Interkingdom and intrakingdom interactions in the microbiome of Heterobasidion fruiting body and associated decayed woody tissues.

IMPORTANCE: We applied macro- (forest stand and forest management) and micro-scale (bacterial and fungal community) analyses for a better understanding of the Heterobasidion pathosystem and associated wood decay process. The core microbiome, as defined by hierarchy analysis and a consistent model, and environmental factors correlation with the community assembly were found to be novel.

Bacteria Community Inhabiting Heterobasidion Fruiting Body and Associated Wood of Different Decay Classes.

The microbiome of Heterobasidion-induced wood decay of living trees has been previously studied; however, less is known about the bacteria biota of its perennial fruiting body and the adhering wood tissue. In this study, we investigated the bacteria biota of the Heterobasidion fruiting body and its adhering deadwood. Out of 7,462 operational taxonomic units (OTUs), about 5,918 OTUs were obtained from the fruiting body and 5,469 OTUs were obtained from the associated dead wood. Interestingly, an average of 52.6% of bacteria biota in the fruiting body was shared with the associated dead wood. The overall and unique OTUs had trends of decreasing from decay classes 1 to 3 but increasing in decay class 4. The fruiting body had the highest overall and unique OTUs number in the fourth decay class, whereas wood had the highest OTU in decay class 1. Sphingomonas spp. was significantly higher in the fruiting body, and phylum Firmicutes was more dominant in wood tissue. The FAPROTAX functional structure analysis revealed nutrition, energy, degradation, and plant-pathogen-related functions of the communities. Our results also showed that bacteria communities in both substrates experienced a process of a new community reconstruction through the various decay stages. The process was not synchronic in the two substrates, but the community structures and functions were well-differentiated in the final decay class. The bacteria community was highly dynamic; the microbiota activeness, community stability, and functions changed with the decay process. The third decay class was an important turning point for community restructuring. Host properties, environmental factors, and microbial interactions jointly influenced the final community structure. Bacteria community in the fruiting body attached to the living standing tree was suppressed compared with those associated with dead wood. Bacteria appear to spread from wood tissue of the standing living tree to the fruiting body, but after the tree is killed, bacteria moved from fruiting body to wood. It is most likely that some of the resident endophytic bacteria within the fruiting body are either parasitic, depending on it for their nutrition, or are mutualistic symbionts.

Is Decreased Xylem Sap Surface Tension Associated With Embolism and Loss of Xylem Hydraulic Conductivity in Pathogen-Infected Norway Spruce Saplings?

Increased abiotic stress along with increasing temperatures, dry periods and forest disturbances may favor biotic stressors such as simultaneous invasion of bark beetle and ophiostomatoid fungi. It is not fully understood how tree desiccation is associated with colonization of sapwood by fungi. A decrease in xylem sap surface tension (σxylem) as a result of infection has been hypothesized to cause xylem embolism by lowering the threshold for air-seeding at the pits between conduits and disruptions in tree water transport. However, this hypothesis has not yet been tested. We investigated tree water relations by measuring the stem xylem hydraulic conductivity (Kstem), σxylem, stem relative water content (RWCstem), and water potential (Ψstem), and canopy conductance (gcanopy), as well as the compound composition in xylem sap in Norway spruce (Picea abies) saplings. We conducted our measurements at the later stage of Endoconidiophora polonica infection when visible symptoms had occurred in xylem. Saplings of two clones (44 trees altogether) were allocated to treatments of inoculated, wounded control and intact control trees in a greenhouse. The saplings were destructively sampled every second week during summer 2016. σxylem, Kstem and RWCstem decreased following the inoculation, which may indicate that decreased σxylem resulted in increased embolism. gcanopy did not differ between treatments indicating that stomata responded to Ψstem rather than to embolism formation. Concentrations of quinic acid, myo-inositol, sucrose and alkylphenol increased in the xylem sap of inoculated trees. Myo-inositol concentrations also correlated negatively with σxylem and Kstem. Our study is a preliminary investigation of the role of σxylem in E. polonica infected trees based on previous hypotheses. The results suggest that E. polonica infection can lead to a simultaneous decrease in xylem sap surface tension and a decline in tree hydraulic conductivity, thus hampering tree water transport.

Dual RNA-seq analysis provides new insights into interactions between Norway spruce and necrotrophic pathogen Heterobasidion annosum s.l.

BACKGROUND: Root and butt rot of conifer trees caused by fungi belonging to the Heterobasidion annosum species complex is one of the most economically important fungal diseases in commercial conifer plantations throughout the Northern hemisphere. We investigated the interactions between Heterobasidion fungi and their host by conducting dual RNA-seq and chemical analysis on Norway spruce trees naturally infected by Heterobasidion spp. We analyzed host and pathogen transcriptome and phenolic and terpenoid contents of the spruce trees. RESULTS: Presented results emphasize the role of the phenylpropanoid and flavonoid pathways in the chemical defense of Norway spruce trees. Accumulation of lignans was observed in trees displaying symptoms of wood decay. A number of candidate genes with a predicted role in the higher level regulation of spruce defense responses were identified. Our data indicate a possible role of abscisic acid (ABA) signaling in the spruce defense against Heterobasidion infection. Fungal transcripts corresponding to genes encoding carbohydrate- and lignin-degrading enzymes, secondary metabolism genes and effector-like genes were expressed during the host colonization. CONCLUSIONS: Our results provide additional insight into defense strategies employed by Norway spruce trees against Heterobasidion infection. The potential applications of the identified candidate genes as markers for higher resistance against root and butt rot deserve further evaluation.

Cadophora margaritata sp. nov. and other fungi associated with the longhorn beetles Anoplophora glabripennis and Saperda carcharias in Finland.

Symbiosis with microbes is crucial for survival and development of wood-inhabiting longhorn beetles (Coleoptera: Cerambycidae). Thus, knowledge of the endemic fungal associates of insects would facilitate risk assessment in cases where a new invasive pest occupies the same ecological niche. However, the diversity of fungi associated with insects remains poorly understood. The aim of this study was to investigate fungi associated with the native large poplar longhorn beetle (Saperda carcharias) and the recently introduced Asian longhorn beetle (Anoplophora glabripennis) infesting hardwood trees in Finland. We studied the cultivable fungal associates obtained from Populus tremula colonised by S. carcharias, and Betula pendula and Salix caprea infested by A. glabripennis, and compared these to the samples collected from intact wood material. This study detected a number of plant pathogenic and saprotrophic fungi, and species with known potential for enzymatic degradation of wood components. Phylogenetic analyses of the most commonly encountered fungi isolated from the longhorn beetles revealed an association with fungi residing in the Cadophora-Mollisia species complex. A commonly encountered fungus was Cadophora spadicis, a recently described fungus associated with wood-decay. In addition, a novel species of Cadophora, for which the name Cadophora margaritata sp. nov. is provided, was isolated from the colonised wood.

Mycobiome analysis of asymptomatic and symptomatic Norway spruce trees naturally infected by the conifer pathogens Heterobasidion spp.

Plant microbiome plays an important role in maintaining the host fitness. Despite a significant progress in our understanding of the plant microbiome achieved in the recent years, very little is known about the effect of plant pathogens on composition of microbial communities associated with trees. In this study, we analysed the mycobiome of different anatomic parts of asymptomatic and symptomatic Norway spruce trees naturally infected by Heterobasidion spp. We also investigated the primary impact of the disease on the fungal communities, which are associated with Norway spruce trees. Our results demonstrate that the structure of fungal communities residing in the wood differed significantly among symptomatic and asymptomatic Heterobasidion-infected trees. However, no significant differences were found in the other anatomic regions of the trees. The results also show that not only each of individual tree tissues (wood, bark, needles and roots) harbours a unique fungal community, but also that symptomatic trees were more susceptible to co-infection by other wood-degrading fungi compared to the asymptomatic ones.

Dominant Tree Species and Soil Type Affect the Fungal Community Structure in a Boreal Peatland Forest.

Boreal peatlands play a crucial role in global carbon cycling, acting as an important carbon reservoir. However, little information is available on how peatland microbial communities are influenced by natural variability or human-induced disturbances. In this study, we have investigated the fungal diversity and community structure of both the organic soil layer and buried wood in boreal forest soils using high-throughput sequencing of the internal transcribed spacer (ITS) region. We have also compared the fungal communities during the primary colonization of wood with those of the surrounding soils. A permutational multivariate analysis of variance (PERMANOVA) confirmed that the community composition significantly differed between soil types (P< 0.001) and tree species (P< 0.001). The distance-based linear models analysis showed that environmental variables were significantly correlated with community structure (P< 0.04). The availability of soil nutrients (Ca [P= 0.002], Fe [P= 0.003], and P [P= 0.003]) within the site was an important factor in the fungal community composition. The species richness in wood was significantly lower than in the corresponding soil (P< 0.004). The results of the molecular identification were supplemented by fruiting body surveys. Seven of the genera of Agaricomycotina identified in our surveys were among the top 20 genera observed in pyrosequencing data. Our study is the first, to our knowledge, fungal high-throughput next-generation sequencing study performed on peatlands; it further provides a baseline for the investigation of the dynamics of the fungal community in the boreal peatlands.

Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch in a field trial.

Heterogenous chitinases have been introduced in many plant species with the aim to increase the resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal communities in the field trial of 15 transgenic and 8 wild-type silver birch (Betula pendula Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis and identified by sequencing the ITS1 region to reveal the operational taxonomic units. ECM colonization was less intense in 7 out of 15 transgenic lines than in the corresponding non-transgenic control plants, but the slight decrease in overall ECM colonization in transgenic lines could not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line showing fairly weak sugar beet chitinase IV expression without significantly increased total endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria) were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that genotype was a more important factor determining the structure of fungal communities than the transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal community was found.

Use of a breeding approach for improving biocontrol efficacy of Phlebiopsis gigantea strains against Heterobasidion infection of Norway spruce stumps.

Sixty-four wild heterokaryotic isolates of Phlebiopsis gigantea were analysed for asexual spore production, growth rate and competitive ability against Heterobasidion in vitro, as well as growth rate in Norway spruce wood. These P. gigantea traits were considered important for controlling infection of Norway spruce stumps by spores of Heterobasidion spp. Ten most promising P. gigantea isolates were crossed with each other and 172 F(1) progeny heterokaryons were analysed for the above-mentioned traits. Thirteen most promising progeny heterokaryons were selected and their biocontrol ability against infection by Heterobasidion was compared with the parental isolates in stem pieces of Norway spruce. The results indicated that the progeny strains had generally better traits and control efficacy than the parental strains. The genetic effects accounted for a part of the variations between progeny and parental strains. This further suggests that there is a potential to improve the biocontrol properties of P. gigantea through breeding.

North American populations of Entoleuca mammata are genetically more variable than populations in Europe.

Entoleuca mammata (syn. Hypoxylon mammatum) is a damaging pathogen of Populus tremuloides and P. grandidentata in North America and P. tremula in Europe, where the fungus occurs only sporadically in alpine regions and Scandinavia. It has been hypothesized that E. mammata was introduced to Europe from North America. In this study, E. mammata isolates collected from Europe and North America were compared by a sequence analysis of two DNA markers derived from DNA fingerprints. The objective of the study was to elucidate the relationship between North American and European E. mammata populations by testing two hypotheses: (1) North American and European isolates are conspecific; and (2) the fungus was introduced between continents causing both a founder effect and a genetic bottleneck. North American populations were found to be more polymorphic, but no major phylogenetic differences between fungal isolates collected from different continents were found. This result combined with the historical observations of the disease in Europe implies that E. mammata was introduced to Europe several centuries ago.

Migrational capacity of Fennoscandian populations of Venturia tremulae.

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