytic communities were characterized using culture-dependent sequencing. We found that N treatment reduced foliar concentrations of CT precursor catechin but not that of CTs. Nitrogen treatment also increased the concentrations of the amino acids and reduced the concentration of some sugars. We introduced beetle herbivores (H) as a second treatment but found no rapid changes in chemical traits nor strong effect on the diversity of endophytes induced by herbivores. A few rare fungi were associated with and potentially vectored by the beetle herbivores. Our findings indicate that in a controlled environment, the externally induced changes did not strongly alter endophyte diversity in aspen leaves.

Biotic threats for 23 major non-native tree species in Europe.

For non-native tree species with an origin outside of Europe a detailed compilation of enemy species including the severity of their attack is lacking up to now. We collected information on native and non-native species attacking non-native trees, i.e. type, extent and time of first observation of damage for 23 important non-native trees in 27 European countries. Our database includes about 2300 synthesised attack records (synthesised per biotic threat, tree and country) from over 800 species. Insects (49%) and fungi (45%) are the main observed biotic threats, but also arachnids, bacteria including phytoplasmas, mammals, nematodes, plants and viruses have been recorded. This information will be valuable to identify patterns and drivers of attacks, and trees with a lower current health risk to be considered for planting. In addition, our database will provide a baseline to which future impacts on non-native tree species could be compared with and thus will allow to analyse temporal trends of impacts.

The tree species matters: Biodiversity and ecosystem service implications of replacing Scots pine production stands with Norway spruce.

The choice of tree species used in production forests matters for biodiversity and ecosystem services. In Sweden, damage to young production forests by large browsing herbivores is helping to drive a development where sites traditionally regenerated with Scots pine (Pinus sylvestris) are instead being regenerated with Norway spruce (Picea abies). We provide a condensed synthesis of the available evidence regarding the likely resultant implications for forest biodiversity and ecosystem services from this change in tree species. Apart from some benefits (e.g. reduced stand-level browsing damage), we identified a range of negative outcomes for biodiversity, production, esthetic and recreational values, as well as increased stand vulnerability to storm, frost, and drought damage, and potentially higher risks of pest and pathogen outbreak. Our results are directly relevant to forest owners and policy-makers seeking information regarding the uncertainties, risks, and trade-offs likely to result from changing the tree species in production forests.

Pedunculate Oaks (Quercus robur L.) Differing in Vitality as Reservoirs for Fungal Biodiversity.

Ecological significance of trees growing in urban and peri-urban settings is likely to increase in future land-use regimes, calling for better understanding of their role as potential reservoirs or stepping stones for associated biodiversity. We studied the diversity of fungal endophytes in woody tissues of asymptomatic even aged pedunculate oak trees, growing as amenity trees in a peri-urban setting. The trees were classified into three groups according to their phenotypic vitality (high, medium, and low). Endophytes were cultured on potato dextrose media from surface sterilized twigs and DNA sequencing was performed to reveal the taxonomic identity of the morphotypes. In xylem tissues, the frequency and diversity of endophytes was highest in oak trees showing reduced vitality. This difference was not found for bark samples, in which the endophyte infections were more frequent and communities more diverse than in xylem. In general, most taxa were shared across the samples with few morphotypes being recovered in unique samples. Leaf phenolic profiles were found to accurately classify the trees according to their phenotypic vitality. Our results confirm that xylem is more selective substrate for endophytes than bark and that endophyte assemblages in xylem are correlated to the degree of host vitality. Thus, high vitality of trees may be associated with reduced habitat quality to wood-associated endophytes.

Draft genome of the oomycete pathogen Phytophthora cactorum strain LV007 isolated from European beech (Fagus sylvatica).

Phytophthora cactorum is a broad host range phytopathogenic oomycete. P. cactorum strain LV007 was isolated from a diseased European Beech (Fagus sylvatica) in Malmö, Sweden in 2016. The draft genome of P. cactorum strain LV007 is 67.81 Mb. It contains 15,567 contigs and 21,876 predicted protein-coding genes. As reported for other phytopathogenic Phytophthora species, cytoplasmic effector proteins including RxLR and CRN families were identified. The genome sequence has been deposited at DDBJ/ENA/GenBank under the accession NBIJ00000000. The version described in this paper is version NBIJ01000000.

Risk of False Positives during Sampling for Heterobasidion annosum s.l.

A standard method to detect infection by Heterobasidion annosum sensu lato (s.l.) in stumps or stems is to cut a disc and examine it under a microscope. Concerns have been raised that spores can be transferred from the bark to the cut surface, thus contaminating the sample. The aims of this study were to test whether viable basidiospores of H. annosum s.l. can be transferred from the bark onto disc surfaces by a chainsaw and to investigate the impacts of different sampling procedures on the extent of contaminations. Logs were cut with or without adding basidiospores to the bark prior to the cut. Infection measurements were significantly greater for discs with treated bark (100% infected, infection coverage 40 cm2 dm-2 of disc area) compared with control discs (47% infected, infection coverage 0.2 to 0.3 cm2 dm-2). In addition, trees were sampled under authentic field conditions using different procedures. The infection measurements differed significantly depending on the procedure; sampling involving debarking or disinfection of the bark with 70% ethanol prior to cutting had lower measurements (6 to 19% and 13% infected, respectively) compared with leaving the bark on untreated (63 to 75% infected). Consideration of the contamination risk is warranted when evaluating the results of earlier studies and when planning new experiments.

Phenotype MicroArrays as a complementary tool to next generation sequencing for characterization of tree endophytes.

There is an increasing need to calibrate microbial community profiles obtained through next generation sequencing (NGS) with relevant taxonomic identities of the microbes, and to further associate these identities with phenotypic attributes. Phenotype MicroArray (PM) techniques provide a semi-high throughput assay for characterization and monitoring the microbial cellular phenotypes. Here, we present detailed descriptions of two different PM protocols used in our recent studies on fungal endophytes of forest trees, and highlight the benefits and limitations of this technique. We found that the PM approach enables effective screening of substrate utilization by endophytes. However, the technical limitations are multifaceted and the interpretation of the PM data challenging. For the best result, we recommend that the growth conditions for the fungi are carefully standardized. In addition, rigorous replication and control strategies should be employed whether using pre-configured, commercial microwell-plates or in-house designed PM plates for targeted substrate analyses. With these precautions, the PM technique is a valuable tool to characterize the metabolic capabilities of individual endophyte isolates, or successional endophyte communities identified by NGS, allowing a functional interpretation of the taxonomic data. Thus, PM approaches can provide valuable complementary information for NGS studies of fungal endophytes in forest trees.

Resistance to Dutch elm disease reduces presence of xylem endophytic fungi in Elms (Ulmus spp.).

Efforts to introduce pathogen resistance into landscape tree species by breeding may have unintended consequences for fungal diversity. To address this issue, we compared the frequency and diversity of endophytic fungi and defensive phenolic metabolites in elm (Ulmus spp.) trees with genotypes known to differ in resistance to Dutch elm disease. Our results indicate that resistant U. minor and U. pumila genotypes exhibit a lower frequency and diversity of fungal endophytes in the xylem than susceptible U. minor genotypes. However, resistant and susceptible genotypes showed a similar frequency and diversity of endophytes in the leaves and bark. The resistant and susceptible genotypes could be discriminated on the basis of the phenolic profile of the xylem, but not on basis of phenolics in the leaves or bark. As the Dutch elm disease pathogen develops within xylem tissues, the defensive chemistry of resistant elm genotypes thus appears to be one of the factors that may limit colonization by both the pathogen and endophytes. We discuss a potential trade-off between the benefits of breeding resistance into tree species, versus concomitant losses of fungal endophytes and the ecosystem services they provide.

Novel bioassay demonstrates attraction of the white potato cyst nematode Globodera pallida (Stone) to non-volatile and volatile host plant cues.

Potato cyst nematodes (PCNs) are a major pest of solanaceous crops such as potatoes, tomatoes, and eggplants and have been widely studied over the last 30 years, with the majority of earlier studies focusing on the identification of natural hatching factors. As a novel approach, we focused instead on chemicals involved in nematode orientation towards its host plant. A new dual choice sand bioassay was designed to study nematode responses to potato root exudates (PRE). This bioassay, conducted together with a traditional hatching bioassay, showed that biologically active compounds that induce both hatching and attraction of PCNs can be collected by water extraction of incised potato roots. Furthermore, our results demonstrated that PCN also were attracted by potato root volatiles. Further work is needed to fully understand how PCNs use host plant chemical cues to orientate towards hosts. Nevertheless, the simple attraction assay used in this study provides an important tool for the identification of host-emitted attractants.

Inducibility of chemical defenses in Norway spruce bark is correlated with unsuccessful mass attacks by the spruce bark beetle.

Secondary attraction to aggregation pheromones plays a central role in the host colonization behavior of the European spruce bark beetle Ips typographus. However, it is largely unknown how the beetles pioneering an attack locate suitable host trees, and eventually accept or reject them. To find possible biomarkers for host choice by I. typographus, we analyzed the chemistry of 58 Norway spruce (Picea abies) trees that were subsequently either (1) successfully attacked and killed, (2) unsuccessfully attacked, or (3) left unattacked. The trees were sampled before the main beetle flight in a natural Norway spruce-dominated forest. No pheromones were used to attract beetles to the experimental trees. To test the trees' defense potential, each tree was treated in a local area with the defense hormone methyl jasmonate (MeJ), and treated and untreated bark were analyzed for 66 different compounds, including terpenes, phenolics and alkaloids. The chemistry of MeJ-treated bark correlated strongly with the success of I. typographus attack, revealing major chemical differences between killed trees and unsuccessfully attacked trees. Surviving trees produced significantly higher amounts of most of the 39 analyzed mono-, sesqui-, and diterpenes and of 4 of 20 phenolics. Alkaloids showed no clear pattern. Differences in untreated bark were less pronounced, where only 1,8-cineole and (-)-limonene were significantly higher in unsuccessfully attacked trees. Our results show that the potential of individual P. abies trees for inducing defense compounds upon I. typographus attack may partly determine tree resistance to this bark beetle by inhibiting its mass attack.

Does temperature regime govern the establishment of Heterobasidion annosum in Scandinavia?

We explored the reasons underlying the biogeographic distribution patterns of the economically important, wood-rotting basidiomycete Heterobasidion annosum in Sweden. Despite the commonness of suitable host trees, Heterobasidion annosum has not been recorded in the north of Sweden, whereas its relative, H. parviporum, is present throughout the country. To test the hypothesis that H. annosum has not spread to the north because of the effect of climate, mainly differences in the general temperature regime, we inoculated Norway spruce stumps and standing trees with H. annosum and H. parviporum at six field sites, three in the south and three in the north of Sweden. Three strains of both species were used in random combinations, so that each selected stump and tree was inoculated with both species at the same time. At 2 and 10 months after the inoculations, we compared the frequencies of detection of H. annosum and H. parviporum colonies at different distances from inoculation points in the stumps and in trees. The H. annosum colonies were detected only infrequently on disks cut from the inoculated stumps (0-4% of re-isolations) in both areas, whereas H. parviporum was detected much more frequently (26-47% of re-isolations). In standing trees, colonies belonging to H. annosum could be detected up to 210 cm (south) and 80 cm (north) and those belonging to H. parviporum up to 210 cm (south) and 140 cm (north) above the inoculation points. Our results suggest that difference in temperature regime does not provide an explanation for the distribution limit of H. annosum.

Downregulation of high-isoelectric-point extracellular superoxide dismutase mediates alterations in the metabolism of reactive oxygen species and developmental disturbances in hybrid aspen.

Transgenic hybrid aspen (Populus tremula L. x P. tremuloides Michx.) plants expressing a high-isoelectric-point superoxide dismutase (hipI-SOD) gene in antisense orientation were generated to investigate its function. Immunolocalization studies showed the enzyme to be localized extracellularly, in the secondary cell wall of xylem vessels and phloem fibers. The antisense lines of hipI-SOD exhibited a distinct phenotype; growth rate was reduced, stems were thinner and leaves smaller than in wild-type (WT) plants. The abundance of hipI-SOD was reduced in the bark and xylem of plants from these antisense lines. The vascular tissue of transgenic lines became lignified earlier than in WT plants and also showed an increased accumulation of reactive oxygen species (ROS). Xylem fibers and vessels were shorter and thinner in the transgenic lines than in WT plants. The total phenolic content was enhanced in the antisense lines. Furthermore, microarray analysis indicated that several enzymes involved in cell signaling, lignin biosynthesis and stress responses were upregulated in apical vascular tissues of transgenic plants. The upregulation of selected genes involved in lignin biosynthesis was also verified by real-time PCR. The results suggest that, in the transgenic plants, a premature transition into maturation occurs and the process is discussed in terms of the effects of increased accumulation of ROS due to reduced expression of hipI-SOD during development and differentiation.

Tall herb herbivory resistance reflects historic exposure to leaf beetles in a boreal archipelago age-gradient.

In this paper, we introduce the coevolution-by-coexistence hypothesis which predicts that the strength of a coevolutionary adaptation will become increasingly apparent as long as the corresponding selection from an interacting counterpart continues. Hence, evolutionary interactions between plants and their herbivores can be studied by comparing discrete plant populations with known history of herbivore colonization. We studied populations of the host plant, Filipendula ulmaria (meadow sweet), on six islands, in a Bothnian archipelago subject to isostatic rebound, that represent a spatio-temporal gradient of coexistence with its two major herbivores, the specialist leaf beetles Galerucella tenella and Altica engstroemi. Regression analyses showed that a number of traits important for insect-plant interactions (leaf concentrations of individual phenolics and condensed tannins, plant height, G. tenella adult feeding and oviposition) were significantly correlated with island age. First, leaf concentrations of condensed tannins and individual phenolics were positively correlated with island age, suggesting that plant resistance increased after herbivore colonization and continued to increase in parallel to increasing time of past coexistence, while plant height showed a reverse negative correlation. Second, a multi-choice experiment with G. tenella showed that both oviposition and leaf consumption of the host plants were negatively correlated with island age. Third, larvae performed poorly on well-defended, older host populations and well on less-defended, younger populations. Thus, no parameter assessed in this study falsifies the coevolution-by-coexistence hypothesis. We conclude that spatio-temporal gradients present in rising archipelagos offer unique opportunities to address evolutionary interactions, but care has to be taken as abiotic (and other biotic) factors may interact in a complicated way.

Polyamine profiles of healthy and parasite-infected Vaccinium myrtillus plants under nitrogen enrichment.

Addition of nitrogen (N) to the field layer of boreal forests has been shown to increase the occurrence of the parasitic fungus Valdensia heterodoxa on Vaccinium myrtillus plants. We investigated whether N addition to soil alters the levels of polyamines in V. myrtillus shoots, and discuss here whether such changes could promote the spread of the parasitic fungus on V myrtillus. Using HPLC, we analyzed the concentrations of free and conjugated polyamines in healthy and naturally V. heterodoxa-infected V. myrtillus plants, which had received a moderate or high dose of N fertilizer, or no additional N. Fertilization with N increased the concentrations of free diamines (putrescine and diaminopropane), but had no significant effect on conjugated amines. Thus, N-induced changes in the constitutive levels of soluble conjugated amines do not seem to explain the increased parasite susceptibility of V. myrtillus under N enrichment. Generally, the concentrations of free diamines and insoluble conjugated putrescine were higher in diseased than in healthy shoots, suggesting parasite-induced accumulation of diamines. Free spermine seemed to accumulate in unfertilized, diseased plants, but in fertilized plants this induction was dampened, suggesting that N-induced alterations in spermine metabolism may promote the spread of parasites on V. myrtillus under N-enrichment.

Nitrogen deposition and the biodiversity of boreal forests: implications for the nitrogen critical load.

The critical load concept is used to establish the deposition levels which ecosystems can tolerate without significant harmful effects. Here we summarize work within the Swedish research program Abatement Strategies for Transboundary Air Pollution (ASTA) assessing the critical load of N for boreal forests. Results from both field experiments in an area with low background N deposition in northern Sweden, and from a large-scale monitoring study, show that important vegetational changes start to take place when adding low N doses and that recovery of the vegetation after ceasing N input is a very slow process. The data presented indicate that changes in key ecosystem components occur even at a lower rate of N input than the present recommended empirical critical load for boreal forest understorey vegetation of 10-15 kg N ha(-1) yr(-1). Based on the data presented, we suggest that the critical load should be lowered to 6 kg N ha(-1) yr(-1).

Do multitrophic interactions override N fertilization effects on Operophtera larvae?

We examined how performance of Operophtera brumata (Lepidoptera) larvae was affected by nitrogen (N) fertilization of boreal forest understorey vegetation. We monitored larval densities on Vaccinium myrtillus plants for a period of 7 years in a field experiment. Preliminary results indicated that the N effect on larval densities was weak. To examine if this was due to indirect interactions with a plant pathogen, Valdensia heterodoxa, that share the same host plant, or due to top-down effects of predation, we performed both a laboratory feeding experiment (individual level) and a bird exclusion experiment (population level) in the field. At the individual level, altered food plant quality (changes in plant concentration of carbon, N, phenolics, or condensed tannins) due to repeated infection by the pathogen had no effect on larval performance, but both survival to the adult stage and adult weight were positively affected by N fertilization. Exclusion of insectivorous birds increased the frequency of larval damage on V. myrtillus shoots, indicating higher larval densities. This effect was stronger in fertilized than in unfertilized plots, indicating higher bird predation in fertilized plots. Predation may thus explain the lack of fertilization effect on larval densities in the field experiment. Our results suggest that top-down effects are more important for larval densities than bottom-up effects, and that bird predation may play an important role in population regulation of O. brumata in boreal forests.