Association genetics identifies a specifically regulated Norway spruce laccase gene,PaLAC5, linked to Heterobasidion parviporum resistance

It is important to improve the understanding of the interactions between the trees and pathogens and integrate this knowledge about disease resistance into tree breeding programs. The conifer Norway spruce (Picea abies) is an important species for the forest industry in Europe. Its major pathogen is Heterobasidion parviporum, causing stem and root rot. In this study, we identified 11 Norway spruce QTLs (Quantitative trait loci) that correlate with variation in resistance to H. parviporum in a population of 466 trees by association genetics. Individual QTLs explained between 2.1 and 5.2% of the phenotypic variance. The expression of candidate genes associated with the QTLs was analysed in silico and in response to H. parviporum hypothesizing that (a) candidate genes linked to control of fungal sapwood growth are more commonly expressed in sapwood, and; (b) candidate genes associated with induced defences are respond to H. parviporum inoculation. The Norway spruce laccase PaLAC5 associated with control of lesion length development is likely to be involved in the induced defences. Expression analyses showed that PaLAC5 responds specifically and strongly in close proximity to the H. parviporum inoculation. Thus, PaLAC5 may be associated with the lignosuberized boundary zone formation in bark adjacent to the inoculation site.     

Community assembly of a euryhaline fish microbiome during salinity acclimation

Microbiomes play a critical role in promoting a range of host functions. Microbiome function, in turn, is dependent on its community composition. Yet, how microbiome taxa are assembled from their regional species pool remains unclear. Many possible drivers have been hypothesized, including deterministic processes of competition, stochastic processes of colonization and migration, and physiological ‘host‐effect’ habitat filters. The contribution of each to assembly in nascent or perturbed microbiomes is important for understanding host–microbe interactions and host health. In this study, we characterized the bacterial communities in a euryhaline fish and the surrounding tank water during salinity acclimation. To assess the relative influence of stochastic versus deterministic processes in fish microbiome assembly, we manipulated the bacterial species pool around each fish by changing the salinity of aquarium water. Our results show a complete and repeatable turnover of dominant bacterial taxa in the microbiomes from individuals of the same species after acclimation to the same salinity. We show that changes in fish microbiomes are not correlated with corresponding changes to abundant taxa in tank water communities and that the dominant taxa in fish microbiomes are rare in the aquatic surroundings, and vice versa. Our results suggest that bacterial taxa best able to compete within the unique host environment at a given salinity appropriate the most niche space, independent of their relative abundance in tank water communities. In this experiment, deterministic processes appear to drive fish microbiome assembly, with little evidence for stochastic colonization.     

Amplicon‐pyrosequencing‐based detection of compositional shifts in bryophyte‐associated fungal communities along an elevation gradient

Although bryophytes are a dominant vegetation component of boreal and alpine ecosystems, little is known about their associated fungal communities. HPLC assays of ergosterol (fungal biomass) and amplicon pyrosequencing of the ITS2 region of rDNA were used to investigate how the fungal communities associated with four bryophyte species changed across an elevational gradient transitioning from conifer forest to the low‐alpine. Fungal biomass and OTU richness associated with the four moss hosts did not vary significantly across the gradient (P > 0.05), and both were more strongly affected by host and tissue type. Despite largely constant levels of fungal biomass, distinct shifts in community composition of fungi associated with Hylocomium, Pleurozium and Polytrichum occurred between the elevation zones of the gradient. This likely is a result of influence on fungal communities by major environmental factors such as temperature, directly or indirectly mediated by, or interacting with, the response of other components of the vegetation (i.e. the dominant trees). Fungal communities associated with Dicranum were an exception, exhibiting spatial autocorrelation between plots, and no significant structuring by elevation. Nevertheless, the detection of distinct fungal assemblages associated with a single host growing in different elevation zones along an elevational gradient is of particular relevance in the light of the ongoing changes in vegetation patterns in boreal and alpine systems due to global climate warming.     

Relationships among wood‐boring beetles,fungi, and the decomposition of forest biomass

A prevailing paradigm in forest ecology is that wood‐boring beetles facilitate wood decay and carbon cycling, but empirical tests have yielded mixed results. We experimentally determined the effects of wood borers on fungal community assembly and wood decay within pine trunks in the southeastern United States. Pine trunks were made either beetle‐accessible or inaccessible. Fungal communities were compared using culturing and high‐throughput amplicon sequencing (HTAS) of DNA and RNA. Prior to beetle infestation, living pines had diverse fungal endophyte communities. Endophytes were displaced by beetle‐associated fungi in beetle‐accessible trees, whereas some endophytes persisted as saprotrophs in beetle‐excluded trees. Beetles increased fungal diversity several fold. Over forty taxa of Ascomycota were significantly associated with beetles, but beetles were not consistently associated with any known wood‐decaying fungi. Instead, increasing ambrosia beetle infestations caused reduced decay, consistent with previous in vitro experiments that showed beetle‐associated fungi reduce decay rates by competing with decay fungi. No effect of bark‐inhabiting beetles on decay was detected. Platypodines carried significantly more fungal taxa than scolytines. Molecular results were validated by synthetic and biological mock communities and were consistent across methodologies. RNA sequencing confirmed that beetle‐associated fungi were biologically active in the wood. Metabarcode sequencing of the LSU/28S marker recovered important fungal symbionts that were missed by ITS2, though community‐level effects were similar between markers. In contrast to the current paradigm, our results indicate ambrosia beetles introduce diverse fungal communities that do not extensively decay wood, but instead reduce decay rates by competing with wood decay fungi.     

Competition and facilitation in synthetic communities of arbuscular mycorrhizal fungi

Interactions between arbuscular mycorrhizal fungal (AMF) species cocolonizing the same host plant are still little understood in spite of major ecological significance of mycorrhizal symbiosis and widespread occurrence of these fungi in communities rather than alone. Furthermore, shifting the composition of AMF communities has demonstrated consequences for the provision of symbiotic benefits to the host as well as for the qualities of ecosystem services. Therefore, here we addressed the nature and strength of interactions between three different AMF species in all possible two‐species combinations on a gradient of inoculation densities. Fungal communities were established in pots with Medicago truncatula plants, and their composition was assessed with taxon‐specific real‐time PCR markers. Nature of interactions between the fungi was varying from competition to facilitation and was influenced by both the identity and relative abundance of the coinoculated fungi. Plants coinoculated with Claroideoglomus and Rhizophagus grew bigger and contained more phosphorus than with any of these two fungi separately, although these fungi obviously competed for root colonization. On the other hand, plants coinoculated with Gigaspora and Rhizophagus, which facilitated each other's root colonization, grew smaller than with any of these fungi separately. Our results point to as yet little understood complexity of interactions in plant‐associated symbiotic fungal communities, which, depending on their composition, can induce significant changes in plant host growth and/or phosphorus acquisition in either direction.     

Host plant genetic control of associated fungal and insect species in a Populus hybrid cross

Plants employ a diverse set of defense mechanisms to mediate interactions with insects and fungi. These relationships can leave lasting impacts on host plant genome structure such as rapid expansion of gene families through tandem duplication. These genomic signatures provide important clues about the complexities of plant/biotic stress interactions and evolution. We used a pseudo‐backcross hybrid family to identify quantitative trait loci (QTL) controlling associations between Populus trees and several common Populus diseases and insects. Using whole‐genome sequences from each parent, we identified candidate genes that may mediate these interactions. Candidates were partially validated using mass spectrometry to identify corresponding QTL for defensive compounds. We detected significant QTL for two interacting fungal pathogens and three insects. The QTL intervals contained candidate genes potentially involved in physical and chemical mechanisms of host–plant resistance and susceptibility. In particular, we identified adjoining QTLs for a phenolic glycoside and Phyllocolpa sawfly abundance. There was also significant enrichment of recent tandem duplications in the genomic intervals of the native parent, but not the exotic parent. Tandem gene duplication may be an important mechanism for rapid response to biotic stressors, enabling trees with long juvenile periods to reach maturity despite many coevolving biotic stressors.     

Plant host and soil origin influence fungal and bacterial assemblages in the roots of woody plants

Microbial communities in plant roots provide critical links between above‐ and belowground processes in terrestrial ecosystems. Variation in root communities has been attributed to plant host effects and microbial host preferences, as well as to factors pertaining to soil conditions, microbial biogeography and the presence of viable microbial propagules. To address hypotheses regarding the influence of plant host and soil biogeography on root fungal and bacterial communities, we designed a trap‐plant bioassay experiment. Replicate Populus, Quercus and Pinus plants were grown in three soils originating from alternate field sites. Fungal and bacterial community profiles in the root of each replicate were assessed through multiplex 454 amplicon sequencing of four loci (i.e., 16S, SSU, ITS, LSU rDNA). Soil origin had a larger effect on fungal community composition than did host species, but the opposite was true for bacterial communities. Populus hosted the highest diversity of rhizospheric fungi and bacteria. Root communities on Quercus and Pinus were more similar to each other than to Populus. Overall, fungal root symbionts appear to be more constrained by dispersal and biogeography than by host availability.     

Specificity of root microbiomes in native‐grown Nicotiana attenuata and plant responses to UVB increase Deinococcus colonization

Plants recruit microbial communities from the soil in which they germinate. Our understanding of the recruitment process and the factors affecting it is still limited for most microbial taxa. We analysed several factors potentially affecting root microbiome structure – the importance of geographic location of natural populations, the microbiome of native seeds as putative source of colonization and the effect of a plant's response to UVB exposure on root colonization of highly abundant species. The microbiome of Nicotiana attenuata seeds was determined by a culture‐dependent and culture‐independent approach, and the root microbiome of natural N. attenuata populations from five different locations was analysed using 454‐pyrosequencing. To specifically address the influence of UVB light on root colonization by Deinococcus, a genus abundant and consistently present in N. attenuata roots, transgenic lines impaired in UVB perception (irUVR8) and response (irCHAL) were investigated in a microcosm experiment with/without UVB supplementation using a synthetic bacterial community. The seed microbiome analysis indicated that N. attenuata seeds are sterile. Alpha and beta diversities of native root bacterial communities differed significantly between soil and root, while location had only a significant effect on the fungal but not the bacterial root communities. With UVB supplementation, root colonization of Deinococcus increased in wild type, but decreased in irUVR8 and irCHAL plants compared to nontreated plants. Our results suggest that N. attenuata recruits a core root microbiome exclusively from soil, with fungal root colonization being less selective than bacterial colonization. Root colonization by Deinococcus depends on the plant's response to UVB.     

Genetic control of α‐farnesene production in apple fruit and its role in fungal pathogenesis

Terpenes are important compounds in plant trophic interactions. A meta‐analysis of GC‐MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)‐α‐farnesene. Four quantitative trait loci (QTLs) for α‐farnesene production in ripe fruit were identified in a segregating ‘Royal Gala’ (RG) × ‘Granny Smith’ (GS) population with one major QTL on linkage group 10 co‐locating with the MdAFS1 (α‐farnesene synthase‐1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC‐MS analysis of headspace and solvent‐extracted terpenes showing that cold‐treated GS apples produced higher levels of (E,E)‐α‐farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)‐α‐farnesene. To evaluate the role of (E,E)‐α‐farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post‐harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)‐α‐farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post‐inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)‐α‐farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.     

Silver fir and Douglas fir are more tolerant to extreme droughts than Norway spruce in south‐western Germany

Improving our understanding of the potential of forest adaptation is an urgent task in the light of predicted climate change. Long‐term alternatives for susceptible yet economically important tree species such as Norway spruce (Picea abies) are required, if the frequency and intensity of summer droughts will continue to increase. Although Silver fir (Abies alba) and Douglas fir (Pseudotsuga menziesii) have both been described as drought‐tolerant species, our understanding of their growth responses to drought extremes is still limited. Here, we use a dendroecological approach to assess the resistance, resilience, and recovery of these important central Europe to conifer species the exceptional droughts in 1976 and 2003. A total of 270 trees per species were sampled in 18 managed mixed‐species stands along an altitudinal gradient (400–1200 m a.s.l.) at the western slopes of the southern and central Black Forest in southwest Germany. While radial growth in all species responded similarly to the 1976 drought, Norway spruce was least resistant and resilient to the 2003 summer drought. Silver fir showed the overall highest resistance to drought, similarly to Douglas fir, which exhibited the widest growth rings. Silver fir trees from lower elevations were more drought prone than trees at higher elevations. Douglas fir and Norway spruce, however, revealed lower drought resilience at higher altitudes. Although the 1976 and 2003 drought extremes were quite different, Douglas fir maintained consistently the highest radial growth. Although our study did not examine population‐level responses, it clearly indicates that Silver fir and Douglas fir are generally more resistant and resilient to previous drought extremes and are therefore suitable alternatives to Norway spruce; Silver fir more so at higher altitudes. Cultivating these species instead of Norway spruce will contribute to maintaining a high level of productivity across many Central European mountain forests under future climate change.     

Host and tissue variations overshadow the response of boreal moss‐associated fungal communities to increased nitrogen load

Human activity has more than doubled the amount of nitrogen entering the global nitrogen cycle, and the boreal forest biome is a nitrogen‐limited ecosystem sensitive to nitrogen load perturbation. Although bryophyte‐associated microbes contribute significantly to boreal forest ecosystem function, particularly in carbon and nitrogen cycling, little is known about their responses to anthropogenic global change. Amplicon pyrosequencing of the ITS2 region of rDNA was used to investigate how fungal communities associated with three bryophyte species responded to increased nitrogen loads in a long‐term fertilization experiment in a boreal Picea abies forest in southern Norway. Overall, OTU richness, community composition and the relative abundance of specific ecological guilds were primarily influenced by host species identity and tissue type. Although not the primary factor affecting fungal communities, nitrogen addition did impact the abundance of specific guilds of fungi and the resulting overall community composition. Increased nitrogen loads decreased ectomycorrhizal abundance, with Amphinema, Cortinarius, Russula and Tylospora OTUs responding negatively to fertilization. Pathogen abundance increased with fertilization, particularly in the moss pathogen Eocronartium. Saprophytic fungi were both positively and negatively impacted by the nitrogen addition, indicating a complex community level response. The overshadowing of the effects of increased nitrogen loads by variation related to host and tissue type highlights the complexity of bryophyte‐associated microbial communities and the intricate nature of their responses to anthropogenic global change.     

Induction of Adventitious Buds on Buds of Norway Spruce (Picea abies) Grown in vitro

Resting vegetative buds of Norway spruce, Picea abies (L.) Karst., were induced to form adventitious bud primordia when cultured on medium -containing cytokinin. After transfer of the induced buds to medium lacking cytokinin, adventitious buds developed. The adventitious buds arose from meristems formed de novo in the needle primordia. No differences were found in the ability to form adventitious buds among buds collected from trees ranging from 5–50 years old.     

Diversity of fungal latent pathogens and true endophytes associated with fruit trees in Uruguay

We have explored the fungal diversity in asymptomatic twigs of apple, peach, pear and blueberry trees, with the objective of discerning between true endophytes and latent pathogens. Several fungal genera containing known bark pathogens were found. Seven Diaporthe species—D. oxe, D. infecunda, D. serafiniae, D. phaseolorum, D. terebinthifolii, D. foeniculina and D. brasiliensis—were identified, along with Botryosphaeria dothidea, Neofusicoccum parvum, Neofusicoccum australe, Cytospora sp., Cytospora acaciae and Pestalotiopsis spp. A pathogenicity trial was undertaken to determine the role of these species on apple, pear, blueberry and peach shoots. Diaporthe brasiliensis, D. foeniculina, Diaporthe inconspicua, D. terebinthifolii, Diaporthe sp.1, Cytospora‐like isolates and Pestalotiopsis spp. isolates produced no lesions on inoculated shoots, suggesting that they could be considered true endophytes on their respective hosts. Meanwhile, some of the isolates of Diaporthe—D. oxe, Diaporthe sp.2, D. infecunda and D. serafiniae, B. dothidea, N. parvum and N. australe could be regarded as latent pathogens in their respective hosts as they produced sunken cankers and necrosis on inoculated shoots. These results demonstrate that apple, pear, blueberry and peach healthy shoots can host many known endophytic fungi along with potential wood disease‐causing fungi that should be regarded as latent pathogens.     

Characterization of a Heterobasidion irregulare endo‐rhamnogalacturonase that mediate growth on pectin

Heterobasidion irregulare is one of five Heterobasidion annosum sensu lato (s.l.) species, which are destructive pathogens in boreal and temperate forests of the northern hemisphere that causes root and butt rot in conifer. A gene encoding endo‐rhamnogalacturonase (HIRHG), which belongs to the glycoside hydrolase family 28 (GH28), was found in a quantitative trait loci (QTL) region for virulence in Heterobasidion. In this study, we showed that HIRHG is highly upregulated during necrotrophic infection of Norway spruce compared with growth in liquid culture and that the HIRHG encoded protein is produced during fungal growth on complex carbon sources. Phylogenetic analysis of endo‐rhamnogalacturonases revealed that rhamnogalacturonase genes have been lost in most of the biotrophic and hemibiotrophic plant pathogens investigated but were common in necrotrophic pathogens and saprophytic fungi. Heterologous expression of the HIRHG gene in the hemibiotrophic fungus Magnaporthe oryzae increased its capacity to grow on pectin; however, the transformed M. oryzae isolates showed significant less infection of rice leaves compared to the wild type.     

Fungi Vectored by the Bark Beetle Ips typographus Following Hibernation Under the Bark of Standing Trees and in the Forest Litter

The bark beetle Ips typographus has different hibernation environments, under the bark of standing trees or in the forest litter, which is likely to affect the beetle-associated fungal flora. We isolated fungi from beetles, standing I. typographus-attacked trees, and forest litter below the attacked trees. Fungal identification was done using cultural and molecular methods. The results of the two methods in detecting fungal species were compared. Fungal communities associated with I. typographus differed considerably depending on the hibernation environment. In addition to seven taxa of known ophiostomoid I. typographus-associated fungi, we detected 18 ascomycetes and anamorphic fungi, five wood-decaying basidomycetes, 11 yeasts, and four zygomycetes. Of those, 14 fungal taxa were detected exclusively from beetles that hibernated under bark, and six taxa were detected exclusively from beetles hibernating in forest litter. The spruce pathogen, Ceratocystis polonica, was detected occasionally in bark, while another spruce pathogen, Grosmannia europhioides, was detected more often from beetles hibernating under the bark as compared to litter. The identification method had a significant impact on which taxa were detected. Rapidly growing fungal taxa, e.g. Penicillium, Trichoderma, and Ophiostoma, dominated pure culture isolations; while yeasts dominated the communities detected using molecular methods. The study also demonstrated low frequencies of tree pathogenic fungi carried by I. typographus during its outbreaks and that the beetle does not require them to successfully attack and kill trees.     

Endophyte communities vary in the needles of Norway spruce clones

Endophytic fungi show no symptoms of their presence but can influence the performance and vitality of host trees. The potential use of endophytes to indicate vitality has been previously realized, but a standard protocol has yet to be developed due to an incomplete understanding of the factors that regulate endophyte communities. Using a culture-free molecular approach, we examined the extent to which host genotype influences the abundance, species richness, and community composition of endophytic fungi in Norway spruce needles. Briefly, total DNA was extracted from the surface-sterilized needles of 30 clones grown in a nursery field and the copy number of the fungal internal transcribed spacer (ITS) region of ribosomal DNA was estimated by quantitative PCR. Fungal species richness and community composition were determined by denaturing gradient gel electrophoresis and DNA sequencing. We found that community structure and ITS copy number varied among spruce clones, whereas species richness did not. Host traits interacting with endophyte communities included needle surface area and the location of cuttings in the experimental area. Although Lophodermium piceae is considered the dominant needle endophyte of Norway spruce, we detected this species in only 33 % of samples. The most frequently observed fungus (66 %) was the potentially pathogenic Phoma herbarum. Interestingly, ITS copy number of endophytic fungi correlated negatively with the richness of ectomycorrhizal fungi and thus potential interactions between fungal communities and their influence on the host tree are discussed. Our results suggest that in addition to environmental factors, endophyte communities of spruce needles are determined by host tree identity and needle surface area.     

Do foliar fungal communities of Norway spruce shift along a tree species diversity gradient in mature European forests?

Foliar fungal species are diverse and colonize all plants, though whether forest tree species composition influences the distribution of these fungal communities remains unclear. Fungal communities include quiescent taxa and the functionally important and metabolically active taxa that respond to changes in the environment. To determine fungal community shifts along a tree species diversity gradient, needles of Norway spruce were sampled from trees from four mature European forests. We hypothesized that the fungal communities and specific fungal taxa would correlate with tree species diversity. Furthermore, the active fungal community, and not the total community, would shift along the tree diversity gradient. High-throughput sequencing showed significant differences in the fungal communities in the different forests, and in one forest, tree diversity effects were observed, though this was not a general phenomenon. Our study also suggests that studying the metabolically active community may not provide additional information about community composition or diversity.     

Diversity and pathogenicity of ophiostomatoid fungi associated with <Emphasis Type="Italic">Tetropium</Emphasis> species colonizing <Emphasis Type="Italic">Picea abies</Emphasis> in Poland

The ophiostomatoid fungi associated with cerambycid beetles Tetropium spp. (their symbiotic vectors) colonizing Norway spruce in Poland (six species collected) were isolated. The virulence of representative isolates was evaluated through inoculations using 2-year-old Norway spruce seedlings. A total of 1325 isolates (Ophiostoma piceae, O. tetropii, O. minus, Grosmannia piceiperda, G. cucullata, and five other less frequent taxa) were obtained. Tetropium castaneum and T. fuscum were vectors of similar spectra of ophiostomatoid fungi although some differences in fungal frequency between these Tetropium spp. were found. Among the fungal associates of the Tetropium spp. collected only G. piceiperda was pathogenic, which suggests that it can play a role in the death of spruce trees following attack by Tetropium spp.     

Ectomycorrhizal fungal community structure across a bog-forest ecotone in southeastern Alaska

We examined the ectomycorrhizal (ECM) fungal community across a bog-forest ecotone in southeastern Alaska. The bog and edge were both characterized by poorly drained Histosols and a continuous layer of Sphagnum species, ericaceous shrubs, Carex species, and shore pine [Pinus contorta Dougl. ex Loud. var. contorta]. The forest had better-drained Inceptisols and Spodosols, a tree community comprised of western hemlock [Tsuga heterophylla (Raf.) Sarg.], yellow cedar (Thuja plicata Donn ex D. Don.), Sitka spruce [Picea sitchensis (Bong.) Carr.] and shore pine, and an understorey of ericaceous shrubs and herbs. ECM root tip density (tips cm^–3 soil) was significantly greater in the forest than the edge or bog and ECM colonization was significantly different in all three plant communities. The below ground ECM fungal taxa were analyzed using molecular techniques (PCR-RFLP and DNA sequencing). Three ECM fungal taxa, Suillus tomentosus (Kauffman) Singer, Cenococcum geophilum Fr.:Fr, and a Russula species, differed in relative frequency, yet were among the four most frequent in all three plant communities. Although differences in ECM fungal richness were observed across plant communities, unequal sampling of ECM roots due to root density and colonization differences confounded richness comparisons. Using resampling procedures for creating taxon-accumulation curves as a function of sampled ECM roots revealed similarities in cumulative ECM fungal taxa richness across the ecotone.